δ-内毒素基因及其使用方法

专利名称：：δ-内毒素基因及其使用方法5-内毒素基因及其使用方法本申请是申请日为2004年02月20日、申请号为200480007563.8、发明名称为"5-内毒素基因及其使用方法"的发明专利申请的分案申请。发明领域本发明涉及分子生物学领域。所提供的是编码杀虫蛋白质的新基因。这些蛋白质及编码它们的核酸序列可用于制备杀虫制剂以及生产转基因的抗害虫植物。发明背景苏云金芽孢杆菌是形成孢子的革兰氏阳性土壤细菌，特征在于它能产生对某些目和种的昆虫有特异性毒性、但对植物和其它非靶向生物体无害的晶状内含物。因此，含苏云金芽孢杆菌菌林或它们的杀虫蛋白质的组合物可用作环境可接受的杀虫剂，以抑制农业有害昆虫或针对多种人或动物疾病的昆虫载体。来自苏云金芽孢杆菌的晶体(Cry)蛋白质(5-内毒素)具有主要针对鳞翅类、双翅类和鞘翅类幼虫的有效杀虫活性。这些蛋白质还显示出抗膜翅目、同翅目、虱、食毛目和螨等有害目以及诸如线虫、扁形动物(Platyhelminthes)和Sarcomastigorphora等其它无脊推动物目的活性(Feitelson(1993)苏云金芽孢杆菌家族树，高级基因工程杀虫剂中(TheBacillusThuringiensisfamilytree,AdvancedEngineeredPesticides)MarcelDekker，Inc.,NewYork,N.Y.)。这些蛋白质主要基于其杀虫活性而最初被划分为Cryl至CryV。主要的种类是鳞翅类特异的(I)、鳞翅类和双翅类特异的(II)、鞘翅类特异的(III)、双翅类特异的(IV)以及线虫类特异的(V)和(VI)。所述蛋白质进一步被划分为亚家族，各个家族内更密切相关的蛋白质被指派上分类字母，诸如CrylA、CrylB、CrylC等。在各亚类内更紧密相关的蛋白质则命名为诸如CrylCl、CrylC2等。最近出现了一种针对Cry基因的新命名法，其基于的是氨基酸序列同源性而非昆虫靼特异性(Crickmore爭(1998)#/cro6/o/.Wo厶We7.62:807-813)。在该新的分类法中，每种毒素被分配了一个独特的名称，其中合并了第一等级(阿拉伯数字)、第二等级(大写字母)、第三等级(小写字母)和第四等级(另一阿拉伯数字)。在新的分类中，罗马数字被转换成第一等级的阿拉伯数字。序列同一性小于45%的蛋白质具有不同的第一等级，而对于第二和第三等级而言这一标准分别为78%和95%。晶体蛋白质在被昆虫中肠摄取和溶解前不呈现杀虫活性。被摄取的毒素原在昆虫消化道内被蛋白酶水解成活性的有毒分子。(貼fte和Whiteley(1989)飾ro6/o人紐53:242-255)。此毒素与乾目标幼虫中肠内的顶端刷状边缘受体结合并插入顶端膜中，产生离子通道或孔，导致幼虫死亡。5-内毒素通常具有5个保守的序列结构域和三个保守的结构性结构域(参阅，例ft口，deMaagd爭(2001)rre/7dsCe/7ef/cs17:193-199)。第一个保守的结构性结构域由7个ot螺旋组成，并涉及膜插入和孔形成。结构域II由排列成希腊语钥匙构形的三个P折叠片组成，而结构域III由"果冻-巻"形式的两个反向平行卩折叠组成(deMaagd爭(2001)同上文)。结构域II和III涉及受体识别和结合，并因此被认为是毒素特异性的决定区。因为昆虫可造成的破坏，所以一直需要发现新形式的苏云金芽孢杆菌5-内毒素。发明简述本发明提供了赋予细菌、植物、植物细胞、组织和种子以杀虫剂抗性的组合物和方法。组合物包括编码5-内毒素和5-内毒素相关多肽序列的分离核酸分子、包含这些核酸分子的载体和包含这些载体的宿主细胞。组合物还包括内毒素的分离或重组多肽序列、包含这些多肽的组合物以及针对那些多肽的抗体。所述核酸序列可用于DNA构建体或表达盒中，用于生物体中的转化和表达，包括微生物和植物。所述核苷酸或氨基酸序列可以是已被设计成最适于在生物体中表达的合成序列，包括但不局限于，微生物或植物。组合物还包括被转化的细菌、植物、植物细胞、组织和种子。具体而言，本发明提供了含有编码SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29中所示氨基酸序列的核苷酸序列或SEQIDN0:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28中所给出的核苷酸序列的分离核酸分子以及它们的变体序列也包括在内。还提供了可用于产生本发明的多肽的方法，以及利用那些多肽来控制或杀死鳞翅类或鞘翅类害虫的方法。其是细菌和植物。这些生物体及其来源的组合物是农业用途所需要的。素或5-内毒素相关蛋白质，或用于检测产品或生物体中5-内毒素或5-内毒素相关蛋白质或者核酸的存在。附图描述图1显示了AXMI-004(SEQIDNO:3)与crylAc(SEQIDNO:31)、crylCa卿IDNO:32)、cry2Aa(SEQIDNO:34)、cry3Aal(SEQIDNO:35)、crylla(SEQIDNO:33)和cry7Aa(SEQIDN0:41)的序列比对。具有C-末端非毒性结构域的毒素如图所示被人为截短。该序列比对显示了以黑色标示的最高度保守氨基酸残基和以灰色标示的高度保守氨基酸残基。发现保守组l是从SEQIDN0:3的大约第174位至大约第196位氨基酸残基。发现保守组2是从SEQIDNO:3的大约第250位至大约笫292位氨基酸残基。发现保守组3是从SEQIDNO:3的大约第476位至大约第521位氨基酸残基。发现保守组4是从SEQIDNO:3的大约第542位至大约552位氨基酸残基。发现保守组5是从SEQIDNO:3的大约第618位至大约第628位氨基酸残基。图2A、B和C显示了AXMI-006(SEQIDNO:7)与crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylla(SEQIDNO:33)、cry3Aal(SEQIDNO:35)、cry3Ba(SEQIDNO:36)、cry4Aa(SEQIDNO:38)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)和cry24Aa(SEQIDNO:47)的序列比对。具有C-末端非毒性结构域的毒素如图所示被人为截短。该序列比对显示了以黑色标示的最高度保守氨基酸残基和以灰色标示的高度保守氨基酸残基。发现保守组1是从SEQIDNO:7的大约第218位至大约第239位氨基酸残基。发现保守组2是从SEQIDNO:7的大约第300位至大约第350位氨基酸残基。发现保守组3是从SEQIDNO:7的大约第547位至大约第592位氨基酸残基。发现保守组4是从SEQIDN0:7的大约笫611位至大约621位氨基酸残基。发现保守组5是从SEQIDNO:7的大约第694位至大约第704位氨基酸残基。图3A、B和C显示了AXMI-007(SEQIDNO:9)与crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylla(SEQIDNO:33)、cry3Aal(SEQIDNO:35)、cry3Ba(SEQIDNO:36)、cry4Aa(SEQIDNO:38)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa卿IDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)和cry24Aa(SEQIDNO:47)的序列比对。具有C-末端非毒性结构域的毒素如图所示被人为截短。该序列比对显示了以黑色标示的最高度保守氨基酸残基和以灰色标示的高度保守氨基酸残基。发现保守组l是从SEQIDN0:9的大约第217位至大约第238位氨基酸残基。发现保守组2是从SEQIDNO:9的大约第299位至大约笫347位氨基酸残基。发现保守组3是从SEQIDNO:9的大约第445位至大约笫590位氨基酸残基。发现保守组4是从SEQIDN0:9的大约笫609位至大约619位氨基酸残基。发现保守组5是从SEQIDNO:9的大约第692位至大约第702位氨基酸残基。图4A、B和C显示了AXMI-008(SEQIDNO:14)与crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylla(SEQIDNO:33)、cry2Aa(SEQIDNO:34)、cry3Aal(SEQIDNO:35)、cry3Bb(SEQIDNO:37)、cry4Aa(SEQIDNO:38)、cry4Ba(SEQIDNO:39)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)、cry24Aa(SEQIDNO:47)、cry25Aa(SEQIDNO:48)、cry39Aal(SEQIDN0:49)和cry楊al(SEQIDN0:51)的序列比对。具有C-末端非毒性结构域的毒素如图所示被人为截短。该序列比对显示了以黑色标示的最高度保守氨基酸残基和以灰色标示的高度保守氨基酸残基。发现保守组1是从SEQIDNO:14的大约第185位至大约第206位氨基酸残基。发现保守组2是从SEQIDNO:14的大约第276位至大约第318位氨基酸残基。发现保守组3是从SEQIDN0:14的大约第497位至大约第547位氨基酸残基。发现保守组4是从SEQIDNO:14的大约第576位至大约586位氨基酸残基。发现保守组5是从SEQIDNO:14的大约第657位至大约第667位氨基酸残基。图5A和B显示了AXMI-008orf2(SEQIDNO:18)与cryl9Aa-orf2(SEQIDNO:46)、crybun2-orf2卿IDNO:50)、crybun3-orf2(SEQIDNO:52)、cry4Aa(SEQIDNO:38)和cry4Ba(SEQIDNO:39)的序列比对。该序列比对显示了以黑色标示的最高度保守氨基酸残基和以灰色标示的高度保守氨基酸残基。图6A、B和C显示了AXMI-009(SEQIDNO:20)与crylAa(SEQIDNO:30)、crylAc(SEQIDNO:31)、crylCa(SEQIDNO:32)、crylla(SEQIDNO:33)、cry3Aal(SEQIDNO:35)、cry3Ba(SEQIDNO:36)、cry3Bb(SEQIDNO:37)、cry4Aa(SEQIDNO:38)、cry6Aa(SEQIDNO:40)、cry7Aa卿IDNO:41)、cry8Aa卿IDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)、cry24Aa(SEQIDNO:47)、cry25Aa(SEQIDNO:48)和cry40Aal(SEQIDNO:51)的序列比对。具有C-末端非毒性结构域的毒素如图所示被人为截短。该序列比对显示了以黑色标示的最高度保守氨基酸残基和以灰色标示的高度保守氨基酸残基。发现保守组1是从SEQIDNO:20的大约笫196位至大约第217位氨基酸残基。发现保守组2是从SEQIDNO:20的大约第269位至大约第311位氨基酸残基。发现保守组3是从SEQIDNO:20的大约第514位至大约第556位氨基酸残基。发现保守组4是从SEQIDN0:20的大约第574位至大约584位氨基酸残基。发现保守组5是从SEQIDNO:20的大约第651位至大约第661位氨基酸残基。图7A、B和C显示了AXMI-014(SEQIDNO:27)与crylAa(SEQIDNO:30)、crylAc卿IDNO:31)、crylla(SEQIDNO:33)、cry2Aa(SEQIDNO:34)、cry3Aal(SEQIDNO:35)、cry3Bb(SEQIDNO:37)、cry4Aa(SEQIDNO:38)、cry4Ba(SEQIDNO:39)、cry6Aa(SEQIDNO:40)、cry7Aa(SEQIDNO:41)、cry8Aa(SEQIDNO:42)、crylOAa(SEQIDNO:43)、cryl6Aa(SEQIDNO:44)、cryl9Ba(SEQIDNO:45)、cry24Aa(SEQIDNO:47)、cry25Aa(SEQIDNO:48)、cry39Aal(SEQIDN0:49)和cry锡al(SEQIDN0:51)的序列比对。具有C-末端非毒性结构域的毒素如图所示被人为截短。该序列比对显示了以黑色标示的最高度保守氨基酸残基和以灰色标示的高度保守氨基酸残基。发现保守组1是从SEQIDN0:27的大约第177位至大约第188位氨基酸残基。发现保守组2是从SEQIDN0:27的大约第251位至大约第293位氨基酸残基。发现保守组3是从SEQIDNO:27的大约第483位至大约第533位氨基酸残基。发现保守组4是从SEQIDNO:27的大约第552位至大约562位氨基酸残基。图8显示了4-20%梯度SDS丙烯酰胺凝胶的照片。泳道1-4包括表达69kDAXMI-004蛋白质的各种浓度的形成孢子的芽孢杆菌细胞培养物。泳道5-8包括各种浓度的BSA。泳道9包含分子量标记物。箭头指示69kD的带。发明详述本发明针对的是用于调控生物体、尤其是植物或植物细胞中害虫抗性的组合物和方法。所述方法包括用编码本发明5-内毒素或5-内毒素相关蛋白质的核苷酸序列转化生物体。具体而言，本发明的核苷酸序列可用于制备具有杀虫活性的植物和微生物。因而提供了已转化的细菌、植物、植物细胞、植物组织和种子。组合物是苏云金芽孢杆菌的5-内毒素或5-内毒素相关核酸和蛋白质。所述序列可用于构建表达载体用于随后转化入目的生物体内，作为探针用于分离其它的5-内毒素或5-内毒素相关基因，以及用本领域已知方法产生改变的杀虫蛋白质，诸如结构域交换或DM改组。所述蛋白质可用于抑制或杀死鳞翅类或鞘翅类有害种群以及用于产生具有杀虫活性的组合物。定义"5-内毒素"指来自苏云金芽孢杆菌且对一种或多种害虫具有毒性的毒素，所述害虫包括、但不局限于鳞翅目、双翅目和鞘翅目等的种类。在某些情况下，5-内毒素蛋白质已从其它生物体分离出来了，包括双酶梭状芽孢杆菌(C/os"/d/"/z76/尸e/7Z7e/^a/zs)和？类芽孢杆菌(户ae"^ac27/^/o/7//7/ae)。5—内毒素蛋白质包含由本文所公布的全长核苷酸序列推导出来的氨基酸序列，以及由于使用备选的下游起始位点或者由于产生具有杀虫活性的较短蛋白质的加工所造成的比全长序列更短的氨基酸序列。加工可发生在所述蛋白质所表达的生物体内，或摄取所述蛋白质后在害虫体内。5-内毒素包括被确认为cryl至cry43、cytl和cyt2的蛋白质以及Cyt-类毒素。目前有超过250种具有广泛特异性和毒性的已知5-内毒素。更全面的名单参阅Crickmore爭(1998)，#/cro6/o/,#。人齡.62:807-813，而定期的更新资料参阅画.biols.susx.ac.uk/Home/Neil-Crickmore/Bt/index上的Crickmore爭(20(J3)"苏云金芽孢杆菌毒素命名(丑ac/7/usf力uri/^/e/7s/stoxinnomenclature)"。细菌基因，诸如本发明的AXMI基因，通常具有多个甲硫氨酸起始密码子接近开发阅读框架的起始点。经常地，开始于一个或多个这些起始密码子的翻译会导致产生功能性蛋白质。这些起始密码子可包括ATG密码子。不过，诸如芽孢杆菌等的细菌还将密码子GTG识别为起始密码子，而翻译起始于GTG密码子的蛋白质在笫一个氨基酸处含有曱硫氨酸。此外，并非常常可以争先确定这些密码子中的哪一个在细菌中天然使用。因此，可以理解，利用这些变通性曱疏氨酸密码子之一还可导致产生编码杀虫活性的5-内毒素蛋白质。例如，本发明AXMI-0045-内毒素的一个备选起始位点是SEQIDNO:1第385位碱基对处。从此备选起始位点开始的翻译产生了SEQIDNO:5中所显示的氨基酸序列。本发明AXMI-0075-内毒素蛋白质的一个备选起始位点可以是SEQIDN0:8第151位碱基对处。从此备选起始位点开始的翻译产生了SEQIDNO:11中所显示的氨基酸序列。本发明的AXMI-0085-内毒素蛋白质的一个备选起始位点可以是SEQIDNO:12的第177位核苷酸处。从此备选起始位点开始的翻译产生了SEQIDN0:16中所示的氨基酸序列。本发明的AXMI-0095-内毒素蛋白质的一个备选起始位点可以是SEQIDN0:19的第34位核苷酸处。从此备选起始位点开始的翻译产生了SEQIDNO:22中所示的氨基酸序列。本发明的AXMI-0095-内毒素蛋白质的另一个备选起始位点可以是SEQIDNO:1的第64位核苷酸处。从此备选起始位点开始的翻译产生了SEQIDNO:24中所示的氨基酸序列。本发明的AXMI-0145-内毒素蛋白质的一个备选起始位点可以是SEQIDN0:25的第136位核苷酸处。从此备选起始位点开始的翻译产生了SEQIDNO:29中所示的氨基酸序列。这些5-内毒素蛋白质都包括在本发明的范围内，可用于本发明的方法中。此外，在所公布的编码一个或多个5-内毒素相关蛋白质的核普酸序列中可以有一个或多个另外的开发阅读框架。"5-内毒素相关蛋白质"指用不同于本发明5-内毒素所用，使用另一开发阅读框架由此处所公布的核苷酸序列编码的蛋白质。诸如此类的蛋白质在本领域中已知可作为辅助蛋白质、稳定序列或5-内毒素相关蛋白质。这些5-内毒素相关蛋白质可具有杀虫活性，或可在促进5-内毒素蛋白质表达中起重要作用。用于检测杀虫活性以及用于测定5-内毒素相关蛋白质对5-内毒素蛋白质表达和晶体形成的影响的方法是本领域已知的(参阅，例如，Park爭(1999)尸i^51V/c/"o6/。/.181:319-327;Ge爭(1998)層S165:35-41;Rosso和Delecluse(1997)j/7/7/.i"/2"'ro/7.63:4449—4455)。这些5—内毒素相关蛋白质包括在本发明范围内，并可单独或与已知的5-内毒素蛋白质联合用于本文所公布的方法中。在某一实施方案中，5-内毒素相关蛋白质具有存在于SEQIDNO:18中的氨基酸序列，由SEQIDNO:17的核苷酸序列编码。"植物细胞"指所有已知形式的植物，包括未分化的组织(如愈伤组织)、悬浮培养细胞、原生质体、叶细胞、根细胞、韧皮部细胞、植物种子、花粉、繁殖体、胚等等。"植物表达盒"指能导致蛋白质在植物细胞中从开放阅读框架表达的DNA构建体。一般其中包含启动子和编码序列。经常地，这样的构建体还包含3，非翻译区。所述构建体可包含"信号序列"或"前导序列"，以促进肽在翻译的同时或翻译后转运至某些细胞内结构，诸如叶绿体(或其它质体)、内质网或高尔基体。"信号序列"指已知或猜测会导致翻译时或翻译后的跨细胞膜肽转运的序列。在真核细胞中，这通常包括分泌入高尔基体内，具有某些由此产生的糖基化作用。"前导序列"指在翻译时产生的氨基酸序列足以引起肽链共翻译转运至亚细胞器的任何序列。因此，这包括通过进入内质网、穿越液泡、包括叶绿体、线粒体等在内的质体而耙向转运和/或糖基化作用的前导序列。"植物转化栽体"指植物细胞有效转化所必需的DM分子。这样的分子可由一个或多个植物表达盒组成，且可组装入一个以上的"载体"DNA分子中。例如，二元载体是利用两个非毗连的DNA载体编码(Hellens和Mullineaux(2000)7ye/7ffs//尸7fl/2f5We/7ce5:446-451)。"栽体"指设计成在不同宿主细胞之间转移的核酸构建体."表达载体"指能在外来细胞中掺入、整合并表达异源DNA序列或片段的载体。"转基因植物"或"已转化的植物"或"稳定转化的植物或细胞组织"指在植物细胞中已掺入或整合了外源核酸序列或DNA片段的植酸序列，以及可能是内源的或存在于未转化的植物细胞内的那些核酸序列。"异源的"通常指对于它们所存在的细胞或部分天然基因组而言不是内源性的，且已通过感染、转染、显微注射、电穿孔、显微投景^(microprojection)等方式力口入细胞内的核酸序歹'J。"启动子"指起到指导下游编码序列转录的功能的核酸序列。启动子与其它转录和翻译调节核酸序列(也称"调控序列")一起是目的DM序列表达所必需的。本文提供了赋予杀虫活性的新的分离核苷酸序列。还提供了5-内毒素和5-内毒素相关蛋白质的氨基酸序列。由此基因翻译所产生的蛋白质可使细胞抑制或杀死摄取它的害虫。"分离的"或"纯化的"核酸分子或蛋白质或者其生物学活性部分指的是，在用重组技术生产时基本上无其它细胞物质或培养基，或者在化学合成时基本上无化学前体或其它化学物质。优选的，"分离核酸两侧(即，位于所述核酸5，和3，末端的序列)的序列(优选蛋白质编码序列)。就本发明的目的而言，"分离的"用于描述核酸分子时，并不包括分离的染色体。例如，在各种实施方案中，分离的编码5-内毒素或5-内毒素相关蛋白的核酸分子可包含天然存在于所述核酸来源的细胞的基因组DNA中该核酸分子两侧的小于大约5kb、4kb、3kb、2kb、1kb、0.5kb或0.1kb的核苷酸序列。基本上无细胞物质的5-内毒素或5-内毒素相关蛋白质包括具有少于大约30%、20%、10%或5%(千重)的非5-内毒素或非5-内毒素相关蛋白质(在此也称为"污染蛋白质")的蛋白质制品。本发明的各个方面在以下部分有更进一步的详述。分离的核酸分子及其变体和片段本发明的一方面是关于含有编码5-内毒素或5-内毒素相关蛋白质和多肽或其生物学活性部分的核苷酸序列的分离核酸分子，以及足以用作杂交探针来鉴定编码5-内毒素或5-内毒素相关蛋白质的核酸分子。用于此处时，术语"核酸分子"意在包括DNA分子(如，cDNA或基因组DNA)和RNA分子(如，mRNA)和用核苷酸类似物产生的所述DNA或RNA的类似物。所述核酸分子可以是单链或双链的，但优选是双链DNA。编码本发明蛋白质的核普酸序列包括SEQIDN0S:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26和28中所提出的序列及其互补序列。"互补序列"意指足以与给定核苷酸序列互补、从而可与该给定核苷酸序列杂交形成稳定的双联体的核普酸序列。由这些核苷酸序列编码的5-内毒素或5-内毒素相关蛋白质的相应氨基酸序列如SEQIDN0S:3、5、7、9、11、14、16、18、20、22、24、27和29中所示。作为这些5-内毒素或5-内毒素相关蛋白质编码核苷酸序列的片段的核酸分子也包括在本发明内。"片段"指编码5-内毒素或5-内毒素相关蛋白质的核苷酸序列的一部分。核苷酸序列的片段可编码5-内毒素或5-内毒素相关蛋白质的生物学活性部分，或可能是可以用下文所述方法作为杂交探针或PCR引物的片段。作为5-内毒素或5-内毒素相关核苷酸序列的片段的核酸分子包含至少约15、20、50、75、100、200、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1100、1200、1300、1400、1500、1600、1700、1800、1900、2000、2100、2200、2300、2400、2500、3000、3500、4000、4500、5000、5500个核苷酸，或者多达本文所公布的全长5-内毒素或5-内毒素相关蛋白质编码核苷酸序列的核苷酸数目(例如，对于SEQIDNO:1而言是2190个核苷酸，对于SEQIDNO:2而言是1890个核苷酸，等等)，这将取决于目的用途。本发明的核苷酸序列的片段将编码保留5-内毒素或5-内毒素相关蛋白质的生物学活性、从而分别保持了杀虫活性或5-内毒素相关蛋白质活性的蛋白质片段。"5-内毒素活性"指杀虫活性。"5-内毒素相关蛋白质活性"指所述蛋白质具有杀虫活性，或所述蛋白质可提高5-内毒素蛋白质的表达。这种蛋白质表达的提高可以以任何机制发生。"保持活性"指所述片段具有5-内毒素或5-内毒素相关蛋白质的活性的至少约30%、优选至少约50%、更优选至少约70%、还更优选至少约80%。用于测定5-内毒素相关蛋白质对5-内毒素蛋白质表达和晶体形成的影响的方法是本领域所已知的(参阅，例如，Park爭(1999)尸^/y181:319-327;Ge爭(1998)#/cro6/o/.丄e〃.165:35-41;Rosso和Delecluse(1997)铷/.63:4449-4455)。用于测定杀虫活性的方法在本领域内是众所周知的。参阅，例如，Czapla和Lang(1990)/.fco/.射画/.83(6):2480-2485;Andrews爭(1988)肠c力e瓜/.252:199-206;Marrone爭(1985)/.o/*Aco/70/z/c^^o/z7o/o^f78:290-293;和美国专利5,743,477，所有这些都全文收编于此作为参考。编码本发明蛋白质生物学活性部分的5-内毒素或5-内毒素相关蛋白质编码核苷酸序列片段将编码本发明全长5-内毒素或5-内毒素相关蛋白质中的至少约15、25、30、50、75、100、125、150、175、200、250、300、350、400、450、500、550、600或65。个连续的氨基酸，或最多可达其中所存在的总氨基酸数目(例如，对于SEQIDNO:3而言是629个氨基酸，对于SEQIDNO:5而言是601个氨基酸，等等)。优选的本发明5-内毒素或5-内毒素相关蛋白质是由与SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29中的核苷酸序列足够一致的核苷酸序列编码的。"足够一致的"指用标准参数，通过本文所述序列比对程序之一与参考序列比较而言具有至少约60%或65%序列同一性、优选至少约70%或75%序列同一性、更优选至少约稀或85%序列同一性、最优选至少约90%、91%、92%、93%、94%、95%、96%、97%、98%或99%序列同一性的氨基酸或核苷酸序列。本领域技术人员应认识到，这些数值可适当调整，以确定由于密码子简并性、氨基酸相似性、阅读框架定位等原因而由两种核苷酸序列编码的蛋白质的相应同一性。为了确定两种氨基酸序列或两种核酸的同一性百分比，将这些序列进行比对以达到最佳的比较效果。两个序列之间的同一性百分比是所述序列共有的相同位置数目的函数(即，同一性百分比=相同位置的数目/总位置数目(如重叠位置)x100)。在某一实施方案中，所述的两个序列长度相同。两个序列之间的同一性百分比可以用类似于下文所述方法的技术确定，允许或不允许有缺口。在计算同一性百分比时，通常严格匹配的才被计算在内。两个序列之间同一性百分比的确定可用数学运算法则完成。用于比较两个序列的数学运算法则的非局限性例子是文献Karlin和Altschul(1990)尸roc.#<2〃.87:2264中提出的运算法则，根据文献Karlin和Altschul(1993)尸roc.Ms〃.Jcat/.5W.90:5873-5877进行修正。这样的运算法则被合并入文献Altschul爭(1990)/.215:403的BLASTN和BLASTX程序中。BLAST核苷酸搜索可用BLASTN软件进行(得分=100，词长=12)，以获得与本发明5-内毒素或5-内毒素相关核酸分子同源的核苷酸序列。BLAST蛋白质搜索可用BLASTN软件进行(得分=50，词长=3)，以获得与本发明5-内毒素或5-内毒素相关蛋白质分子同源的氨基酸序列。为了获得带缺口的序刊比对以进行比较，可如文献Altschul爭(1997)肠/e/cJci&組25:3389中所述利用GappedBLAST。或者，可用PSI-Blast进行检测两个分子之间疏远关联的反复搜索。参阅，Altschul爭(1997),同上文。在应用BLAST、GappedBLAST和PSI-Blast程序时，可使用各个程序(如，BLASTX和BLASTN)默认的参数。见丽.ncbi.nlm.nih.gov,另一用于比较序列的数学运算法则的非限制性例子是ClustalW运算法则(Higgins爭(1994)肠/e/"c油ies.22:4673-4680)。ClustalW可比较序列，并将氨基酸或DNA序列全部比对，因此可提供有关整个氨基酸序列的序列保守性的资料.ClustalW运算法则被用于数个商品化的DNA/氨基酸分析软件包中，诸如栽体NTiProgramSuite(Informax，Inc)的ALIGNX才莫块。用ClustalW进行氨基酸序列的比对后，可评估氨基酸同一性百分比。可用于ClustalW序列比对分析的软件程序的非局限性例子是GeneDocTM。GenedocTM(KarlNicholas)可评估多个蛋白质之间的氨基酸(或DNA)相似性和同一性。用于序列比较的数学运算法则的另一非限制性例子是文献Myers和Miller(1988)""i7^4:11-17中的运算法则。这样的运算法则被引入ALIGN程序(版本2.0)中，它是GCG序列比对软件包的一部分(可获自Accelrys，Inc.,9865Sc訓tonRd.，SanDiego,California,USA)。当应用ALIGN程序来比较氨基酸序列时，可使用PAM120加权残基表、缺口长度罚分12和缺口罚分4。本发明还包括变体核酸分子。5-内毒素或5-内毒素相关蛋白质编码核苷酸序列的"变体"包括编码本文所公布的5-内毒素或5-内毒素相关蛋白质、但由于遗传密码简并性而有保守性差别的序列，以及如上所述足够相似的那些序列。天然存在的等位基因变体可用众所周知的分子生物学技术鉴定，诸如聚合酶链式反应(PCR)和下文概述的杂交技术。变体核苷酸序列还包括合成来源的核普酸序列，例如如下所述通过定位诱变产生、但仍然编码本发明公布的5-内毒素或5-内毒素相关蛋白质的序列。包含在本发明范围内的变体蛋白质是有生物学活性的，也就是说，它们继续具有天然蛋白质的目的生物学活性，即保持杀虫活性。"保持活性"是指变体具有5-内毒素或5-内毒素相关蛋白质的活性的至少约30%、优选至少约50%、更优选至少约70%、还更优选至少约80%。用于测量杀虫活性的方法在本领域中是众所周知的。参阅，例如，Czapla和Lang(1990)/.fco/7.f/^咖o/.83(6):2480-2485;Andrews爭(1988)"/oc/e瓜/252:199-206;Marrone爭(1985)/.fco/7咖/cf/^咖o/og/78:290-293;以及美国专利5，743，477，所有这些均全部收编于此作为参考。本发明还包括变体核酸分子。&-内毒素或5-内毒素相关编码核苷酸序列的"变体"包括那些编码本文所公布的5-内毒素或5-内毒素相关蛋白质、但由于遗传密码简并性而有保守性差异的序列以及如上所述充分相同的那些序列。天然存在的等位基因变体可用众所周知的分子生物学技术鉴定，诸如聚合酶链式反应(PCR)和下述杂交技术。变体核苷酸序列还包括合成来源的核苷酸序列，例如如下所述通过定位诱变产生、但仍然编码本发明公布的5-内毒素或5-内毒素相关蛋白质的序列。技术熟练人员应认识到，可通过突变在本发明的核苷酸序列中引入变异，从而导致所编码的5-内毒素或5-内毒素相关蛋白质的氨基酸序列中的变化，但不改变所述蛋白质的生物学活性。因此，可通过在本^#会布的相应核苷酸序列中引入一个或多个核苷酸取代、添加或缺失而制备变异的分离核酸分子，从而在所编码的蛋白质中引入一个或多个氨基酸取代、添加或缺失。突变可通过标准技术引入，诸如定位诱变和PCR介导诱变。这样的变异核苷酸序列也包括在本发明的范围内。例如，优选的，可以在一个或多个预知的、优选非必需氨基酸残基处进行保守氨基酸取代。"非必需的"氨基酸残基是可以在5-内毒素或5-内毒素相关蛋白质序列的野生型序列中发生改变、而不改变生物学活性的残基，而"必需的"氨基酸残基是生物学活性所必需的。"保守的氨基酸取代"是用具有相似侧链的氨基酸残基替换某一氨基酸残基。具有相似侧链的氨基酸残基家族在本领域中已有定义。这些家族包括具有碱性侧链的氨基酸(如，赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(如，天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有P-分支侧链的氨基酸(如，苏氨酸、缬氨酸、异亮氨酸)和具有芳香族侧链的氨基酸(如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。在5-内毒素蛋白质之间通常有5个高度保守的区域，主要集中在结构域的中心或结构域之间的连接处(Rajamohan爭(1998)户/"^A7/c/e/cJc/VM/.S/o/.60:1-23)。多种5-内毒素的保守氨基酸块以及共有序列可参阅以下文献Schnepf爭(1998)Vo/.》io/.TeK.62:775-806和Lereclus爭(1989)，编码苏云金芽孢杆菌类芽孢5-内毒素的基因的作用、结构和分子组成(Role，Structure,andMolecularOrganizationoftheGenesCodingfortheParasporald-endotoxinsof^3c///t/stA￡yr//^7e/2s/s)。在"原核发育的调控(RegulationofProcaryoticDevelopment)"一书中，IssarSmit，Slepecky，R.A.，Setlow，P.美国孩i生物学会，Washington,D.C.20006。已提出具有这些保守区的5-内毒素可共享相似的结构，由三个结构域组成(Li爭(199DA^^re353:815-821)。结构域I在5-内毒素之间具有最高的相似性(Bravo(1997)/.5sc"n'o/.179:2793—2801)。可在非保守区内进行保持活性的氨基酸取代。一般而言，不对保守氨基酸残基、或定位在保守基元内的氨基酸残基进行这样的取代，其中所述的残基对于蛋白质活性是必需的。保守的、并且可能为蛋白质活性所必需的残基例子包括，例如，在所提供的序列比对中含有的所有蛋白质之间均相同的残基。保守的、但可能允许发生保守性氨基酸取代而仍保持活性的残基的例子包括例如在所提供的序列比对中所包括的所有蛋白质之间仅有保守性取代的残基。不过，本领域技术人员应理解，功能性变体可在保守残基中具有微小的保守或非保守变异。或者，可通过诸如饱和诱变等方法沿着所有或部分编码序列随机引入突变而制备变异核苷酸序列，并可对所产生的突变体筛选赋予5-内毒素或5-内毒素相关活性的能力，以鉴别保持活性的突变体。诱变后，所编码的蛋白质可重组表达，而蛋白质的活性可用标准检测技术测定。用诸如PCR、杂交等方法可鉴定相应的5-内毒素或5-内毒素相关序列，这样的序列与本发明的序列基本上相同。参阅，例如，SambrookJ.和Russell,D.W.(2001)分子克隆实验室手册M/"f/"rC7o/2i/^:JZa6orator7Va/zi/aA(冷，良港实验室出版;土，冷，良港，)PCR流程方法和应用指南户^尸ro&coh;爿Cu2efo#<^力0&a/7t/Jp/7//c<3〃o"51(AcademicPress,NY)。在杂交方法中，可用全部或部分5-内毒素或5-内毒素相关核苷酸序列筛选cDNA或基因组文库。所述cDM和基因组文库的构建方法是本领域普遍已知的，并被描述在Sambrook和Russell，2001中。所谓的杂交探针可以是基因组DNA片段、cDNA片段、RNA片段或其它寡核苷酸，它们可用诸如"P等可检测基团或诸如其它放射性同位素、焚光化合物、酶或酶辅因子等任何其它可检测标记物进行标记。可通过标记基于本文所公布的已知5-内毒素或5-内毒素相关编码核苷酸序列的合成寡核苷酸制备用于杂交的探针。此外，还可使用基于核苷酸序列或所编码的氨基酸序列中的保守核苷酸或氨基酸残基设计的简并引物。所述探针通常包含在严谨条件下与本发明5-内毒素或5-内毒素相关编码核苷酸序列或其片段或变体的至少约12个、优选约25个、更优选至少约50、75、100、125、150、175、200、250、300、350或400个连续核苷酸杂交的核苷酸序列区。用于杂交的探针的制备是本领域所普遍已知的，并且公开在此处收编作为参考的文献Sambrook和Russell，2001中。在杂交技术中，已知的核苷酸序列的全部或部分被用作与来自选定生物体的克隆基因组DNA片段或cDNA片段群(即，基因组或cDNA文库)中存在的其它相应核苷酸序列选择性杂交的探针。杂交探针可以是基因组DM片段、cDNA片段、RM片段或其它寡核苷酸，且可用诸如"P等可检测基团或任何其它可检测标记物标记。因此，例如，用于杂交的探针可通过标记基于本发明的5-内毒素或5-内毒素相关序列的合成寡核苷酸而制备。杂交探针的制备方法以及cDNA和基因组文库的构建方法是本领域通常已知的，并公开在Sambrook爭(1989)分子克隆实验室手册腺ecw/arC7o/7一"a6or"or/#2細/(第二版，冷泉港实验室出版社，Plainview，纽约)中。例如，本文所公布的整个5-内毒素或5-内毒素相关序列或其一个或多个部分可用作能特异性地与相应5-内毒素或5-内毒素相关样序探针。为了实现各种条件下的特异杂交，所述探针包含独特的序列，并且优选长度为至少约IO个核苷酸、最优选至少约20个核苷酸。这样的探针可用于通过PCR从选定的生物体中扩增相应的5-内毒素或5-内毒素相关序列。这一技术可用于从目的生物体中分离额外的编码序列，或作为诊断检测法用于确定生物体中编码序列的存在与否。杂交技术包括铺板的DNA文库(或者噬斑或者菌落，参阅，例如，Sarabrook爭(1989)分子克隆实验室手册(第二版，冷泉港实验室出版社，Plainview，纽约))的杂交筛选。这些序列的杂交可在严谨条件下进行。"严谨条件"或"严谨的杂交条件"指，在此条件下，探针与其靶序列的杂交程度可检测性地高于其它序列(例如，至少高于背景2倍)。严谨条件是随序列而定的，在不同的环境下有所不同。通过控制杂交和/或漂洗条件的严谨性，可鉴别与探针100%互补的靶序列(同源探测)。或者，可将严谨条件调整成允许序列中有一些错配，从而检测较低程度的相似性(异源探测)。通常，探针的长度小于约1000个核苷酸，优选长度小于500个核苷酸。通常，严谨条件是这样的条件，其中在pH7.0至8.3时盐浓度小于约1.5MNa离子、通常约0.01至1.0MNa离子浓度(或其它盐)，并且对于短探针(如10-50个核苷酸)而言温度至少约30°C，而对于长探针(例如，大于50个核苷酸)而言至少约60°C。还可通过添加诸如甲酰胺等去稳定剂来达到严谨条件。示范性的低严谨条件包括用30-35%甲酰胺、1MNaCl、1%SDS(十二烷基硫酸钠)的緩沖液在37。C进行杂交，在IX至2XSSC(20XSSC=3.0MNaC1/0.3M柠檬酸三钠)在50-55。C漂洗。示范性的中等严谨条件包括在40-45%甲酰胺、1.0MNaCl、1%SDS中于37°C杂交，并在0.5X至IXSSC于55-60。C漂洗。示范性的高严谨条件包括在50%甲酰胺、1MNaCl、1%SDS中于37°C进行杂交，并在0.IXSSC中于60-65°C漂洗。任选的，漂洗緩冲液中可包含约0.1%至约1°/。SDS。杂交的持续时间一般少于大约24小时，通常约为4至12小时。一般而言，特异性是杂交后漂洗条件的函数，关鍵因素是最终漂洗溶液的离子强度和温度。对于DNA-DNA杂交，L可用Meinkoth等Wahl(1984)細/.扁c力e迈.138:267-284中的公式估计:Tra=81.50C+16.6(logM)+0.41(%GC)—0.61(%form)-500/L;其中的M是单价阳离子的摩尔浓度，％GC是DNA中鸟噤呤和胞嘧啶核苷酸的百分比，°/。form是杂交溶液中曱酰胺的百分比，而L是碱基对中杂交体的长度。L是互补靶序列的50%与完全匹配的探针杂交的温度(在确定的离子强度和pH下)。对于每1%的错配，L下降约1。C;因此，为了与所期望的同一性的序列杂交，可以调节乙、杂交和/或漂洗条件。例如，如果搜寻具有^90%同一性的序列，乙可下降10。C。通常，将严谨条件选择成比该具体序列与其互补序列在确定的离子强度和pH的热融点(L)低约5°C。不过，严格的严谨条件可利用比热融点(Tm)低l、2、3或4°C的条件进行杂交和/或漂洗；中等的严谨条件可利用比热融点(TJ低6、7、8、9或10°C的条件进行杂交和/或漂洗；低严谨条件可利用可利用比热融点(TJ低11、12、13、14、15或20"C的条件进行杂交和/或漂洗。利用所述公式、杂交和漂洗组合物以及预期的乙，普通技术人员可理解杂交和/或漂洗溶液的严谨性的变化已得到了本质的描述。如果所需程度的错配导致L小于45°C(水溶液)或32。C(曱酰胺溶液)，则优选提高SSC浓度，以利用较高的温度。有关核酸杂交的详尽指南可参阅文献Tijssen(1993)生化和分子生物学实验技术-用核酸探针进行杂交(Ls6or"or/7ecA/2/《"es//Jc/dZ^o6e",第I部分，第二章(Elsevier,纽约)；和Ausubel爭薦禪(1995)分子生物学通用方法(a/rreW尸r^oco/s//7#o/ecu/ar)，第二章(GreenePublishingandWiley-Interscience，NewYork)。参阅Sambrook爭(1989)分子克隆实验室手册(淑ecw/ar"a6or"c/r#雄")(第二版，冷泉港实验室出版社，Plainview，NewYork)。分离蛋白质及其变体和片段5-内毒素和5-内毒素相关蛋白质也包含在本发明的范围内。"5-内毒素蛋白质"指具有SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29中所示氨基酸序列的蛋白质。"5-内毒素相关蛋白质"指具有SEQIDNO:18中所示氨基酸序列的蛋白质。同时还提供了它们的片段、生物学活性部分和变体，它们可用于实践本发明的方法。"片段"或"生物学活性部分"包括含有编码如SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29中所示5-内毒素或5-内毒素相关蛋白质的氨基酸序列的一部分且保持5-内毒素活性或5一内毒素相关活性的多肽片段。5-内毒素或5-内毒素相关蛋白质的生物学活性部分可以是例如10、25、50、IOO或更多个氨基酸长的多肽。这样的生物学活性部分可通过重组技术制备，并评估其5-内毒素或5-内毒素相关活性。检测杀虫活性的方法是本领域众所周知的。参阅，例如，Czapla和Lang(1990)/.肠/7.艇画/.83(6):2480-2485;Andrews爭(1988)飾c力e瓜/.252:199-206;Marrone爭(1985)/.o,fco;30iZ7/cA/2&边o/o^F78:290-293;以及美国专利5，743，477，所有这些均全部收编于此作为参考。用于此处时，片段含有SEQIDN0:3、5、7、9、11、14、16、18、20、22、24、27或29中的至少8个毗连氨基酸。不过，本发明还包含其它的片段，诸如该蛋白质中的任何大于约10、20、30、50、100、150、200、250、300、350、400、450、500、550、600和650个氨基酸的片段。"变体"指具有与SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列至少约60°/。、65°/。、优选至少约70%、75%、更优选至少约80°/。、85%、最优选至少约90%、91%、92%、93%、94%、95%、96%、97%、98°/4或99%相同的氨基酸序列的蛋白质或多肽。变体还包括在严谨条件下与SEQIDNO:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28的核酸分子或其互补分子杂交的核酸分子编码的多肽。这样的变体通常保持5-内毒素或5-内毒素相关活性。变体包括由于诱变而氨基酸序列有所区别的多肽。本发明所包含的变体蛋白质是有生物学活性的，即，它们继续具有天然蛋白质的所需生物学活性，即保持杀虫活性。检测杀虫活性的方法是本领域众所周知的。参阅，例如，Czapla和Lang(1990)/.fco几iW咖o/.83(6):2480-2485;Andrews爭(1988)扁c力e迈./.252:199-206;Marrone爭(1985)/.o尸fcw2c迈/cj^M迈o7(^/78:290-293;以及美国专利5,743,477,所有这些均全部收编于此作为参考。改变或改良的变体已认识到可用各种方法改变5-内毒素或5-内毒素相关蛋白质的DNA序列，并且这些改变可能产生所编码的蛋白质氨基酸序列不同于本发明5-内毒素或5-内毒素相关蛋白质氨基酸序列的DNA序列。这一蛋白质可以多种方式改变，包括氨基酸取代、缺失、截短和插入。这些操作方法是本领域通常已知的。例如，5-内毒素或5-内毒素相关蛋白质的氨基酸序列变体可通过DNA中的突变来制备。这还可以通过数种诱变形式之一和/或定向进化来完成。在某些方面，氨基酸序列中编码的改变基本上不会影响蛋白质的功能。这样的变体将具有所需的杀虫活性。不过，应理解5-内毒素或5-内毒素相关蛋白质赋予杀虫活性的能力可基于本发明组合物通过使用所述技术得以提高。例如，可以在于DNA复制期间呈现出高比率碱基错误掺入的宿主细胞中表达5-内毒素或5-内毒素相关蛋白质，诸如XL-1Red(Stratagene)。在所述林系中增殖后，可分离5-内毒素或5-内毒素相关DM(例如通过制备质粒，或通过PCR扩增并将产生的PCR片段克隆入载体中)，在非诱变性菌林中培养5-内毒素或5-内毒素相关突变，鉴定具有杀虫活性的突变的5-内毒素或5-内毒素相关基因，例如进行检测来测试杀虫活性。通常，将所述蛋白质混和并用于喂服试验中。参阅，例如，Marrone爭(1985)/.o尸fco/20迈yc^7^/zzo/c^/78:290-293。这样的试验可包括使植物接触一种或多种害虫并测定植物存活的能力和/或引起害虫死亡的能力。导致毒性增强的突变的例子参阅Schnepf爭(1998)#/cro6/o/."/'o人yer.62:775-806。或者，可在氛基或羧基末端对许多蛋白质的序列进行改变而基本上不影响其活性。这可包括利用现代分子方法引入的插入、删除或改变，诸如PCR,包括由于在PCR扩增所用的寡核苷酸中含有氨基酸编码序列而改变或延长蛋白质编码序列的PCR扩增。或者，所加入的蛋白质序列可包括完整的蛋白质编码序列，诸如那些在本领域中通常用于产生蛋白质融合体的序列。这样的融合蛋白质常常用于(1)增加目的蛋白质的表达，(2)引入结合结构域、酶促活性或表位以方便蛋白质纯化、蛋白质检测或本领域已知的其它实验性应用，(3)使蛋白质的分泌或翻译定向于亚细胞性细胞器，诸如革兰氏阴性细菌的周质空间，或真核细胞的内质网，后者常常导致蛋白质的糖基化。本发明的变体核苷酸和氨基酸序列还包括来自诸如DNA改组等诱变和重组方法的序列。通过这样的步骤，可利用一个或多个不同的5-内毒素或5-内毒素相关蛋白质编码区构建具有所需特性的新的5-内毒素或5-内毒素相关蛋白质。以此方式，由含有具有充分的序列同一性且可体外或体内同源重组的序列区的相关序列多核苷酸群产生重组多核苷酸文库。例如，用此方法，可在本发明的5-内毒素或5-内毒素相关基因与其它已知5-内毒素或5-内毒素相关基因之间对编码目的结构域的序列基元进行改组，以获得编码具有改良目的特性的蛋白质的新基因，诸如提高杀虫活性。所述的DM改组策略是本领域所已知的。参阅，例如，Stemmer(1994)户roc.Jcad.Sc/.^S^91:10747-10751;Ste咖er(1994)Y"i/re370:389-391;Crameri爭(1997)5/由c//.15:436-438;Moore爭(1997)/.Wo/.272:336-347;Zhapg夢(7W7J户roc.^caA^SVi94:4504-4509;Crameri爭(1998)iV"ure391:288-291;以及美国专利5，605，793和5，837，458。结构域交换或改组是产生改变的5-内毒素或5-内毒素相关蛋白质的另一机制。结构域II和III可在5-内毒素蛋白质之间进行交换，产生具有改良的杀虫活性或目标谱的杂合或嵌合毒素。产生重组蛋白质并检测其杀虫活性的方法在本领域中是众所周知的(参阅，例如，Naimov爭(200D铷/.紐/r肌Wcro6/o入67:5328-5330;deMaagd爭(1996)f脂'層.飾roWo厶62:1537-1543;Ge爭(1991)/.C力e瓜266:17954-17958;Schnepf爭(1990j/.A/o人C力e迈.265:20923-20930;Rang爭91999)^2"'r0".#/cr/oWo/.65:2918-2925)。植物转化可通过本领域已知的数种技术之一完成植物细胞的转化。首先，以允许其在植物细胞中表达的方式改造5-内毒素或5-内毒素相关基因。通常表达这种蛋白质的构建体应含有驱使基因转录的启动子以及允许转录终止和聚腺苷酸化的3，非翻译区。这样的构建体的组成在本领域中是众所周知的。在某些情况下，可有效改造基因，使得产生的肽被分泌出来，或定向于植物细胞中。例如，可改造基因，使其含有信号肽，以方便所述的肽转移至内质网。还可能优选的是，改造植物表达盒使其含有内含子，从而使内含子的mRNA加工为表达所必需。通常这一"植物表达盒"将被插入"植物转化载体"中。该植物转化栽体可由完成植物转化所需的一个或多个DNA载体组成。例如，本领域的通用惯例是利用由一个以上毗连DNA区段组成的植物转化载体。这些载体在本领域中常常称为"二元载体"。二元栽体以及具有辅助质粒的载体是最常用于农杆菌介导的转化中的，其中完成有效转化所需的DNA区段的大小和复杂性是相当大的，并且将功能分摊到分开的DNA分子上比较有利。二元载体通常包含含有T-DM转移所必需的顺式作用序列(诸如左边界和右边界)、经改造能在植物细胞中表达的可选择标记物以及"目的基因"(经改造能在期望产生转基因植物的植物细胞中表达的基因)的质粒载体。还存在于此质粒载体中的是细菌复制所必需的序列。顺式作用序列以可有效转移入植物细胞并在其中表达的形式安排。例如，可选择标记基因和目的基因定位于左和右边界之间。常常有另一个质粒载体包含介导T-DM从农杆菌转移至植物细胞的的反式作用因子。按照本领域中所理解的，此质粒常常具有使植物细胞可被农杆菌感染并通过在边界序列处的断裂以及病毒介导的DNA转移来转移DNA的致病作用(Vir基因)(Hellens和Mullineaux(2000)7>e/](/s/刀尸/a力tSc/e刀ce，5:446—451)。数种类型的农杆菌菌林(如LBA4404、GV3101、EHAIOI、EHA105等)可用于植物转化。该第二种质粒载体不是用诸如显微投影、显微注射、电穿孔、聚乙二醇等其它方法转化植物所必需的。一般而言，植物转化方法包括将异源DNA转移入靶植物细胞(如，不成熟或成熟的胚、悬浮培养物、未分化的愈伤组织、原生质体，等等)中，随后进行最大极限水平的适当选择(取决于可选择标记基因)，以从未转化细胞集合组中回收转化的植物细胞。通常将外植体转移至新鲜置换的同种培养基中并进行常规培养。随后，在放置于补充了最后将所述的芽转移到选择性i根4养l中以回收生根的芽或苗。然i将转基因苗培养成成熟的植物并产生能繁殖的种子(如Hiei爭(1994)T7e7Vai3//o"r加/6:271-282;Ishida爭(1996)^"wre"/Wec/wo7o^r14:745-750)。通常将外植体转移至新鲜补充的同种培养基中并进行常规培养。产生转基因幼苗的有关技术和方法的全面描述参阅文献Ayres和Park，1994(C"7/ca//e"譜//7尸/fl;2fSc/e腦13:219-239)以及Bommineni和Jauhar，19970Va7力'ca42:107-120)。由于被转化的物质包含许多细胞，转化和未转化的细胞都存在于任何受试靶愈伤片或组织或细胞组中。杀死未转化细胞并允许转化细胞增殖的能力使被转化的植物培养物得以生长。通常，去除未转化细胞的能力是对快速回收被转化植物细胞并成功产生转基因植物的限制。转基因植物的产生可用数种方法之一完成，包括但不局限于通过农杆菌将异源DNA引入植物细胞中(农杆菌介导的转化)、用粘附于颗粒上的外源性外来DNA轰击植物细胞以及各种其它非粒子定向介导的方法(如Hiei爭(1994)r力e户/a^/owr/za/6:271-282;Ishida爭(1996)A"f/reAi"ec力/Jauhar(1997)Va/cTica42:107-120)转移DNA。转化方法以及将核苷酸序列引入植物的方法可能随着待转化的植物或植物细胞的类型(即单子叶或双子叶植物)而变化。将核苷酸(Crossway爭(1986)S/Wec/w/^/es4:320—334)、电穿孔(Riggs爭(1986)尸roc.」cad.Sc厶83:5602-5606)、农杆菌介导的转化(美国专利5,563,055;美国专利5,981,840)、直接基因转移(Paszkowski爭(1984)灘O/,3:2717-2722)和弹道颗粒加速(参阅,例如，美国专利4，945，050;美国专利5，879，918;美国专利5，886，244;美国专利5,932,782;Tomes爭(1995)"通过显微投射轰击将DNA定向转移入完整的植物细胞中(Direct腿TransferintoIntactPlantCellsviaMicroprojectileBombardment)"，在"植物细胞、组织和器官培养基本方法(尸/a/7fCe//，77^we,Orga/iOz〃d/re;尸w3^2/pe/2fa7Vef力od^)"一"^'哞，Gamborg和Phillips编禪(Springer—Verlag，Berlin);McCabe爭(1988)A/ofec力/2o7og/6:923-926)、气溶胶束转化(美国已出版专利申请20010026941;美国专利4,945,050;国际专利申请WO91/00915;美国已出版专利申请2002015066)以及Lecl转化(WO00/28058)。也可参阅Weissinger爭(1988)細.紐6^/7"22:421-477;Sanford爭(1987)*戎j(户a/^/ci;/afeSciencerec/wo/og/)5:27—37;Christou爭(1988)尸/aWM/s/oA87:671-674;McCabe爭(1988)万/o/Tec//7o/0g/6:923—926;Finer和McMullen(1991)ZnP7troCe//Per."/o人27P:175-182;Singh爭(1998)r力eor.Jp;/.96:319-324(大豆)；Datta爭(1990)"/otec力/70/0^78:736-740;Klein爭(1988)尸roc.iVa〃.Jcacf.Sc/.〃W85:4305-4309;美国专利5,240,855;美国专利5，322，783和5，324，646;Tomes等柳""通过微粒轰击将DNA定向转移入完整的植物细胞中(DirectDNATransferintoIntactPlantCellsviaMicroprojec出eBombardment)"，在"植物细胞、组织和器官培养基本方法(/Va射*，薦襌Gamborg和Phillips(Springer-Verlag，Berlin);Klein爭(1988)户/a"f尸//"'o厶91:440-444;Hooykaas—VanSlogteren爭(1984)^""re"o/^oW311:763-764;美国专利5，736,369;ByteMer爭(1987)细c.咖84:5345-5349(Liliaceae);DeWet爭(1985)在"胚珠组织的实验操作(7TeAjr77er/迈e77"/#fl/7//7i//a〃0"o尸6Vw/e77ss"es)"—书中，编辑Chapman爭(Longman,NewYork),笫197-209页；Kaeppler爭(1990)植物细胞报告户/a/^Ce//9:415-418和Kaeppler爭(1992)7力eor.场/.84:560-566;D'Halluin爭(1992)4:1495-1505;Li爭(1993)户/aWCe/77e/7or〃12:250-255以及Christou和Ford(1995)^■/so尸淑<3//75:407-413;0sjoda爭(1996)艇i/reA/ofec/wo/o^F14:745-750，所有这些均收编于此作为参考。在将外源性外来DNA整合入植物细胞中后，在培养基中进行最大极限水平的适当选择，以杀死未转化的细胞，并通过定期转移至新鲜培养基中而分离和增殖在此选择处理中存活的据推定已转化的细胞。通过连续传代并进行适当的选择，可识别并增殖被所述质粒载体转化的细胞。然后用分子和生化方法证实转基因植物基因组中被整合的异源目的基因的存在。已4皮转化的细胞可以传统方式生长成植物。参阅，例如McCormick爭(1986)P7a;^Ce/7;e;or〃5:81-84。然后可培养这些植物，用同样的被转化抹系或不同的林系授粉，并鉴定具有所需表型特征的组成型表达的所得杂合体。可培养两代或多代，以保证所需表型特征的表达稳定维持和遗传，然后收获种子以保证已实现了所需表型特征的表达。以此方式，本发明提供了在其基因组中稳定掺入了本发明的核苷酸构建体、例如本发明的表达盒的已转化种子(也称为"转基因种子")。本发明的5-内毒素或5-内毒素相关序列可存在于用于在目的植物中表达的表达盒中。所述的盒将包括可操作性的与本发明序列连接的5'和3'调控序列。"可操作性的连接"指在启动子和第二序列之间的功能性联接，其中的启动子序列起始并介导了与第二序列相应的DNA序列的转录。通常，可操作性连接意味着被连接的核酸序列是毗邻的，并且，在需连接两个蛋白质编码区时，则是毗邻且处于同一阅读框架中。所述的盒可另外含有至少一个其它基因以共转化入生物体中。或者，所述的其它基因可存在于多个表达盒中。向所述的表达盒中提供多个限制性位点，以供插入5-内毒素或5-内毒素相关序列，处于调控区的转录调控下。所述的表达盒沿5'-3'转录方向将包含在植物中发挥作用的转录和翻译起始区(即，启动子)、本发明的DNA序列以及转录和翻译终止区(即终止区)。启动子对于植物宿主和/或本发明的DNA序列而言可能是天然的或类似的、或者外源的或异源的。此外，启动子可以是天然序列或可以是合成序列。启动子对于植物宿主而言是"天然的"或"同源的"，指的是启动子在该启动子将被引入的天然植物中存在。启动子对于本发明DNA序列而言是"外源的"或"异源的"，指的是启动子对于可操作性连接的本发明DNA序列而言不是天然或自然存在的启动子。终止区可以是对于转录起始区而言是天然的，可以是就可操作性连接的目的DNA序列而言是天然的，可以是就植物宿主而言是天然的，或者可以来自其它来源(即，对于启动子、目的DNA序列、植物宿主或它们的任何组合而言是外源的或异源的)。方便的终止区可从#癌农杆菌的Ti-质粒获得，诸如章鱼碱合酶和胭脂碱合酶终止区。还可参阅Guerineau爭(1991)Ge/.(e刀".262:141-144;Proudfoot(1991)6W/64:671-674;Sanfacon爭(1991)Ce総紐5:141-149;Mogen爭(1990)尸/aflfCe//2:1261-1272;Munroe爭(1990)Ge/e91:151-158;Ballas爭(1989)^c/e/cJc油紐17:7891-7903;以及Joshi爭(1987)肠/e/.cJc/"es.15:9627-9639。在适当的情况下，可将基因最优化以提高其在被转化宿主细胞中的表达。即，所述基因可以用宿主细胞偏好的密码子合成以促进表达，或者可以宿主偏好的密码子使用频率用密码子合成。例如，有关宿主偏好的密码子使用的讨论参阅Campbell和Gowri(1990)尸/a;^尸力7^'o7.92:1-11。合成植物偏好基因的方法是本领域所已知的。参阅，例如，美国专利6，320，100、6，075，185、5，380，831和5，436，391，美国已出版专利申请20040005600和20010003849，以及Murray爭(1989)A^/c/e/c^c/"/e义17:477-498，在此收编作为参考。在一个实施方案中，目的核酸被定向于叶绿体中表达。以此方式，当目的核酸不是直接插入叶绿体的情况下，表达盒将另外含有编码转运肽的核酸以指导目的基因产物进入叶绿体。这样的转运肽在本领域中是已知的。参阅，例如，VonHeijne爭(199D/Vai^Vo/.飾/.雖9:104-126;Clark爭(1989)/.W/'o/.C力e瓜264:17544-17550;Della-Cioppa爭(1987)户/aW脚Wo/.84:965-968;Romer爭(1993)A/oc力e迈."/o/7力/义Co/zww/7.196:1414—1421;k乂及Shah爭(1986)5We/ce233:478-481。用于叶绿体转化的方法在本领域中是已知的。参阅，例如，Svab爭(1990)户roc.射/.JcacT.5W.〃W87:8526-8530;Svab和Maliga(1993)尸roc.艇/.JcacT.<SW.蹈90:913-917;Svab和Maliga(1993)W仿O/.12:601-606。所述方法依赖于含可选择标记的DNA的粒子枪投递以及通过同源重组将DNA定向于质体基因组。此外，质体转化可通过用核编码及质体指导的RNA聚合酶的組织优选表达而反式激活沉默的质体携带的转基因而完成。这样的体系已被报道于文献McBride爭(1994)iVis".91:7301-7305中。可优化定向于叶绿体的目的核酸以表达于叶绿体中，从而解决植物细胞核和此细胞器之间的密码子使用中的差异。以此方式，目的核酸可以用叶绿体偏好的密码子合成。参阅，例如，美国专利5，380,831,在此收编作为参考。植物转化的评估将异源性的外来DNA引入植物细胞中后，用各种方法证实植物基因组中异源基因的转化或整合，诸如与被整合基因相关的核酸、蛋白质和代谢物的分析。PCR分析PCR分析是在移植至土壤中之前的较早阶段筛选已转化的细胞、组织或芽是否存在被掺入基因的快速方法(Sambrook和Russell，2001)。PCR是用特异于目的基因或农杆菌栽体背景等的寡核苷酸引物进行的。DNA杂交分析植物转化通过基因组DNA的DNA印迹分析证实(Sambrook和Russell,2001)。通常，从转化林中提取总DNA，用适当的限制酶消化，在琼脂糖凝胶中进行分级分离并转移至硝酸纤维素或尼龙膜上。然后依照标准技术，用例如放射性标记了32P的耙DNA片段探测所述的膜或"印迹"，以证实被导入基因在植物基因组中的整合(Sambrook和Russell,2001.分子克隆实验室手册#o/eci//arC7o72//7^vJL260rafor/Va/7i/a/.冷7良港实验室出版^土，冷7良港，纽约)。RNA杂交分析依照本领域中常规使用的标准方法，从转化林的特异组织中分离RNA，在甲醛琼脂糖凝胶中分级分离，印迹至尼龙滤膜上(Sambrook，J.和Russell,D.W.2001.分子克隆实验室手册#o/ecw/ar6V0/z/i2^vjL360rafor/#a/7i/<37.冷7良港实验室出版it，冷泉港，纽约)。然后用本领域已知方法(Sambrook和Russell,2001),通过将所述滤膜与来自5-内毒素或S-内毒素相关蛋白质的放射性探针杂交而测试5-内毒素或与5-内毒素相关基因编码的RNA的表达。蛋白质印迹和生化检测可通过标准步骤(Sambrook,J.和Russell,D.W.2001.分子克隆实验室手册Vo/ecu/drC7oj7/;^:船/n/a/.冷泉港实验室出版社，冷泉港，纽约)，利用结合5-内毒素或5-内毒素相关蛋白质上存在的一个或多个表位的抗体对转基因植物进行蛋白质印迹和生化检测及类似测定，以证实由5-内毒素或5-内毒素相关基因编码的蛋白质的存在。植物中的杀虫活性在本发明的另一方面，可产生表达具有杀虫活性的5-内毒素或5-内毒素相关蛋白质的转基因植物。可利用上述举例说明的方法产生转基因植物，但产生转基因植物细胞的方式对本发明而言不是关键的。可利用诸如农杆菌介导的转化、气溶胶束(aerosolbeam)、biolisUc转化及非粒子介导方法等本领域中已知或描述的方法来进行，具体所用方法依实验者而定。可用本领域中描述的通用方法分离表达5-内毒素或5-内毒素相关蛋白质的植物，例如，通过转化愈伤组织、筛选被转化的愈伤组织以及从所述的转基因愈伤组织再生出可育植物。在所述过程中，可利用任何基因作为可选择标记，只要通过它在植物细胞中的表达能识别或选择已被转化的细胞即可。已开发了许多与植物细胞一起使用的标记物，诸如对氯霉素、氨基糖苷类G418、潮霉素等等的抗性。编码叶绿体代谢中所涉及的产物的其它基因也可用作选择标记。例如，能提供对诸如草甘膦、溴苯腈(bromoxynil)或口米峻淋酉同(imidazolinone)等植物除草剂的抗'f生的基因可能有具体用途。这样的基因已有报道(Stalker爭(1985)/.万/o人C力e瓜263:6310-6314(溴苯腈抗性腈水解酶基因)；以及Sathasivan爭(199(J)^uc/.」c/'^ies.18:2188(AHAS咪唑啉酮抗性基因)。可对表达5-内毒素或S-内毒素相关蛋白质的可育植物测试杀虫活性，并选择显示出最佳活性的植物进行进一步的培育。本领域中有可供利用的方法能够用于检测对害虫的活性。通常，所述蛋白质被混合并用于喂服试验中。参阅，例如，Marrone爭(1985)/.o尸fco/70迈/c78:290—293。杀虫控制中的应用般方法是本领域所已知的。参阅，例如，美国专利5,039,523和EP0480762A2。本发明的芽孢杆菌林系或经遗传改造而含有杀虫基因和蛋白质的微生物可用于保护农作品和产品免受害虫的侵袭。在本发明的一个方面，当将产生毒素的细胞施用于有目标害虫的环境中时，用延长所述细胞中产生的毒素的活性的试剂处理生产毒素(杀虫剂)的生物体的完整(即未裂解)细胞。或者，通过将异源基因引入细胞宿主而产生杀虫剂。异源基因的表达直接或间接的导致了杀虫剂在细胞内的产生和保持。在本发明的一个方面，当将所述细胞施用于有目标害虫的环境中时，在延长该细胞所产生的毒素的活性的条件下处理这些细胞。所产生的产物保持了毒素的毒性。然后可依照常规技术配制这些自然封装的杀虫剂，以便施用于含有目标害虫的环境中，如，土壤、水和植物的叶子。参阅，例如EPA0192319以及其中引用的文献。或者，可对表达本发明基因的细胞进行配制，以便可将所产生的物质作为杀虫剂施用。本发明的活性成分通常以组合物的形式施用，并可与其它化合物同时或接连地施用于待处理的农作物地区或植物。这些化合物可以是肥料、除草剂、防冻剂、表面活性剂、去垢剂、杀虫皂、休眠油、聚合体和/或在单次施用制剂后可长期对目标区域进行给药的随时间释放型或可生物降解型载体制剂。它们也可以是选择性除草剂、化学杀虫剂、杀病毒剂、杀微生物剂、杀变形虫剂、杀虫剂、杀真菌剂、杀菌剂、杀线虫剂、杀软体动物剂或数种这些制剂的混合物，如果需要，可进一步与农业可接受载体、表面活性剂或制备领域中常规采用的促进应用的佐剂一起施用。合适的载体和佐剂可以是固体或液体，并与制备工艺中通常应用的物质相对应，如，天然或再生的矿物、溶剂、分散剂、增湿剂、粘附剂、粘合剂或肥料。同样的，所述制剂可制备成可食用的"饵"或作成害虫"陷阱"，使得可被杀虫制剂的目标害虫所饲用或摄取。应用本发明活性成分或含本发明细菌菌林所产生的杀虫性蛋白质中至少一种的本发明农用化学品组合物的优选方法是叶片施用、种子包裹和土壤施用。施用次数和施用频率取决于相应害虫的侵袭强度。所述组合物可制备成粉剂、尘剂、丸剂、颗粒剂、喷雾剂、乳剂、胶体、溶液等等，且可用诸如含所述多肽的细胞培养物的干燥、冻干、匀浆、提取、过滤、离心、沉降和浓缩等常规方式制备。在所有含至少一种所述杀虫多肽的所述组合物中，所述多肽可以重量约1%-约99%的浓度存在。利用本发明方法可杀死鳞翅类或鞘翅类害虫，或者减少其在特定区域内的数目，或者可预防性的施用于周围区域以防止易感害虫的蔓延。优选的，所述害虫摄取或接触杀虫有效量的所述多肽。"杀虫有效量"指能导致至少一种害虫死亡或能显著降低害虫的生长、伺用或正常生理发育的杀虫剂用量。这个量将取决于各种因素而变化，诸如待控制的具体目标害虫；待处理的特异环境、位置、植物、农作物或农业场所、环境条件，以及该杀虫有效性多肽组合物施用的方法、速率、浓度、稳定性和用量。制剂还可随气候条件、环境因素和/或施用频率和/或害虫侵袭的严重程度等而发生变化。所述的杀虫剂组合物可通过制备细菌细胞、晶体和/或孢子悬浮液或者与所需的农业可接受栽体有组合的分离蛋白质组分制成。所述组合物可在施用前配制成适当的形式，诸如冻千、冷冻干燥、脱水或在水性载体、培养基或诸如盐水或其它緩冲液等适当的溶剂中。制备好的组合物可以是尘土或颗粒物形式，或者油(植物油或矿物油)中的悬浮液，或者水或油/水乳剂，或作为可湿粉末，或者与适于农业应用的任何其它载体物质相组合。适当的农业载体可以是固体或液体，在本领域中是众所周知的。术语"农业可接受载体"涵盖了通常应用于杀虫剂制备工艺中的所有佐剂、惰性成分、分散剂、表面活性剂、粘着剂、粘合剂等，这些是杀虫剂制备业中的技术人员众所周知的。所述制剂可与一种或多种固体或液体佐剂混合，并通过各种方式制备，例如，用常规的制备工艺将杀虫组合物与适当的佐剂均匀混合、混和和/或碾磨。合适的制剂和应用方法参阅美国专利6,468,523，该文件在此收编作为参考。"害虫"包括但不局限于，昆虫、真菌、细菌、线虫、螨、蜱等等。昆虫类的害虫包括选自以下目的昆虫鞘翅目、双翅目、膜翅目、鳞翅目、食毛目、同翅目、半翅目、直翅目、缨翅目、革翅目、等翅目、虱目、蚤目、毛翅目等，尤其是鞘翅目、鳞翅目和双翅目。昆虫类害虫包括选自以下目的昆虫鞘翅目、双翅目、膜翅目、鳞翅目、食毛目、同翅目、半翅目、直翅目、缨翅目、革翅目、等翅目、虱目、蚤目、毛翅目等，尤其是鞘翅目和鳞翅目。针对主要农作物的本发明昆虫类害虫包括玉米&"//7/3/2"6//a//s，欧洲玉米螟；y4^ro〃s2'/7s/7o/，小地虎；〃e/2'coFer/7flzea，谷实夜蛾；S/7odo/^e/^/Vt/^7/7e油，草地夜蛾；^ra""/^"",巨座玉米螟(西南玉米杆草螟)；fy^/z7，//^705^//ws，南美玉米苗斑螟(小玉米螟)；"/"raeasaccAara//51，小蔗杆草螟；Z/a67"o〃car/r^7/"ers，玉米(幼芽)根叶曱；"/fl6ro〃ca/an^7cori7/s6ar6er/，长角叶甲；"/<a6r<9〃caW2f/ec/'迈/7w/i""flAowrd/，黄瓜十一星叶曱；spp.，才危爪口卩头虫；Cyc/ocep力s/a6orea//s，；i匕方圆头厚金龟(圆头独角仙)；C/c/oce;力a/a/边迈acw7a"，南方圆头犀金龟(圆头无斑独角仙)；尸o/^7/is力;7o/j/ca，弧丽鲅(日本丽金龟)；C力aefoci7e迈ar/a，玉米(铜色)跳曱；5^力e/30/7力o/"s迈a2/，玉米隐咮象(玉米谷象)；^opa7os/;7力wz7鹏yf/2、玉米坊(玉米缢管辨)；J/i/ra/7力/s迈aWrad/c2、玉米根辨；"//^i/s/ewco/^erf/s/ewco/7feri/s，麦长蝽(美洲谷长蝽)；#e/fl/zo/7/f/s尸e迈wrrw6r咖，赤胫黑蝗(赤腿鲊蜢)；#e/a/op/M^/2^///2//es，血黑蝗(迁徙蚱蜢)；外/e迈"/7/"wrs，(玉米)种錄'，^ro迈yzfl/7ar7/cor/7/s，玉米斑潜錄；J/a/7力o^/7;^o6sci7/ri/s，玉米黄呆蓟马(美;川維尾蓟马)；iSb/e/70/7s/s/p//e"a，窃蚊；7Wr<3/zyc/wsi/r〃cae，二斑叶螨(普通红叶螨)；高粱C///o/w"e/风玉米禾螟；5^oc^era/Vwg/per^s,草地夜蛾；〃e//core_rpazea，谷实夜蛾；A/as迈o/a/;7f/51//^720se/7z^，南美玉米苗斑螟(小玉米螟)；尸e/〃aSi/We"a/7ea，粒肤地虎；尸/7//0/7力<3^2cr/"27a，蜂螬(金龟幼虫)；^7eocTes,宽胸口卩甲(C画cTe價)和細/mspp.，金针虫(叩曱幼虫)；迈e/a/o;us，黑角负泥虫(橙足负泥虫)；C/aefoc/e迈apw/2'car2'a，玉米(铜色)刃t曱；5^Ae/o;力or:^迈fl/cT2、玉米隐味象(玉米谷象)；i力o/7a/os/;7力w7/迈a/d/s，玉米坊(玉米纟益管辨)；57/力a/Vara,美甘蔑伪毛辨；"7/'"i/s7eyco/^erws7ewco/^e/^，麦长棒(美洲谷长蝽)；Co/7&r/i3/aso/^力/co/a，高粱瘿蚊；re"a/2/c//^c/w/zahar/zzys,红叶蝶(朱/^,叶竭)，refra/7/c/u^ur^2.cae，叶竭(普通红叶螨)；小麦sewc^s7e〃ai/"/，""a，粘虫；5"/7odo/^era/Vw^7/ercfa，草地夜蛾；^fi7as/z/o/7a/;7ws//g/70se/7iAy，南美玉米苗斑螟(小玉米螟)；^ro〃VoH力^w7/a，西方灰地老虎；f/a57Z70/a/;7W5"//^■/o<ye//w<y，南美玉米苗斑螟；Ou/e则迈e/a/70/ws，黑角负泥虫(橙足负泥虫)；^rperaj3mc""，三叶草叶象(车轴草叶象)；Z/a6ro〃cafy/7￡/ec//z7pi//7""a力(？附rd/，黄瓜十一星叶甲；俄国麦长管坊；i"c力/zap力/s^Ta迈/;3w迈，麦二盆坊；Macros//7力m7are加e，麦长管蚜；〃e/a/3o;7/f^/e迈i/rrz/6r"迈，赤胫黑虫皇(赤腿蚱蛭)；Ve7a/7o/7/w(///Tere/2〃a/2:，异黑虫皇(殊种蚱蛭)；^e/a/o//i^■sa/zgu/zij'pes,血,繁、虫皇(迁徙坤樣)；//^yet/o/acfestrz;cfor，小麦瘦坟；57fod/;7/os/s迈ose/7a/7a，麦红吸装虫；^/isrc>/z7_rzaa辺er/ca/3a，美洲杆蝇(美洲麦杆黄潜蝇)；i^7e/z^acoarc""，麦瘿种蝇(冬作种蝇)；Fra/i7//7/e//fl/7^cfl，烟褐花蓟马(烟草褐蓟马)；Ce/力^c/'/z"i/s，麦茎蜂；Jcer/a^//;7ae，麦癃螨(郁金香瘤螨)；向曰葵5^/e/边fl力e/i'fl/2fAa/za，向日葵芽ii虫；〃o迈oeoso/na向日葵(同)斑螟；^F^0gra迈iz7ae;rc/a迈a〃on/s，向日葵叶甲；g/66o薦，胡萝卜利犀金龟(胡萝卜金龟)；肠/as/一era/nwrf/Wi/〃fl/7fl，向日葵籽癀蚊；棉花〃e7/c^力hF/resce/2s，烟芽夜蛾；〃e7/ccyei77azea，棉铃虫；5^odo/^eraei/^/a,甜菜夜蛾；尸ect/z70/7力oragoss/;2'e/7a，红铃麦域(棉红铃虫)；爿/f力o/2o迈wsgr2/3c^,墨西哥棉铃象(甲)；爿;力/sgos，//，棉坊；尸se:/f/afo迈osce7/sser/aff/s，棉序盲蜂(棉逸匕盲蜂)；rr/a/ei/rodes由〃/画a，苘麻粉虱(结翅粉IU;Z/戸/2'/2eo/ar/、(美国)牧草盲蜂；Ve/a/20//M/e/z7i/rrz/6r"迈，赤胫黑嫂(赤腿蚱蛭)；^/a卿/Md/〃erey7〃a/2、异黑虫皇(殊种蚱蛭)；7Tr/;7S"6aci'，烟蓟马(葱蓟马)；/^2/1///^2'6//3/""a，烟褐花蓟马(烟草褐蓟马)；refra刀/c力w51c/z7/s6arj'/7ws，红叶竭(朱^1^、叶竭)；ref/"a/7/c力w51z;r〃cae，二斑叶螨(普通红叶螨)；稻"/"raeasaccAara/M小蔑軒草螺；5^0do;7fera/Vug/pe/Y/a,草地夜域；〃e//coye_r/7a么ea，冲帛铃虫；Co/as//s6rwinea，葡萄肖叶甲；Z/ssor力o/^rwsor7zop力/7w51，稻才艮象；57^0/7力//"<or/zae，米象；Ae;7力c^eft/jri/^ro/7/cff/s，,、尾叶蜂；"//mws/ewco;^erws/ei/co/7fe/^，麦长蝽(美洲谷长蝽)；Jcro"ei7咖力27are，拟缘椿(喜绿棒)；大豆/^ewdo/7/i/Wa//7c/i/de/^，大豆(尺)夜蛾；J/7〃csr".age鹏afa//51，梨豆夜蛾；尸/af力7Pe/7asca6ra，苜着绿夜域；6^t_r//7/a/7i/627a/2，欧夕川玉米螟；J^ro〃s0/2，小地虎；Spoffo/^eraeu'gf;a，甜菜夜蛾；y/i"esce/s，烟芽夜蛾；〃e//coFer/>azea，棉铃虫'，^/7//ac力/M7ar/resf/s，墨、西哥(大)豆孤虫；/7ers/cae，书匕对；fzz7/oa"a尸a6ae，蚕豆微叶蝉；Jcro"e/72咖力/7are，拟缘蜂(喜绿蝽)；#e/a/2op/i/s/e/z/wrri;6r叫赤胫黑虫皇(赤腿蚱蛭)；t^e7a/o/7wsf/i//ere/7〃a7i、异黑蝗(殊种蛑蜢)；//fl"ra，(玉米)种绳；Ayer2'cof/r//^rar2's6///s，大豆牧草虫；7!ftr//751fa6acy，烟蓟马(葱蓟马)；re"s;/c/"s"rires"i7/，土耳其叶螨(草莓叶螨)；r"ra/z/c力^wr〃cae，二斑叶螨(普通红叶螨)；大麦0s"/zz/a/7u6//a//s，欧、,川玉米螺；J^rof/s/;7s//an，'J、地虎；ScA/zap力/s^Ta边/i3"/zz，麦二盆坊；A/2'^w51/ewco/^erws/ewco/7ferus，麦长棒(美洲谷长蝽)；Jcro"eiV2咖A/7are，拟缘捲(喜绿棒)；^;sc力/"i/51ser^/51，烟草漆(褐臭蝽)；/e//a/77"i/ra，(灰地)种蝇；舱7e〃0/af/e"ru"or，小麦癭蚊；户e"o6/a/"e/s，麦岩螨；油籽油菜Are"'cor//7e6ras"'cae，甘蓝椅(菜奸)；"///c^refa"f;c/尸erae，十字花跳菜曱；船zz/es"ac^7/7^y/""a,蓓带夜蛾(披肩夜蛾)；尸/"e7/s^x/o"e"a，菜蛾；Ze〃assp.，地种竭(花錄)。线虫类包括寄生性线虫，例如根节、胚嚢和损害线虫，包括异皮线虫(#e"refersspp.)、根结线虫(^e/o油，espp.)和球异皮线虫(W^w/eraspp.)，具体而言是胚嚢线虫类，包括但不局限于，ero^/erag/jc/aes(大豆异皮线虫)、Vefero(/erasc力ac力〃/(甜菜胞嚢线虫)、射erc^era<s,ae(燕麦胞嚢线虫)和67o耐eraro"oc力/e脂、(马铃薯金线虫)以及苍白球异皮线虫(67由dera/7a/7/da)。4员害线虫包括短軒线虫(Praf/Ze^c/"-spp.)。以下实施例仅供例证说明而非起限制作用。实施例实施例1:质粒DNA的抽提以如下方式由菌株ATX13026或ATX13002制备质粒DNA。在大量的丰富培养基中培养菌抹ATX13026或ATX13002的纯培养物。将培养液离心以收获细胞沉淀块。然后以本领域已知方法用SDS处理细胞沉淀块，导致细胞壁破损且DNA释放。然后通过高盐浓度沉淀蛋白质和大基因组DNA。通过标准乙醇沉淀来沉淀质粒DM。通过在氯化铯梯度中的高速离心将质粒DNA与任何剩余的染色体DM分开。使用UV光显现梯度中的DNA，并使用注射器吸取较低密度条带(即较低条带)。此条带含有来自菌林(或是ATX13026或是ATX13002)的质粒DM。利用本领域已知方法通过在琼脂糖凝胶上的显影来检查DNA的质量。实施例2:基因的克隆正如本领域已知的，将纯化的质粒DNA剪切成5-10kb大小的片段，并且在存在所有四种dNTP的条件下使用T4DNA聚合酶和Klenow片段修补5'和3'单链突出。然后，如本领域已知的，通过T4多核普酸激酶处理而在5'末端添加磷酸根。如本领域已知的，将修复的DNA片段过夜连接到适当制备以接受插入片段(例如通过限制酶消化而生成平端)的标准高拷贝载体(即pBluescriptSK+)中。通过用已知在克隆位点侧翼具有切割位点的限制酶消化克隆亚群来分析文库的质量。确定了高百分比的克隆含有插入片段，平均长度5-6kb。实施例3:文库平板的高通量测序将通过上述方法制备的文库涂布在含有适当抗生素的丰富培养基上以维持质粒克隆，并将菌落单独挑到装有2ml含有适当抗生素的培养基的96孔板中。将这些板于37。C、以350rpm摇速培养过夜。将板离心而将细胞收集到板底。通过标准的碱裂解法以高通量方式由这些培养物分离质粒DNA。以如下方式对来自每块板的大量克隆测定来自该文库的克隆的末端序列。4吏用荧光染料终止物测序技术(AppliedBiosystems)和位于克隆位点每一侧侧翼的标准引物测定选择用于分析的每个克隆的DNA序列。一旦反应在热循环仪中完成，则4吏用标准乙醇沉淀法来沉淀DNA。将DNA重悬于水，并加载到毛细管测序仪上。由克隆位点的任一侧测定每个文库平板的DM,每个平板的每个插入片段产生两个阅读结果。实施例4:测序数据的装配和筛选将得到的DNA序列汇编成装配方案，并排列在一起以形成毗连序列群。这可以使用电脑程序有效进行，诸如VectorNTi,或者使用DNA比对和分析程序的Pred/Phrap套餐。将这些毗连序列群连同未能添加到毗连序列群中的任何个别阅读序列与所有类型的已知杀虫剂基因的汇编数据库进行比较。进一步分析鉴定为与已知内毒素或杀虫剂基因具有同一性的毗连序列群或个别阅读序列。在得到的序列中，克隆pAX004、pAX006、pAX007、pAX008、pAX009和pAX014含有鉴定为与已知内毒素基因具有同源性的DNA。因此，选择这些克隆用于进一步测序。实施例5:5-内毒素基因的测序和鉴定设计与内毒素基因具有同源性的序列退火的引物，使得由这些引物产生的DNA序列将与克隆中的现有DNA序列交叠。这一称为"寡聚物行走"的方法在本领域是众所周知的。该方法被用于测定显示与已知内毒素基因有同源性的区域的完整DNA序列。在上文所述克隆的情况中，该方法被用于测定整个克隆的DNA序列，从而得到每个基因的单一核苷酸序列。然后将完整DNA序列放回最初的大装配结果中以进一步确认。这能够将更多的DNA序列阅读结果插入毗连序列群，产生覆盖整个区域的多个阅读结果。对与已知内毒素基因同源的每个区域进行的DNA序列分析鉴定得到了与已知5-内毒素基因同源的开放读码框。将由pAX004鉴定得到的开放读码框命名为AXMI-004。将由pAX006鉴定得到的开放读码框命名为AXMI-006。将由pAX007鉴定得到的开放读码框命名为AXMI-007。将由pAX008鉴定得到的开放读码框命名为AXMI-008。将由pAX009鉴定得到的开放读码框命名为AXMI-009。将由pAX014鉴定得到的开放读码框命名为AXMI-014。SEQIDNOS:1和2提供了AXMI-004的DM序列，而SEQIDN0:3提供了预测的AMXI-004蛋白质的氨基酸序列。位于SEQIDN0:1笫385位核苷酸处的AXMI-004备选起始位点产生了SEQIDNO:5中提供的氨基酸序列。SEQIDNOS:6提供了AXMI-006的DNA序列，而SEQIDNO:7提供了预测的AMXI-006蛋白质的氨基酸序列，SEQIDN0:8提供了AXMI-007的DM序列，而SEQIDNO:9提供了预测的AMXI-007蛋白质的氨基酸序列。位于SEQIDNO:8第151位核苷酸处的AXMI-007备选起始位点产生了SEQIDNO:11中提供的氨基酸序列。SEQIDNOS:12和13提供了AXMI-008的DNA序列，而SEQIDNO:M提供了预测的AMXI-008蛋白质的氨基酸序列。位于SEQIDNO:12笫177位核苷酸处的AXMI-008备选起始位点产生了SEQIDN0:16提供中的氨基酸序列。进一步的分析鉴定得到了紧临AXMI-008开放读码框3'末端的开放读码框。SEQIDNO:18中提供了在本文中称为AXMI-008orf2的该开放读码框的这一预测的氨基酸序列。SEQIDNOS:19中提供了AXMI-009的DNA序列，而SEQIDNO:20中提供了预测的AMXI-009蛋白质的氨基酸序列。位于SEQIDNO:19笫34位核苷酸处的AXMI-009备选起始位点产生了SEQIDNO:22中提供的氨基酸序列。位于SEQIDNO:19第64位核苷酸处的另一个AXMI-009备选起始位点产生了SEQIDNO:24中提供的氨基酸序列。SEQIDNOS:25和26中提供了AXMI-014的DM序列，而SEQIDNO:27提供了预测的AMXI-008蛋白质的氨基酸序列。位于SEQIDNO:25第136位核普酸处的AXMI-014备选起始位点产生了SEQIDNO:29中提供的氨基酸序列。实施例6:分离基因与已知内毒素基因的同源性用本发明的DNA序列和氨基酸序列搜索DM和蛋白质数据库揭示出了这些序列与已知内毒素同源。图1显示了AXMI-004与几种内毒素的序列比对。Blast搜索确定了crylCa具有最强的同源区，在毒性结构域的整体序列同一性为43%(见表1)。表1.AXMI-004与示范性内毒素类型的氨基酸同一性<table>tableseeoriginaldocumentpage159</column></row><table>AXMI-006图2显示了AXMI-006与几种内毒素的序列比对。Blast搜索确定了cry4Aa具有最强的同源区，尽管AMXI-006蛋白质(SEQIDNO:7)与一大组内毒素蛋白质的序列比对显示最同源的蛋白质是crylOAa。crylOAa与AXMI-006的整体氨基酸同一性是25%(见表2)。对AXMI-006氨基酸序列的检查提示它不含几种内毒素家族中存在的C-末端非毒性结构域。由序列比对中除去毒素的这个C-末端蛋白质后，序列比对反映了氨基酸同一性只存在于毒素结构域中(见表2，第3列)。图2显示了这一"修剪过的"序列比对。表2.AXMI-006与示范性内毒素类型的氨基酸同一性<table>tableseeoriginaldocumentpage46</column></row><table>AXMI-007Blast搜索确定了c/r^a具有与AXMI-007最强的同源区，尽管AMXI-007蛋白质(SEQIDNO:9)与一大组内毒素蛋白质的序列比对显示最同源的蛋白质是c/r7Wa。cir7Wa与AXMI-007的整体氨基酸同一性是25%(见表3)。对AXMI-007氨基酸序列的检查提示它不含几种内毒素家族中存在的C-末端非毒性结构域。由序列比对中除去毒素的这个C-末端蛋白质后，序列比对反映了氨基酸同一性只存在于毒素结构域中(见表3，第3列)。图3显示了这一"修剪过的"序列比对。表3.AXMI-007与示范性内毒素类型的氨基酸同一性<table>tableseeoriginaldocumentpage47</column></row><table>Blast搜索确定了CiyW^具有与AXMI-008最强的同源区，而且AMXI-008蛋白质(SEQIDNO:14)与一大组内毒素蛋白质的序列比对显示最同源的蛋白质是cry40Aa。crj^Wa与AXMI-008的整体氨基酸同一性是66%(见表4)。对AXMI-008氨基酸序列的检查提示它不含几种内毒素家族中存在的C-末端非毒性结构域。由序列比对中除去毒素的这个C-末端蛋白质后，序列比对反映了氨基酸同一性只存在于毒素结构域中(见表4,第3列)。图4显示了这一"修剪过的"序列比对。表4.AXMI-008与示范性内毒素类型的氨基酸同一性<table>tableseeoriginaldocumentpage48</column></row><table>紧临AXMI-008编码区下游(3')的开放读码框与已知内毒素相关蛋白质具有同源性。Blast搜索确定了c/y6w^r/7(crj^^a的下游开放读码框)具有最强的同源区。数据库中存在几种其它orf-2样蛋白质，而且图5显示了AMXI-008-orf2蛋白质(SEQIDNO:18)与这些蛋白质中的一组的序列比对。这些蛋白质还与cir^a和cr/MaC-末端非毒性结构域享有同源性。AXMI-8-orf2与CTr^^aor/7的整体氨基酸同一性是86%(见表5)。表5.AXMI-008-orf2与相关蛋白质的氨基酸同一性<table>tableseeoriginaldocumentpage49</column></row><table>Blast搜索确定了cryBAa具有与AXMI-009最强的同源区，而且AMXI-009蛋白质(SEQIDN0:20)与一大组内毒素蛋白质的序列比对显示最同源的蛋白质是cry3Ba和cryl6Aa。cry3Ba和cryl6Aa与AXMI-009的整体氨基酸同一性是26%(见表6)。对AXMI-009氨基酸序列的检查提示它不含几种内毒素家族中存在的C-末端非毒性结构域。由序列比对中除去毒素的这个C-末端蛋白质后，序列比对反映了氨基酸同一性只存在于毒素结构域中(见表6，笫3列)。图6显示了这一"修剪过的"序列比对。表6.AXMI-009与示范性内毒素类型的氨基酸同一性<table>tableseeoriginaldocumentpage50</column></row><table>Blast搜索确定了cr7^Ma具有与AXMI-014最强的同源区，而且AMXI-0014蛋白质(SEQIDNO:27)与一大组内毒素蛋白质的序列比对显示最同源的蛋白质是cry40Aa。crr^4a与AXMI-014的整体氨基酸同一性是55%(见表7)。对AXMI-014氨基酸序列的检查提示它不含几种内毒素家族中存在的C-末端非毒性结构域。由序列比对中除去毒素的这个C-末端蛋白质后，序列比对反映了氨基酸同一性只存在于毒素结构域中(见表7，第3列)。图7显示了这一"修剪过的"序列比对。表7.AXMI-014与示范性内毒素类型的氨基酸同一性<table>tableseeoriginaldocumentpage51</column></row><table>实施例7:AXMI-006与AXMI-007之间的同源性对氨基酸序列AXMI-007与AXMI-006的比较显示了这两种毒素共享显著的氨基酸同源性。氨基酸序列AXMI-006(SEQIDNO:7)和AXMI-007(SEQIDNO:9)的比对显示了这两种蛋白质在氨基酸水平上85%相同。因此，AXMI-006和AXMI-007组成一类新的相关内毒素。实施例8:杀虫活性的测定法常常以多种方式评估杀虫剂蛋白质作为杀虫剂作用于害虫的能力。本领域众所周知的一种方法是进行喂食测定法。在这种喂食测定法中，将害虫暴露于含有待测化合物的样品或对照样品。通常，这是通过将待测物质或其合适稀释物置于害虫将要摄食的物质诸如人工词料上来进行的。待测物质可以由液体、固体或浆液构成。可以将待测物质置于表面之上，然后使其干燥。或者，可以将待测物质与熔化的人工饲料混和，然后分装到测定槽中。测定槽可以是例如杯、盘或微量滴定板的孔。针对吸食害虫(例如坊虫)的测定法可以包括使用隔离物将测试物质与昆虫分开，理想的是吸食昆虫的吸食口器能够刺穿的物体，从而能够摄食测试物质。通常，将测试物质与喂食刺激物诸如蔗糖混和以促进测试化合物的摄食。其它类型的测定法可以包括将测试物质显微注射到害虫的口或肠中，以及开发转基因植物，随后测试害虫以转基因植物为食的能力。植物测试可以包括分离通常取食的植物部分，例如将小笼附于叶片上，或者在装有昆虫的笼中分离整个植抹。本领域还知道测定害虫的其它方法和途径，可以在如下文献中找到Robertson,J.L.和H.LPreisler，1992，《4吏用节肢动物的杀虫齐寸生物领1定法》(尸e"j'6/oassa/sw'MartAro/70cfs),CRC，伯克莱屯，佛罗里达州。或者，测定法一般描述于如下期刊中"ArthropodManagementTests"和"JournalofEconomicEntomology"，或者是与美国昆虫学学会(EntomologicalSocietyofAmerica,ESA)成员的讨论。实施例9:克隆AXMI-006用于蛋白质表达如下将AXMI-006克隆到用于大肠杆菌表达的载体中。pAX480含有用于选择转化子的卡那霉素抗性基因，以及可通过IPTG诱导来调控蛋白质表达的"c启动子。通过将编码"His标签区的DNA片段紧临着插入插入片段克隆区的上游来修饰pAX480，使得得到的克隆在所表达的蛋白质的N端含有6xHis标签。本领域众所周知表达含有6xHis-使用尸尸"^"raTM高保真DNA聚合酶(Stratagene)PCR扩增AXMI-006的编码序列。设计寡核苷酸引物，使得得到的PCR产物在两个末端附近含有期望的限制性位点，以便于克隆。用合适的限制酶消化所得到的PCR产物(约2.2kb),并亚克隆到经过修饰的pAX480中。通过限制性分析鉴定含有插入片段的克隆。得到的克隆pAX906含有与6xHis标签融合的AXMI-006开放读码框，其转录和翻译导致生成具有一段六个组氨酸的"融合蛋白"。通过DNA序列分析确认了pAX906的DNA序列，随后如制造商(Stratagene，拉霍亚，加利福尼亚州)所述转化到化学感受态大肠杆菌BL21中。将pAX906在BL21中的单菌落接种到添加了卡那霉素的LB培养基中，并于37。C以剧烈振摇培养几个小时。将这些培养物培养至0D6。。为0.6-0.8，然后通过添加G.1mMIPTG来诱导蛋白质生成。将培养物在i秀导条件下培养3小时，然后通过离心沉淀细胞并重悬于PBS。采用IO秒超声间隔，使用MisonixSonicator3000超声处理细胞，十30秒，并在冰上保温l分钟。"、4实施例10:AXMI-006对烟芽夜蛾和草地夜蛾的活性的生物测定法4吏用人工饲料(多物种饲料，SouthlandProducts,LakeVillage,阿肯色州)进行经过超声处理的pAX906培养物的生物测定法。通过将经过超声处理的pAX906细胞或作为对照的大肠杆菌菌林的细胞施加到饲料表面并使饲料表面干燥来进行生物测定法。在24孔组织培养板中进行生物测定法。生物测定法在黑暗中、25。C和65%相对湿度下进行。用Breathe简易密封带(DiversifiedBiotech,波士顿，马萨诸塞州)密封盘子。笫5天记录结果。表8.AXMI-006对草地夜蛾的生物测定法<table>tableseeoriginaldocumentpage54</column></row><table>阻碍发育被定义为昆虫体形变小，而且一些幼虫摄食减少。发育受阻碍的昆虫还可能表现出相对于未处理饲料而言回避经过处理的饲料。实施例11:AXMI-008、AXMI-009和AXMI-014对粉紋夜蛾的杀虫活性将含有pAX008、pAX009或pAX-014的大肠杆菌菌林，以及未转化的大肠杆菌的培养物在2mlLB培养基(Luria-Bertani肉汤，BectonDickinson&Company,斯帕克斯，马里兰州)中于37。C以250rpm振摇培养24小时。将含有质粒的菌抹在含有适当抗生素的LB培养基中培养，以选择质粒在大肠杆菌中的维持。使用人工饲料(多物种饲料，SouthlandProducts,LakeVillage,阿肯色州)在24孔组织培养板中进行生物测定法。通过将含有pAX-014的大肠杆菌培养物施加到饲料表面并使饲料表面干燥来进行生物测定法。将菌林的整个培养物以每孔40^1培养物的浓度施加于伺料。生物测定法在黑暗中、25°C和65°/。相对湿度下进行。用Breathe简易密封带(DiversifiedBiotech,波士顿，马萨诸塞州)密封盘子。笫5天记录结果。表IO.对粉发在喊的杀虫活性<table>tableseeoriginaldocumentpage55</column></row><table>实施例12:5-内毒素基因在芽孢杆菌中的表达通过PCR由来自实施例4的克隆扩增AXMI-004、AXMI-006、AXMI-007、AXMI-008和AXMI-009,并通过本领域众所周知的方法克隆到芽孢杆菌表达栽体pAX916中。对于AXMI-004，将得到的克隆命名为pAX920。对于AXMI-006，将得到的克隆命名为pAX921。对于AXMI-007，将得到的克隆命名为pAX919。对于AXMI-008,将得到的克隆命名为pAX922。对于AXMI-009，将得到的克隆命名为pAX917。所得到的克隆在转化到c/y(S)苏云金芽孢杆菌菌林的细胞中后表达相应蛋白质。可以在多种常规培养基中培养含有5-内毒素基因且表达5-内毒素蛋白质的芽孢杆菌菌林。在CYS培养基(10g/1Bacto-casitone;3g/1酵母提取物；6g/1KH2P04;14g/1K2HPO"0.5mMMgS04;0.05mMMnCl2;0.05mMFeS04)中培养含有期望基因的芽孢杆菌菌林，直至通过显微镜检查观察到孢子形成。制备样品，并在针对重要昆虫害虫的生物测定法中对5-内毒素蛋白质测试杀虫活性。才法配制CYS培养基10g/1Bacto-casitone;3g/1酵母提取物；6g/1KH2P04;14g/1L线；0.5mMMgS(V'0.05mMMnCl2;0.05mMFeS04。CYS混合物应当是pH7，如果需要就进行调节。优选用NaOH或HC1。然后将培养基高压灭菌，并在高压灭菌后添加100mlIOX经过滤的葡萄糖。如果得到的溶液浑浊，可以在室温搅动至清澈。实施例13:在芽孢杆菌中表达的AXMI-004的N端氨基酸序列对于在芽孢杆菌中表达的AXMI-004的分析说明在这些培养物中检测到的蛋白质产物在长度上相对于全长AXMI-004蛋白质而言可能是缩短的。因为许多内毒素蛋白质在芽孢杆菌中表达后在N端受到切割，我们测定了由芽孢杆菌表达产生的AXMI-004蛋白质的N末端。在PAGE凝胶上分离来自AXMI-004的蛋白质样品，并且通过本领域众所周知的方法将蛋白质转移到PVDF膜上。切下与AXMI-004对应的蛋白质条带。通过本领域已知的连续Edman降解法测定该蛋白质的N端氨基酸序列。得到的序列如下ERFDK画LE该氨基酸序列与全长AXMI-004序列(SEQIDNO:3)的比较证明该氨基酸序列来自AXMI-004在芽孢杆菌中表达后的内在切割，导致N末端与SEQIDNO:3第28位氨基酸相对应的蛋白质(公开于SEQIDNO:5)。实施例14:AXMI-004对昆虫害虫的生物测定法利用公认的生物测定法流程，使用形成孢子的表达AXMI-004的芽孢杆菌细胞培养物裂解物确定AXMI-004的杀虫活性。在50mlCYS培养基中培养芽孢杆菌培养物，以用于标准生物测定法和LCs。生物测定法。然后将培养物于30。C、以250rpm培养2-3天，直至细胞形成孢子。通过显微镜检查孢子的存在来确定孢子形成。通过将形成孢子的培养物以12，000xg离心10分钟来制备AXMI-004蛋白质样品。收集沉淀并重悬于4ml20mMTris-HCl，pH8.0。将管置于水上的同时将悬浮液超声处理20秒(大功率，使用微型探针)。通过在SDS4-20y。梯度丙烯酰胺凝胶上与已知量的牛血清清蛋白(BSA)—起电泳来测定样品的蛋白质浓度(图8)。AXMI-004的浓度经测定是0.4pg/iil。使用按照本领域所知制备的人工饲料(多物种饲料，SouthlandProducts,LakeVillage,阿肯色州)的表面处理生物测定法来测试AXMI-004的杀虫活性。通过将表达AXMI-004的芽孢杆菌培养物施加到饲料表面并使表面通风干燥来进行生物测定法。在标准生物测定法中，每个孔使用5个虫卵，而LCs。生物测定法每个孔使用IO条新生昆虫幼虫。^使用细尖毛刷施加虫卵或幼虫。标准表面生物测定法在24孔组织培养板中进行。将40ul每种样品施加到每个孔中。因为每个孔的表面积是2cm2(板的来源)，40pi细胞裂解物样品含有大约0.4ug/ulAXMI-O(H。测定LCs。的生物测定法在48孔组织培养板中进行，每个孔展示的表面积是1cm2(来源)，每个孔使用大约20ul0.4pg/ulAXMI-004。每个生物测定法中AXMI-004蛋白质的最终量是大约8pg/cm2。用Breathe简易密封带(DiversifiedBiotech,波士顿，马萨诸塞州)密封生物测定法的板。对照样品包括只含培养基的样品，以及未经样品处理的孔。然后将生物测定法在黑暗中于25。C和65%相对湿度下放置5天，并记录结果。表11.AXMI-004的杀虫活性<table>tableseeoriginaldocumentpage57</column></row><table>AXMI-004显示针对欧洲玉米螟和烟芽夜蛾有强杀虫活性(100%致死)。AXMI-004还显示针对红铃麦蛾有50-75%致死的杀虫活性。浓度为43jag/cn^的AXMI-004对红铃麦蛾产生70%死亡率。AXMI-004严重阻碍小地虎、棉铃虫和草地夜蛾的发育。实施例15:AXMI-004针对烟芽夜蛾和欧洲玉米螟的杀虫活性的量化通过在昆虫生物测定法中测试一定范围的AXMI-004蛋白质浓度并将这些蛋白质样品施加到昆虫饲料表面来测定AXMI-004蛋白质对欧洲玉米螟和烟芽夜蛾幼虫的LC5。。记录每个蛋白质浓度的死亡率，并使用Probit分析软件进行分析。结果在95%置信空间是显著的。因为测定是通过表面污染来进行的，在假设全部蛋白质样品在测定过程中都保留在表面上而在昆虫的摄食水平下很少有扩散的前提下测定LC5。。因此，测定的数值可能有些低估AXMI-004蛋白质对测试昆虫的毒性。表12.AXMI-004对欧洲玉米螟的LC<table>tableseeoriginaldocumentpage58</column></row><table>表13.AXMI-004对烟芽夜蛾的LC<table>tableseeoriginaldocumentpage59</column></row><table>LC50=2874ng/cm2;95%CI=2189-3933实施例16:AXMI-004、AXMI-006、AXMI-007和AXMI-009针对牧草盲蝽(Lyguslineolaris)的杀虫活性的量化如下制备细菌裂解物切下芽孢杆菌在50mlCYS培养基中培养60小时。然后将芽孢杆菌培养物以12，000rpm离心IO分钟，并丢弃上清液。将沉淀重悬于5ml20mMTrisHC1pH8。如下进行生物测定法切下Eppendorf管的底尖和盖子，形成喂食槽。将杀虫剂蛋白质或对照以溶液倒入管帽中并覆盖昆虫能够刺穿从而摄食的石蜡膜(PechineyPlasticPackaging,芝加哥，伊利诺斯州)从而呈现给昆虫。将Eppendorf管倒置管帽朝下置于黑暗中，并用细尖毛刷将一龄或二龄Ar^/s/7//z7//^置于Eppendorf槽中。用石蜡膜密封切下的Eppendorf管尖制成测定槽。将得到的测定槽帽端朝下于环境温度保温。在15°/。葡萄糖溶液中以浓度6.6ug/ml测试杀虫剂蛋白质。<table>tableseeoriginaldocumentpage159</column></row><table>实施例17:AXMI-008对黄粉虫(Tenebriomolitor)的生物测定法制备表达AXMI-008的芽孢杆菌培养物的样品，并测试对黄粉虫的杀虫活性。当pAX922制备成pH4.0的不溶组分时，它显示对通常称为大黄粉虫的昆虫有活性。由含有pAX922的芽孢杆菌菌林的培养物制备AXMI-008的样品。如下制备生物测定法样品将培养物在CYS培养基中培养4天，直至孢子形成。将样品以10，000rpm离心10分钟，丟弃上清液。用20mMTrispH8清洗沉淀，并以10,000rpm离心10分钟。丟弃上清液，并将沉淀重悬于3ml50mM柠檬酸钠和25mM氯化钠及2mMDTTpH4。将样品于37。C保温1小时。保温后，将样品以13,000rpm离心10分钟，丟弃上清液。将沉淀重悬于50mM柠檬酸钠和25mM氯化钠pH4。使用人工飼料(SouthernCornRootwormDiet,Bioserv，Frenchtown,新泽西州，#F9757B)在24孔组织培养板中进行样品对黄粉虫的生物测定法。通过表面处理以Axmi008浓度8ug/cm'施加样品，并使之空气干燥。使用细尖毛刷施加昆虫。用Breathe简易密封带(DiversifiedBiotech,波士顿，马萨诸塞州)密封生物测定盘，并在无光条件下于65°/。相对湿度和25。C保温7天并记录结果。表15.AXMI-008对黄粉虫的杀虫活性<table>tableseeoriginaldocumentpage61</column></row><table>*其余黄粉虫发育受到阻碍。发育受阻即观察到幼虫体形较小、生长较慢，而且摄食剧烈减少或仅有最低限度。昆虫还可能表现出相对于未处理饲料而言回避经过处理的饲料。实施例18:AXMI-009蛋白质对鞘翅类害虫的生物测定法通过将40pl不溶组分施加到2cm2饲料表面上、达到最终总蛋白质浓度为8pg/cm2来进行AXMI-009蛋白质制剂的生物测定法。使用南方玉米食虫祠料(Bioserv，Frenchtown，新泽西州，井F9757B)测试玉米根叶甲(Diabroticavirgiferavirgifera)和黄瓜十一星叶曱(Diabroticaundecimpunctata)。通过将表达AXMI-009的芽孢杆菌培养物施加到伺料表面并使饲料表面干燥来进行生物测定法。生物测定法在24孔组织培养板中进行。标准生物测定法每个孔使用25个虫卵。将虫卵施加到含有0.1%琼脂和30ug/ml制霉菌素的溶液中。用Breathe简易密封带(DiversifiedBiotech,波士顿，马萨诸塞州)密封盘子并将盖子盖回盘上。将生物测定法避光于65%相对湿度(RH)和25。C保温7天。对于玉米根叶甲和黄瓜十一星叶甲都看到了不溶组分的活性。对照是培养基、緩冲液1mMTrispH10.5、和芽孢杆菌表达载体pAX916。表16.玉米根叶曱<table>tableseeoriginaldocumentpage61</column></row><table>表17.黄瓜十一星叶曱<table>tableseeoriginaldocumentpage62</column></row><table>实施例19:用于植物表达的栽体将5-内毒素编码区DNA可操作连接用于在植物中表达的合适启动子和终止子序列。这些序列在本领域是众所周知的，而且可以包括用于在单子叶植物中表达的水稻肌动蛋白启动子或玉米泛素启动子、用于在双子叶植物中表达的拟南芥UBQ3启动子或CaMV35S启动子、以及nos或PinII终止子。用于构建和鉴定启动子-基因-终止子构建体的技术在本领域同样是众所周知的。将上文所述植物表达盒与合适的植物选择标记结合使用，以便于选择经转化的细胞和组织，并连接到植物转化载体中。这些载体可以包括用于由农杆菌介导的转化的二元载体或用于气雾剂或biolistic转化的单一质粒载体。实施例20:玉米细胞的转化授粉后8-12天采集玉米穗。由穗分离胚，并将大小为0.8-1.5mm的那些胚用于转化。将胚盾片朝上置于合适的培养基上，诸如DN62A5S培养基(3.98g/LN6盐；1mL/L(1000x储液)N6维生素；800mg/LL-天冬酰胺；100mg/L肌醇；1.4g/LL-脯氨酸；100mg/LCasaminoacids;50g/L蔗糖；1mL/L(1mg/mL储液)2，4-D)，并于25。C在黑暗中保温过夜，将由此产生的外植体转移到筛网上(每块板30-40粒)，转移到渗透培养基上达30-45分钟，然后转移到光照板上(见例如PCT公开号WO/0138514和美国专利号5,240,842)。采用基本上如PCT公开号WO/0138514中描述的条件(aerosolbeamaccelerator),使用气溶胶气流加速器，将设计在植物细胞中表达5-内毒素的DNA构建体加速进入植物组织。光照后，将胚在渗透培养基上保温30分钟，然后置于保温培养基上在黑暗中于25。C保温过夜。为了避免过度损伤经过光照的外植体，在转移至恢复培养基前将它们至少保温24小时。再将胚散布到恢复期培养基上，在黑暗中于25。C保温5天，再转移至选择培养基上。将外植体在选择培养基中保温可长达8周，这取决于所用具体选择方法的本质和特征。选择期后，将由此产生的愈伤组织转移至胚成熟培养基，直至观察到成熟体细胞胚的形成。然后将由此产生的成熟体细胞胚置于低光照下，并通过本领域已知方法启动再生程序。使由此产生的芽在生根培养基上生根，并将由此产生的植抹转移至苗圃罐中，繁殖成转基因植物。辦DN62A5S培养基成分絲来源Chu'SN6M盐^給物(Prod.No.C416)3.98g/LPhytotechnologyLabsChu，sN6维生素溶液(Prod.No.C149)1mL/L(lOOOx储紀PhytotechnologyLabsL-天冬纖800mg/LPhytotechnologyLabs肌醇100mg/LSigmaL-脯氨酸1.4g/LPhytotechnologyLabsCasaminoacids100mg/LFisherScientific庶^^50g/LPhytotechnologyLabs2，4-D(Prod.No.D~7299)1mL/L(1mg/mL#^USigma用1NK0H/1NKC1将溶液的pH调至pH5，8，添加Gelrite(Sigma)至3g/L，并高压灭菌。冷却至50°C后，添加2ml/L硝酸银(PhytotechnologyLabs)的5mg/ml储液。it酉己方帝J备大纟勺20块平板。实施例21:通过由农杆菌介导的转化转化到植物细胞中授粉后8-12天采集穗。由穗分离胚，并将大小为0.8-1.5mm的那些胚用于转化。将胚盾片朝上置于合适的培养基上，并在黑暗中于25。C保温过夜。但是，本质上并不必将胚保温过夜。使胚接触含有用于Ti质粒介导的转移的合适载体的农杆菌菌林达5-10分钟，然后铺板在共培养培养基上，在黑暗中于25。C保温3天。共培养后，将外植体转移至恢复期培养基，在黑暗中于25。C保温5天。将外植体在选择培养基中保温可长达8周，这取决于所用具体选择方法的本质和特征。选择期后，将由此产生的愈伤组织转移至胚成熟培养基，直至观察到成熟体细胞胚的形成。然后将由此产生的成熟体细胞胚置于低光照下，并通过本领域已知方法启动再生程序。使由此产生的芽在生根培养基上生根，并将由此产生的植林转移至苗圃罐中，繁殖成转基因植物。结论正如上文实施例中所示，本发明的5-内毒素蛋白质针对多种害虫具有活性。AXMI-004对包括欧洲玉米螟、小地虎、棉铃虫、草地夜蛾、烟芽夜蛾、红铃麦蛾、烟草天蛾、粉紋夜蛾、和牧草盲蝽在内的害虫有杀虫活性。AXMI-006对包括烟芽夜蛾、草地夜蛾、和牧草盲蝽在内的害虫有杀虫活性。AXMI-007对包括牧草盲蝽在内的害虫有杀虫活性。AXMI-008对包括黄粉虫和粉紋夜蛾在内的害虫有杀虫活性。AXMI-009对包括牧草盲蝽、粉紋夜蛾、玉米根叶甲、和黄瓜十一星叶甲在内的害虫有杀虫活性。AXMI-014对包括粉紋夜蛾在内的害虫有杀虫活性。说明书中提到的所有发表物和专利申请指示了本发明所属领域熟练技术人员的技术水平。本文收录所有发表物和专利申请作为参考，其程度与专门和个别指出收录每一项发表物或专利申请作为参考相同。虽然出于清楚理解的目的通过例示和实施例已经较为详细的描述了上述发明，显然可以在所附权利要求的范围内实践某些改变和修饰。序列表<110>Caxozzi,NadineHargiss,TracyKoziel,MichaelG.Duck,NicholasB.Carr,Brian<i2o>6-内毒素基因及其使用方法<130>045600/274379<150>60/448,632<151>2003-02-20<150>60/448,633<151>2003-02-20<150>60/448,797<131>2003-02-20<:150>60/448,806<151>2003-02-20<150>60/448,810<151>2003-02-20<150>60/448,812<:151>2003-02-<160>S2<170>FastSEQforWindowsVersion4.0<210>1<211>2190<212>DNA<2工3>苏云金芽孢杆菌<400>1tatgatgataELCtttg3caacagccgcatcccaagcttattttgtacaaattacactttagcatataagaaaattttttcatatataagttaggataaactttacttgtaaacacaagtaaaaatatttacgaatccta±ctataaaaatatgagtgaattgaaggggaaatttaagaaaataataaatataggaggaagaggtatgaataatgatttaagtgatcgccaatattaatatgattggtatgtccattgtatctgaacttatttttgtgtttaatcaattgtggtctcgtttacatgt的agg幼ttaattgatactaaaatagaattagcaggtatacaaagaaaccttgaaaatgatattg幼幼ctcaaa的ctcaaggtcaggcggttgaaaggagtstgcctc祖tttactgtctatgtgcaagctgctaatcttcatggaaagtgttggggatggtcggagcagaaatatacccatg站tacacaaatcattgtgtaaaaaataaaggttcttcttatcaagattggactcttactgttttagatatcgttgctttaataacaaccgttgetcagctaacaagggaacctaaattacattctgtgtctcaattacctagaactccacatctgatggaatttttaagagtgggaagaaactattattggggaggacataatataagatcacctctatat的tagagagttttatggacccgtttttaagacgttatcacataacgtactttttcaatattaattgtat60ttgtatgcataatagtttcttattgattga120gaaaatatttttgcttttcttttttttatt180aatagtttattagagatagagcctcatttt240ctaattttaaaattattccttttaaatgta300agtactaatcgaacttgttgtttgctaaaa360tcaaaggasicatgattatctaaaagtttgt420诉acggtttgataagaatgatgcactggaa480ggtatgattccaggcggaacagctttgcaaS40ggtgattctggatggaatgcgttcatggaa600gaagggtatgcaaaaaat站agccttatctacatatatacaattacgt貼tgaatgg诉a720aaggtagctaattactat诉幼gtcttgag780gcagtggagaattttgaagtaccactttta840ttattattattaa诉gatgtttc的tttat900attaaaatttattatgat貼acagattaagS60站ttggtataataaag诉cttgaga诉tta1020tac站ttataatcgtttccgta诉g站at:g1080ttcccgcactELt诉tgtacaLaacttatcca1140gtttatacggatcctttacttaattttaat1200agttttagtgacatgga站atgcaacaatt1260atgctaacaatttatacagattggtatagt1320cgcgtgacgtcttaccatgtaggaggagag1380gcaaatcaagaggttcct的的atttttat"40aagccgactcta的accattacagcagcct1500gcaccagctcggtagtttta33tcce1gttgcgtaaatctgt3tcaaattgtctcasagatagtattaatgsctttsggttgcacaasgcagataaaattgctccttttaatgtatcgtgaggttacctcsggaccccttaccaatacaatggtcaggcaccaggtcctggatcgtgtaagggaccaccatctgagggctatatttagatt&atttattcctttacgtagcaagaggatcgtaaggtatactgaatcaaattactgtaaggaacatttggagattcgttattaat3ttggttagaacagtaaggtatacaatgttgaatagttagagggsggtagattcttagtcaccgttggtgccatctattattacgcgatatgagaagcttctacgaaacttcccgatcgtttagtagcattttctgt￡iga3ttcc3ttaatgagtttatgtcatgctttcttggacgattcacaggtaaatattaacagatttacaGtccatttaggagttcsagacactcctacagccgccttttaacgtttgttacatcgtagtagtsaaagcaaggggatatatgcaccattaatttgtcacgtttctcaaatagtttatgtg15601620:U8Q174018001860192019802040210021602190<210>2<211>1890<212>DNAdlh苏云金芽孢杆菌<220><221>CDS<222>(1)…UB90)<400>23tgagtgaaMetSerGluttgaaggggaaatttaagaaaagtactaatcgaacttgtLeuLysGlyLysPheLysLysSerThrAsnArgThrCys510IS48tgtttgetaaaaataataaatataggaggaagaggtatgaatteaaagCysLeuLeuLyslielieAsnlieGlyGlyArgGlyMetAsnSerLys20253096gaacatgattatetaaaagtttgtaatgatttaagtgacgecaatattG3LuHisAspTyrLeuLysValCysAsiiAspLeuSerAspAlaAsnlie3S4045144aatatggaaeggtttgataagaatgatgcactggaaattggtatgtecAsnMetGluArgPheAspLysAsnAspAlaLeuGlulieGly.MetSer505560192attgtatctgaacttattggtatgattccaggcggaacagetttgcaalieValSerGluLeulieGlyMetlieProGlyGlyThrAlaLeuGin65707580240tttgtgtttaatcaattgt的tctcgtttaggtgattctggatggaatPheValPheAsnGinLeuTrpSerArgLeuGlyAspSerGlyTrpAsn859095288gcgttcatggaacatgtggaggaattaattgatactaaaatsigaag的AlaPheMetGluHisValGluGluLeu工leAspThrLys工leGluGly100105110336tstgcaaaaaataaagecttatctgaattagcaggtatacaaagaaacTyrAlaIiysAsnLysAlaLeuSerGlulieuAlaGlylieGinArgAsn115120125384cttgaaacatatatsicaattacgtaatgaat的gaaaatgatattgaaLeuGluThrTyrlieGinLeuArgAsnGluTrpGluAsnAsplieGlu13013S140432站CtC2L抑3AsnSerLys145getcaaggtaagAlaGinGlyLysISOgtagetaattactatgaaagtcttgagValAlaAsnTyrTyrGluSerLeuGlu155160480caggcggttgaaaggagtatgcctcaatttgcagtggagaattttgaaGinAlaValGluArgSerMetProGinPheAlaValGluAsnPheGlu165170175S28gtaccacttttaactgtctatgtgcaagetgetaatcttcatttatta57SValProLeuLeuThrValTyrValGinAlaAlaAsnLeuHisLeuLeu180185190ttattaagagatgttteagtttatggaaagtgttggggatggteggagLeuLeuArgAspValSerValTyrGlyLysCysTrpGlyTrpSerGlu19520020S624Gin3332103ttlieLys3ttlietatTyrtatTyr215gatAspS33LiysGin3ttlie220tatTyr3CCThrC3tHisgaaGlu672t3CTyr225￡L￡LtestHistgtCys230￡L3tJ\ST\TrptatTyrs的235ggaGlycttLeugagGlu3g3ArgLeu240720As119gtGlytct245tcttatTyrGinAsptggTrp250t3CTyrsatAsnt3tTyr33tAsncgtArg255ttcPhe768cgtArg3g3ArggaaGluatgMet2603Ctct匕lieusetThrgttValttaLeu265ga匕AspateliegttValgetAlatt3lieu270ttcPheccgPro816C5LCHisTyrgatAsp275ValC33GinactThrtatCC3Pro280lieThr3CCThrgttValgetAla28SGinCt3Ieu864顿Argg貼Glu290tatTyrThrAspcctPro295ttacttLeuttt300cctProttalieucatHis912tct30S9tgValtctGinttaLeucctPro310SertttPheagtSerMet315gasGluAsngcaAlaThrattlie320ArgsetThrCCELProcatHisctgLeu325atgMetg站GlutttPhett3LeuagaArg330atgMetetaLeuThr8ttlietatTyr335Thr1008gattggtatagtgtgggaagaaactattattggggaggacatcgcgtgAspTrpTyr*SerValGlyArgAsnTyrTyrGlyGlyHisArgVal3403453501056acgtcttaccatgtaggaggagagaatataagateacctetatatggtThrSerTyrHisValGlyGlyGluAsnlieArgSerPi:oLeuTyrGly3553601104agagaggcaaatcaagaggttcctagagatttttatttttatggacccArgGluAlaAsnGinGluValProArgAspPheTyrPheTyrGlyPro3703753801152gttVal385tttPhe的gacgThrLeutedSer3S0站gccgProactThretaLeu的aArg39SccaProLeuGinGincctPro4001200gfcaccagetcctccttttaatttacgtagettagagggagtagaattcAlaProAlaProProPheAsnLeuArgSerLeuGluGlyValGluPhe40S4104151248cacactccfcacaggtagttttatgtatcgtgaaagaggateggtagatHisThrProThrGlySerPheMetTyrArgGluArgGlySerValAsp42042S4301296tctSetttt站tAsn435弼GluttglieuccgProcctProtttPhe440aatAsnProgttVal卿GlyttaLeu445cctProestHis站g1344gtatacagtca<=cgtttatgtcatgcaacgtttgttcgtaaatctggg1392LeuCysHisAlaThrPheValArgLysSerGly4504554603CCThr465cctProtatTyrttaThra_C3470ggtGlygccAlaatelietttPhetctSer475tggTrpThrC3tHiscgtArgSer4801440getAlagaaGluGlu3CCThr33tAsn485ThrattliegaaGluteaSera_3tAsn4903ttlie3tt工leacgThrGin3tClie495ccgPro1488LeugtaVal335Ala500ta匕TyrGin3tt工legggGlytC3Ser505GlyactThractThrgtaValArg510333Gly1536ProGlyttcPhe515Thr卿GlygggGlygatAsp3t3lie520cttArgArg抓Gly525cctPro卿GlyThr1584t匕tPheggaGly530gatAspatgMet3ga工lesatAsia535attlieAsnAlaCC3Prott3Leu540tctSerC33GinArgtatTyr1S32cgtArg545gtaValArgliecgtArgtatTyr5S0getAlatctSeraegThrThrgatAsp5S5ttaLeuc貼GintttPhegtcValacgThr5601680agtSer3ttlie站tAsng的Gly■accThr5653CCThrattlie€L6ttAsnattlieGly57033CAsnttcPheccgProsetThr5753ttlie1728aataatetaaatactttacggttctgagggctatagaacagtategtttAsnAsnLeuAsnThrLeuGlySerGl\iGlyTyrArgThrValSerPheS805855901776agtSer3CtThrCC3Pro595tttPheSerttcPhetC3Ser￡131600Alac貼GinageSersita_工letttPhe605ttaLieuGly18243t3lieC33GinG10gcaAlatttPhetctGly615C33Ging33GlugttValt3tTyrgtgVal620gatAsp333attliegaaGlu1872ttt.Phe625attliecctProgttValGlutag1890<210>3<211>629<212>PRT<2">苏云金芽孢杆菌<400>3MetSerGlulieuGlyLys1SCyslieuLeuLyslielieAsnlie20GluHisAspTyrlieuLysValCys3S40AsnMetGluArgPheAspLysAsnSOSSlieValSerGlulieulieGlyMetPheValPheAsnGinLeuTrpSer85Ala昨sMetGluBisValGluGlu100Ly曰LysSerThrAsiaArgThrCys1015GlyGlyArgGlyMetAsnSerLys2S30AsnAspLeuSerAspAlaAsnlie4SAspAlalieuGlulieGlyMetSer60lieProGlyGlyThrAlalieuGin75B0ArglieuGlyAspSerGlyTrpAsnSOLeulieAspThrLyslieGluGly<table>tableseeoriginaldocumentpage70</column></row><table><210><211><212><213><220〉<221><222>41806DNA苏云金芽孢杆菌CDS(1).(180S)<400>4atgaatMetAsnteaSer站gljysgaaGlucatHisgatAsptatTyrCt3Leu333Ijys10gttValtgtCys33tAsngatAspLeu15a_gtSer48AspAlaAsialie20AsnMetGlueggArgtttPhe25gatAspLysAsngatAspAla30ctgLieuGlu96attlieggtGlyMet35tecSerattliegt3ValtctSergaaGlu40cttLeu3ttlieGlyMetlie45ProggcGlyGly144ThrgetAla50ttgLieucaia_GintttPhegtgValtttPhe55aatAsnC33GinttgLieutggTrptctSerGOcgtArgttaLeuggtGlyga±Asp192tctSerGlyt的TrpaatAlattcPhe70atgMetgaacatHisVal弼Glu75Glutt3LeuattliegatAspsetThr80240liegaaGlu鹏tatTyr85Ala6L6L3LAsn祖eiAla90ttalieutctSergaaGluIjeugcaAlaGly2B83t3lieGillagaArgAsn100CttlieugaaGluThrtatTyratalie105GinttalieucgtsatAsng的Glu110tggTrpgasGlu33633tgatAspattlie11593aGluteaSeraaggetAla120Gin93tGlyLyeValgetAla125aattacTyrtatTyr384g犯GluagtSer130CttlieugagGluGinAlagttVal13SgaaGluaggArgSerMetcct140c站GintttEheAla9t9Val432gagGlu145AsntttPheg幼GluValPro150cttLeuttaIjeuactgtcThrValtatTyr1559tgValGingetAlagetAlaAsn160480cttcatttattattattaagagatgttteagtttatggaaagtgttggLeuHisLeuLeuLeuLeuArgAspValSerValTyrGlyLysCysTrp16S170175S2BggatggtcggagcagaaaattaaaatttattatgataaacagattaagGlyTrpSerGluGinLyslieLyslieTyrTyrAspLysGinlieLys180185190S76tatacccatgaatacacaaatcattgtgtaaattggtataataaag诉TyrThrHisGluTyrThrAsnHisCysValAsnTrpTyrAsnLysGly195200205cttgagagattaaaaaataaaggttcttcttatcaagattggtacaatLeuGluArgLeuLysAsnLysGlySerSerTyrGinAspTrpTyrAsn210215220€72tataatcgtttccgtagagaaatgactcttactgttttagatategttTyrAsnArgPheArgArgGluMetThrLeuThrValLeuAsplieVal230235240getAlattaLieuttcPheccgProescHis245tatTyrgatAspgtaValGinactThr250tatTyrcca_ProatalieThr3CCThr255gttValgetAlacagGina_c3Thr2603ggArgGlugttValtatTyra_cgThr265gatAspcctPrott￡LLeucttLeu3attAsn270tttPheAsn816cctProIjysttaIjeu275catHistctgtgValtctSerca_3Gin280tt3LeucctProagtSertttPheagtSer285AspatgMetgaaGlu86433tAsngcaAla290ThrattlieagaArg3CtThrCC3Pro295catHisctgIjeuMetGlutttPhe300Leuaga_Arg3tgMetetaLeuacaatttatacagattggtatagtgtgggaagaaactattattggggaThrlieTyrThrAspTrpTyrSerValGlyArgAsnTyrTyrTrpGly305310315320960GlyC3tHiscgcArggtgVal3egThr325tctSert3CTyrestHisgtaValggaGly330GlyGlu33tAsn3t3lieArg335tC3Ser1008cctetatatggtagagaggcaaatcaagaggttcctagagatttttatProLeuTyrGlyArgGluAlaAsnGinGluValProArgAspPheTyx3403453S010S6ttttatTyrGly355cccPro3ttValtttPhesagLysacgThr360tt3LieuteaSerLyaProactThr365etaIjeuagaArgCC3Pro1工04ttacagGin370GincctProgcaAlaCC3ProgetAla375cctProcctProtttPhe33tAsnttalieu380cgtageSerlieugagGlu1152Gly385gtaValGluttcescHis3CtThr390cctProThrggtGly3gtSerttt395atgMetTyrcgtArgGluArg4001200ggateggtagattcttttaatgagttgccgccttttaatccagttgggGlySerValAspSerPheAsnGluLeuProProPheAsnProValGly4054104151248ttacctcataaggtatacagtcaccgtttatgtcatgcaacgtttgttLeuProHisLysValTyrSerHisArgLeuCysHisAlaThrPheVal4204254301296cgtArgtct435的gGlyThrcctProTyrttsi440ThrThr的tGlygecAlaatelie445ttttctSertggTrp1344acacatcgtagtgetgaagaaaccaatacaattgaateaaatattattThrHisArgSerAlaGluGluThrAsnThrlieGluSerAsnlielie4504S54601392acgcaaateccgttagtaaaagcatatcaaattgggteaggcactactThrGinlieProlieuValLysAlaTyrGinlieGlySerGlyThrThr4654704754801440gtaaggaaaggaccaggattcacaggaggggatatacttcgaagaacaValArgLysGlyProGlyPteThrGlyGlyAsplieLeuArgArgThr4854904SS1488ggtcctggaacattt的agatatgagaataaatatt站tgcaccattaGlyProGlyThrPheGlyAspMetArglieAsnlieAsnAlaProLeu153€510tctcaaagatatcgtgtaaggattcgttatgettctacgacagatttaSerGinArgTyrArgValArglieArgTyrAlaSerThrThrAspLeu5155205251584caatttgtcacgagtattaatgggaccaccattaatattggtaacttcGinPheValThrSerlieAsnGlyThrThrlieAsnlieGlyAsnPhe5305355401632ccgaaaactattaataatetaaatactttaggttctgagggctatagaProLysThrlieAsnAsnLeuAsnThrLeuGlySerGluGlyTyxArg5455505555601680acagtategtttagtactccatttagtttcteaaatgcacaaageataThrValSerPheSerThrProPheSerPheSerAsnAlaGinSerlie5655705751728tttagattaggtatacaagcattttctggagttcaagaagtttatgtgPheArgLeuGlylieGinAlaPheSerGlyValGinGluValTyrVal5805855901776gataaaattgaatttattcctgttgaatagAspLyslieGluPhelieProValGlu*5956001806<210>5<211>601<212>PRT<"3>苏云金芽孢杆菌<400>MetAsnSerLysGluHisAspTyrlieuLysValCysAsnAspLeuSer1510ISAspAlaAsnlie20AsnMetGluArgPhe25AspUysAsnAspAla30LeuGlulieGlyMet35Ser工leValSerGlu40IjeulieGlyMetlie45ProGlyGlyThrAla50IjeuGinPheValPhe55AsilGinLeuTrpSer60ArgLeuGlyAspSerGlyTrpAsnAlaPheMetGluHisValGluGlulieu工leAspThr65'707580LysneGluGlyTyr85AlaLysAsnLiysAla90lieuSerGluLeuAla95GlylieGluArgAsn100LeuGluThrTyrlie105GinlieuArgAsnGlu110TrpGluAsilAsplie115GluAsnSerLysAla120GinGlyLysValAla125AsnTyrTyrGluSer130lieuGluGinAlaVal135GluArgSerMetPro140GinPheAlaValGluAsnGluValProLeulieuThrvalTyrValGinAlaAlaAsn145ISO155160lieuHisLeulieuIjeuLeuArgAspValSerValTyrGlyLysCysTrp1653L70GlyTrpSerGluGinLyslieLyslieTyrTyrAspLysGilllieLys180185TyrThrHisGluTyrThrAsnHiaCysValAsnTrpTyrAsnLysGly195200205IjeuGluArglieuLysAsnLysGlySerSerTyrGinAspTrpiyrAsa210215220TyrAsnArgPheArgArgGluMetThrLeuThrValLeuAsplieVal225230235240AlaLeuPheProHis245TyrAspValGinThr2S0TyrProlieThrThr2SSValAlaGinUeuThr260ArgGluValTyrThr265AspProlieulieuAsn270PheAsnProLyslieuHisSerValSerGinLeuProSerPheAspMetGlu275280285AsnAlaThrlieArgThrProHisLeuMetGluPheLeuArgMetLeu290295300ThrlieTyrThrAspTrpTyrSerValGlyArgAsnTyrTyrTrpGly305310315320GlyHisArgValThrSerTyrHisValGlyGlyGluAsnlieArgSer325330335ProLeuTyrGlyArgGluAlaAsnGinGluValProArgAspPheTyr340345350PheTyrGlyProValPheLysThrIjeuSerLysProThrIjeuArgPro355360365IjeuGluGinProAlaProAlaProProPheAsnIjeuArgSerLeuGlu370375380GlyValGluPheHisThrProThrGlySerPheMetTyrArgGluArg385390395400GlySerValAspSerPheAsnGluIj已UProProPheAsnProValGly405410UeuProHisLysValTyxSerHisArgLeuCysHisAlaThrPheVal420425430ArgLysSerGlyThrProTyrLeuThrThrGlyAlaliePheSerTrp435440445ThrHisArgSerAlaGluGluThrAsnThr工leGluSerAsnlielie450455460ThrGin工leProIjeuValIjysAlaTyrGinlieGlySerGlyThrThr465470475480ValArgLysGlyProGlyPheThrGlyGlyAsplielieuArgArgThr485490495GlyProGlyThrPheGlyAspMetA巧lieAsn工leAs:nAlaProIjeu500505510SerGinArgTyrArgValArglieArgTyrAlaSerThrThrAspLeu515520525GinPheValThrSerlieAsnGlyThrThrlieAsnlieGlyAsnPhe530S35S40ProLysThrlieAsnAsnlieuAsnThrUeuGlySer*GluGlyTyrArg545550555560ThrValSerPheSerThrProPheSerPheSerAsnAlaGinSerlie56S570575PheArglieuGlylieGinAlaPheSerGlyValGinGluValTyrVal580585590AspLyslieGluPhelieProValGlu595<210>6<211>2208<212>DNA<2工3>苏云金芽孢杆菌<220><221>CDS<222>(1)-.，(2208)<400>6atgcaaaat幼cgataat站cgaatatgaaELttattgattegcatMetAsnGinAsnAsnA6pAsnAsnGluTyrGlulielieAspSerHis151015ccttattttccgagaaacagtaatgattctagataccctThrSerProTyxProAanArgAsnSerAsnAspSerArgTyrPro202S30t3CaatSLSlt站tcaattacaa站c站ttsc站￡1弼TyrThrAsnAsnProAsnGinProLeuGinAsaThrAsnTyrLysGlu354045tggetcaatatgtgtc站gggcaatatggtgataatttc诉gTrpLeuMetCysGinGlyAsnTharGinTyrGlyAspAsnPheGlu505560acatttgetagtgetgatacaattgetgcagtt的tgcaggtactattThrPheAlaSerAlaAspThrlieAlaAlaValSerAlaGlyThrlie65707580"0gtatecValSerggtactctgGlyThrLeuB5ttalieugecAlaggtGly3t3lie抓Gly90gggGlyetcLieuactThrtctSer3ta_工le95tecSer28BggaccgGlyProataggaatalieGlylie100ggtGlygetAla3teilie105lieatalietctSertttPheGly110a_ccThrcta3363tC3CtlieThrgtcttttggValPheTrpliscccProAlaGly120GluGingacAsp3333C3Thr12SgtaValtggTrpThr384catstttGinPhe130attaaastg工leLysMetGlyGlu135attlietttPhegttValgatAsp3C3Thr140ccgPrott3LeuThrgaaGlu432ageataSerlie145aaacagctaLysGinLeusagLys150ttalieuGinsetThrttaUeu9站Glu155的3Glyttt3g3ArgGinlie160480ttacaaLeuGinagetataatSerTyxAsn165ThrgcaAlaLteu93tAspgatAsp170tggTrp3g3Arg3站LysttaLeu333Lys175Arg528Ct3C33IjeuGingetcctggaAlaProGly180tt3IjeuCC3ProCC3ProtC3Ser185tC3SerAlattaLeuC33GinGin190getAlaAla576ttgactLeuThrcttaaaatalieuLyslie19Scga_ArgtttgagGlu200站tgttC3CHis站tgatAsp20stttPheattliecgaArg€24g33a_ta>Glulie210cctggtttcProGlyPheGincttIjeu215GluactThrt&tTyracgThr220LeuIjeuCt3cctPro672atttatlieTyr225gcgcastgetAlaGinAlagetAla230sattttPhecatHisttaUeuaatAsn235ttaLeutt3Ginc貼GinGly240720getgaattggetgatgaatggaatgcagatatacatcctteacaaattAlaGluLeuAlaAspGluTrpAsnAlaAsplieHisProSerGinlie245250255768gaacctaatgetggaacateagatgactattataaacttttaaaagaaGluProAsnAlaGlyThrSerAspAspTyxTyrLysLeuLeuLysGlu2602S5270816aatatacctaaatatagtaactattgtgcaaatacctatagaacaggaAsnlieProLysTyrSerAsnTyrCysAlaAsnThrTyrArgThrGly27S2802858"ctaaaaaatcttagagacgaaccaaatatgaaatggagtatatttaatLeuLysAsnLeuArgAspGluProAsnMetLysTrpSer工lePheAsn29029S300312诉ctatcgaagatatatgaccattactgtatta诉taccatetctcaaAspTyrArgArgTyrMetTfarlieThrVallieuAspThrlieSerGin30S310315320960ttttctttatatgatataaaaagatatagagatteaataggaggaataPheSerLeuTyrAsplieLysArgTyrArgAspSerlieGlyGlylie3253303351008gaagtaaaaggcattaagaatgaaetcacaagagaa_atttatacaactGluValLysGly工leLysAsnGlulieuThrArgGlulieTyrThrThr340345350GlulieaatAsn355tttgatcgtcttPheAspArgLeucctPro360caa_ctta_gaGinJjeuArggttcaaValGin365cccPro33tAsnctalieu1104getacgatggaatataatttaacacgtgcaagttttaaattatttteaAlaThrMetGluTyrAsnLeuThrArgAlaSerPheLysLeuPheSer3703753801152tttttagaacaa匕ttattttttatacagaaaatacaaatttcgggaatPheLeuGluGinPheliePheTyrThrGluAsnThrAsnPheGlyAsn3853903954001200cgtttagttggtatttctaa匕cgtgatgcacctacttatageaatactArgLeuValGlylieSerAsnArgAspAlaProThrTyrSerAsnThr4054104151248ata工leactThrgaaGluactttatatggagaaThrLeuTyrGlyGlu420agaacaggtArgThr*Gly425tca_cccSerPro5LCELThr4306LC8LThrEL貼1296aca^taagaccatttgaatcttataaagttteaattgtaactgatagaThrlieArgProPheGluSerTyrLysValSerlieValThrAspArg4354404451344caateacctcctgttteccctattcaaccacactttataattaatcaaGinSerProProValSerPro工leGinProHisPhelielieAsnGin450455"01392attgaactttatttaaatggcteatctaacaacacaetcaaatattealieGlulieuTyrlieuAsnGlySerSerAsnAsnThrLeuLysTyrSer4654704754B01440gcaAlag9a999tctttatctaattatcaaaacacaactttttttcaatttGlyGlySerLeuSerAsnTyrGinAsnThrThrPhePheGinPhe4854904951488cctagaaaaaaagactgcaatctagttattgatccaggttgtteaccaProArgLysLysAspCysAsnLeuVallieAspProGlyCysSerPro5005055101536aactttaataactatagtcatattttateccattttteattatttactAsnPheAsnAsnTyrSerHislieLeuSerHisPheSerLeuPheThrSISS20S251584tattectatgtgattggattacagctacaaatattagatacaggtgtaTyxSerTyxVallieGlyLeuGinLeuGinlieLeuAspThrGlyVal53053S5401632ttaggatggacacacagtagtgttgatagatataafcgcaatateagatLeuGlyTrpThrHisSerSerValAspArgTyrAsnAlalieSerAsp5455505555601680aaaataattacaatgateccagcaateaaaggtaacaatcttgatacaLyslielieThrMetlieProAlalieLysGlyAsnAsnlieuAspThr5655705751728aactctaaggtaatt诉aggacctggtcatacaggaggaaacttggttAsnSerLysVallieGluGlyProGlyHisThrGlyGlyAsnLeuVal58058SS901776tatttacaaagtcaagggcgtttagaaattacatgtgaaactcctaatTyrLeuGinSerGinGlyArgLeuGlulieThrCysGluThrProAsnS356006051824tcttctttcattagaicttcgaitatgetgetSerThrGinSerTyrPhelieArgLeuTyrAlaThrAsnGlyAla610615cttcctaatatatctctta_taccaigta的3GlyAsnThrLeuPro工leSerLeuThrlieProGlyVallieGly625630635640CC3cctcgaetcactttttctggttat站tlieProProGinArgLeuAsnAshThrPheSerGlyAsnTyrAsn645650655ttatacgattttgggtatttctt匕CC3gtalieuGinTyrGlyAspPheGlyTyrPheGinPheProSerThrVal"5670ttacctttsaatCC3ttttttcgtgcagatThrLieuProIjeuAsnArgAsnlieProPheliePheAsnArgAlaA印675680685gtateatceiattttsatesttgatattgaatttataccaiValSerAsnl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lieHisProSerGinlieGluProAsn1S5200205624getggsacateagatgactattataaacttttaga_aaa_tstacctAlaGlyThrSerAspAspTyrTyxLysLeuLeuLysGluAsnliePro21021S220672aaatatagtaactattgtgcaaatacctatagagaaggaetaaataaaLysTyxSerAsnTyrCysAlaAsnThrTyxArgGluGlyLeuAsntiys225230235240720cttcgaaacgaacctaatatgagatggagtatatttaatgattatcgaLeuArgAsnGluProAsnMetArgTrpSerliePheAsnAspTyrArg2452S0255768agatatatgactattactgtattagatactategetcaattttcttttArgTyrMetThrlieThrValLeuAspThrlieAlaGinPheSerPlie2602S5270816tatgettataaagagataca_aagatteaataggaagaataggtggcattTyrAsplieLysArgTyrLysAspSerlieGlyArglieGly*Glylie2752802858S4aaaactgaaLysThrGlu290cttacaagagaaattlieuThrArgGlulie295tatacaactgaaataaattttgacTyrThrThrGlulieAsnPheAsp300912cgtcttacttaccttgaaattcaaArgIjeuThrTyrlieuGlulieGin305310cccaatetcgetataatggaatatProAsnLeu^lalieMetGluTyx315320960aatttaacacgtteagggcttagattaAsnLeuThrArgSerGlyljeuArg:Leu325tttteatttttagatgaacttPheSerPhelieuAspGluIjeu3303351008atattttatacaaaaaatgaaacgtacg的aatcgtttagttggtattliePheTyrThrLysAsnGluThrTyrGlyAsnArgLeuValGlylie34034S3501056gcgaatcgtaatagatctacttatgetacgaca的aactgaaattataAlaAsnArgAsnArgSerThrTyrAlaThrThrGlyThrGlulielie3S53603651104tstgga诉a的aacaggtccacccacaacaaaasetttaatacceitttTyrGlyGluArgThrGlyProProThrThrLysThrlieulieProPhe37037538011S2gaatectataaagttteaattgtaact诉tagacaagtaactcctactGluSerTyrLysValSerlieValThrAapArg<31nValThrProThr*1200385390395400tecccttttcctaacatatactttacaattaatcaaattgaactttatSerProPheProAsnlieTyrPheThrlieAsnGinlieGluLeuTyr40541041S1248ttaaataatteacctagtaataaattaacatatteagetggggggaatUeuAsnAsnSerProSerAsnLysLeuThrTyrSerAlaGlyGlyAsn4204254301296ttatctaatgataaaaaaacaactgattttcaatttcctgtaaaaaaaLieuSerAsnAspLysLysThrThrAspPheGinPheProValLysLys4354404451344gactgtaaaAspCysLys450CC3Pro3tt3tt工le工leaatccaaattgtAsnProAsnCys455tt5LCC3LeuPro460ageSertatTyr3a±Asn1392tatsgtestTyrSerHis465liettatec:LeuSer470cagttttctttaGinPheSerLeutttaatPheAsn475TyrtecSerTyr4B01440attggattagcgetaaatatattatatacaggtgcattaggatggacalieGlyLeuAlalieuAsnlieTbeuTyrThrGlyAlaIjeuGlyTrpThr4854904S51488ca_cagtagtHisSerSergttVal500aatagaaataatgcaataAsnAsnAlalieS05teagatSerAspa祖Lys3tslieS103tt工le3C3ThratgateccaMetliePro515Alaateaa>3lieLysggtaacagtcttGlyAsnSerLeu520gatacaAspThrAsn525tctSersagVal1584attgaaggalieGluGly530cctProggtcatGlyHisseaggagg3aacThrGlyGlyAsn535ttggttLeuVal540t3tttalieuGinSei:1632csagggcgtGinGlyArg545gagattGlulie550acatgtagaactThrCysArgThrcctaieitProAsn555tctSera_caGintctSer5601680teittacattTyrTyrlieArgcttcgaUeuArg565tacgetacaaatTyrAlaThrAsn570ggtgetGlyAla99333tactThr575cttlieu1728cctsatataProAsnlietctcttacaSerlieuThr5803t3ccaggagtalieProGlyVal585atagga>lieGly3t3lieCC3Pro590cctProGin1776cgaetcaacaacactttttctggtacaaattataataatttacaatacArgLeuAenAsnThrPheSearGlyThrAsnTyrAsnAsnLeu<31nTyr59560060$1824ggagattttgggtatttccaatttccaagtacagt汰acattacctttaGlyAepPhe'GlyTyrPheGinPheProSerTlirValThrLeuPro3Jeu€10615€201872aatc9a幼cataccatttatatttaatcgtgcagatgtateaaatteaAsnArgAsnlieProPheliePheAsnArgAlaAspValSerA8iiSer625630635640attttaateattgataaaattgaatttataccaattacttectctgbalielieulielieAspLyslieGluPhelieProlieThrSerSer*Val"S650cgccaaaat的agaaaaacaaaaatta.gaaactatecaaacaa—aataArgGinAenArgGluLysGinLysLeuGluThrlie61nThrLyslie:2016"06S5670aatacatttttcacaaatcatacaaaaaatactttaaatatagaagccAsnThrPhePheThrAsnHisThrLysAsnThrLeuAsnlieGluAla67568068520643caaactatThrAsnTyr690gatattgattaaAsplieAsp*2085<210>11<211>694<212>PRT<2">苏云金芽孢杆菌<400>11MetCysGinGlyThrGinGlyAspAsnPheGlqTh;rPheAla151015SerAlaAspThr20lieAlaAlaVal25GlyThrlieVal30SerGlyThrLeuAlaGlylieGlyGlyLeuThrSerlieSerGlyProlie35404SGlylie50lieGlyAlalielie55lieSerPheGlyThrIjeu60lieThrValiPheTrpProAlaGlyGluGinAspLysThrValTrpThrGinPhelie65707580LysMetGlyGlulie85PheValAspThrPro90LeuThrGluSerlie9SLysGinlieuLysLeu100GinThrlieuGluGlyPhe105ArgGinlieLeu110GinSerTyrAsnThrAlalieuAspAspTrpArgLysLeuLysArgIjeuGinAla115120125ProGlyProProSerSerAlaIjeuGinGluAlSAl2LLieuTlirlieu130135140ljysliePheGluAsnValHisAsnAspPhelieArgGluliePro145150155160GlyPheGinlieuGlu165ThrTyrLysThrIjeu170LeuUeuProlieTyr175AlaGinAlaAlaAsn180PheHisIjeuAsnLeuLieu185GinGinGlyAla190GluUeuAlaAspGlu195TrpAsnAlaAsplie200HisProSerGinlie205GluProAsnAlaGly210ThrSerAspAspTyr215TyrLysIjeuLeuLysGlu220AsnlieProLysTyrSerAsnTyrCysAlaAsnThrTyrArgGluGlyIjeuAsnLys225230235240IjeuArgAsnGluPro245AsnMetArgTrpSer250liePheAsnAspTyr255ArgArgTyrMetThrlieThrValLeuAspThr*lieAlaGinPheSerPhe260265270TyrAsplieLysArgTyrLysAspSerlieGlyArglieGlyGlylie275280285LysThrGluIjeuThrArgGlulieTyrThrThrGlulieAsnPheAsb235300ArgLeuThrTyrLeuGlulieGinProAsnlieuAlalieMetGluTyr30531031S320AsnlieuThrArgSer32SGlyLeuArgLeuPhe330SerPhelieuAspGlu335lieuliePheTyrThi1LysAsnGluThrTyrGlyAsnArgLeuValGlylie34034S350AlaAspArgAsnArgSerThrTyrAlaThrThrGlyThrGlulielie355365TyrGlyGluArgThrGlyProProThrThrLysThrLeulieProPhe37037S380GluSerTyrValSerlieValThrAspArgGinValThrProThr385330400SerProPheProAsnUeTyrThrlieAsnGinlieGluLeuTyr40S410415IjeuAsnAsnSerProSerAsnLysLeuThrTyrSerAlaGlyGlyAsn420425430LeuSerAsnAspLys:LysThrThrAspPheGinPheProValLysLys435440AspCysLysProlielieAsnProAsnCysIjeuProSerTyrAsnSer450455460TyrSerHislieLeuSerGinPheSerLeuPheAsnTyrSerTyrIiys470475480lieGlyIjeuAlaIjeuAsnlieLeuTyrThrGlyAlaIjeuGlyTrpThr485490495HisSerSerValAsnArgAsnAsnAlalieSerAsplielieThr500505510Met工leProAla工leLysGlyLeuAspThrAsnSerLysVal515520S25lieGluGlyProGlyHisThrGlyGlyAsnLeuValTyi:LeuGinSer530535540GinGlyArgLieuGVulieThrCysArgThrProAsnSerThrGinSer545550555TyrTyrlieArgLeuArgTyrAlaThrAsnGlyAlaGlyAsnThrLeu565570575ProAsnlieSerLeuThrlieProGlyVallieGly工leProProGin580585ArgLeuAsnAsnThrPheSerGlyThrAsnTyrAsnAsnIjeuGinTyr5S5600605GlyAspPheGlyTyrPheGinPheProSerThrValTharLeuProLeu615AsnArgAsnlieProPheliePheAsnArgAlaAsjpValSerAsnSer62S635"0lielieulielieAspIiyslieGluPhelieProlieThrSerSerVal65SArgGinAsnArgGluLysGinLysLeuGluThrlieGinThrLyslie665670AsnThrPhePheThrAsnHisThrLysAsnThrLeuAsnlieGluAla675680685ThrAsnTyrAsplieAsp690<210>12<211>5，<212>DNA。lh苏云金芽孢杆菌<400>12tacatgcaataceitaaagagaaggtttaaatatttgtagaaacattgttacaggaatacact站caLtatatttaLttaggtgtcttaaaaagtccatatcaaaataaaaatgaatatgaaacgccaaacagatatccttttgcaa汰taatcgtcagggaatctcat的gatgaaatttgggtaaatcttat3gagtttgtgatagaacctataatagga^aactaAttccgactaatcgtctattatctataattcgftaaagaggtagccgttgacggtaaattga站aattatagagagtaagacggtaaaccacttcaa站gacaaatattaaactttcagaaagagatttcaatgaaactcaagtattgttattacctacttttgcacgggettgcagttcaatataaagcac站t的tcg诉att站tatcacctaaceigtggttgtg站tgcaa幼cggcagagtataccaattattagat站aacagg的的gacaiggtgctgacaaaatgacgttggcggtattggatattatcgatccattgccaacacatgtagagtt诉ctecatcgggaacttatagttggttacggaattatggaacacggg3acct33tttagttacttatgtttcgagatattttgccggctg93taggtaacggtatttttcaacgtatgtctggakaaatcaatacctcaccaaaaataatgggtt60ttggggtactacgaatatatagaaagstcac120taaggactaitataaggagtgaaaaa诉tgsi180tattggaatcctcatcgaataacacaaata240gggatatgtctactatgtcttggaatgatt300aatcagtcgaaacgataacaagtattggga360gtttgggtggaaittaat3C3ctattatc貼420aaactgtgtcagcactttctatatgtgatt480atagtgttttaagtgatgcgattgcagattS40attatctttcttatcttggggcttggctta600attctgatatcggacaattsigtttattatt660ttctagga的gtcattgtcsLagaaacaatg720aagctgcaaatgtgcagttattactattaa780tcccatttttgagtgcagagaatgtaagat840attttacc的tgattactatgagc9attasL300gtttatattggtatcaggtaggtttaaatccttggtcgaaattt站taaatttcgtagag1020ctat汰tttccaacttatgattttgagaaat1080gggaaatttatac的atgcagtgggatatt1140ggcctaatactttt站tgggttagaggcta1200ggcttagcaaaata诉tatatataatgagt1260g站ctc9tcattatgaagactacacaaagg1320ctacgagtaatgatctacgtaatattgatt1380atttaaattacacttgtattgtgttgtatagtgaaaatatttcaa^SLtts3ttt33tccgttagattgatgttttgtatcttaaattcgctcccaagtaaacgcggtgaagctgtttaactacgttasaatatgtcagatgacattcggtEicagatgcgtaatagatgagtagtaaagattgttccasatatcatcgtcttatgtatgcaatttgattttcttattaeiaaacaacacstgtgtagatgcatceiaLaeLcgcttgctccaggtta3tttatatatggcscgcaatcaaattgggttacgtgtgagtagaggtaattttatatagcscacgggttgtgtattacaaattatacttggctgtacatgcga站站ttaacggacatccccaatatttaaatgttaagtgaatgcaattttgttcgt的tacataaccgggttaaatgttttcccagaattcacaaataattcttgattcgtttgactcttgcaggtttcgstcat3cttgccatttgagctttttttttgaattaagataatcttggtttcttttcacggtctgtacgatgt3tataccagagtattgatgcaggtgcactctaattggaaactccaattgaagcctcttgctaatttttttaagaagctgctttcgcttgaaaaatgaaattatcgaatagttttcgatctgtttaaataagcgaatagaaacgagaacaaaatcagtcagtctcaatgatattaggagcacgagtctaaattaagatttggcctcagtatgtgatgcccgcaaacatattgeicaatttctaatcctaacaggtggagaaaaaacttattttacaagatcgactggatgaactatggatgcacgaaacaggaaatgagcgcgggggattgccaaaa一caggei站cacgatagcatcgaatatgtatgcaagggtataaacaatgatgtgtaatcaagaaaascctacgattaatgaacssttcctgattttggtatttgatatgagcaacagtcggcctagttcatctcccttgtttcttgatatcctgtgttcttggccsttttsa诉gattcaattttcatcatattcgttgtatttgctgaagattgtggatacc幼ccattctBtattt诉atascccgctgaccaatcttcattctgtaaaasaaattacttcgtgtttcaaaataatgggcggtgttagagagagttaacggcctstacctacaaaatcaga_aaactgcagtttagtgtattcttactctgacacctccatcaatcagatgttagaatatgsccaattaaaaaaaggttgtcagattatgagtaatctacttcataggatcacatatatggaaccatatactggtaacacgatatgcagccgccaagggtaatatag&tagccgg3g站gtcagatggataaagcattggc9ttgg9貼acatgaacaagatacagtcgatatgtatatcaatgtcataacccgcggtELCstatctc33a3ttaagttcacgtgatcgtgtagttgctttgagcaaagttacaasc5t3Ct3acctgcattcgatataaccgtcac幼幼tcaggtctccttaatactcctttccagaag3tstgatggaccaataatatgataaaattaattaaagcctcttccaagtaatattcgttgtactctcgaaatatggtctatacataatgagatctgtttcgtgtcacttgtttcattctctacctgttaaacggtaaatatta幼atctcttatgttctcgccattagctataggcagattttaagcaggatgaccctctcatatatggttgatgtaataaagacaaagttaatctttatccgasattatgatacctagttcagggagaacggagtcctatgaatagatcaaaatastgttaccaaaatggaCS3t33tCCtBactct3tttacgttatcgattacgggaaa_taLaitccttC3LtCCt3C3CC3aaagcatgtgagatacaaatcgctacagta3catgtg站agttacactattccctatgtaagagttaaacaaatggtgaccttgcatgctttcttgcgaaacggattgaatatgcaaggataatggaaatcggttgcsLcattaacgattctattgcat5Lt站agcatttcaaacggtcca站ttctaagtaataggagctaaatcacaggtctaELt的acgccttc3貼caaa的attttttcgctttctatatagcttaatgaacccctaatctattaagagtctgcaaacagtaagtgaagcttttcttgaaa站tctcccatagaatcttctgcggtcatgaacctatcgtagggtagacaeLttgaagacacatacaggcggtgaagaggcsctcatagtttatgcaccgtattcgtcgatttaaaaaaacattcagtctcga_cateLa_ggacctagaattactagaattttacaagtagcggccattttgggatgaaagacttttctgatttttaaaggtaagagggtgaaattgatgaagtgtcacgacaaatgtaggggaatctagtatgatccagcatattacttteLCcatasittgcacgtatcatttacacaagggagcttcsigcaagatcatctatgtaacgagaaggatataataggagaaatctagtaatatgtaaccaagta貼taagaacataacataataaggagatttttaggctgag的gcatgcagtaaacgataccaatgcagattgaagtaacccgctccttgcttcaatttgctactaaaitcct站tgctgtgtttcccttcgaatttgttctgattaagttgcagaaattgaatgcagt诉ttctaaagttaacaggtggctatgatatcgtagctgctgtttcatccttaagaataacgacggcctactcgtaggagagatctacgttccaatgcggcatagtttaaaacagttattaccacsggtggggtttcgttatgggttcaaatctatagtgatt3tggatgtggattgaattccaggtggtatttaaaactggcggaatgattggaaaatttcttctgtcaaaattgtgggaagctatcatgaaaitcgctgtgaatgctcccgaatcgtcatccgtattgatgtaagtcattgasg站atggaacaaaacaggctagatcatatggttaccg站tgcaaggttagattacagggtaittagttctatggatatgttgatggattgatactagcgatgtataatcaggcataactctcaacatcattttttcgtttttggtgttttgctactttttactttcacctsgtaagccggtatgacacttcttttgsaatcttcttgatgagcta站tgt3幼站3tcgcactccattcatgctacttttttctttggtttttacaggcgggacaaagctgtatggttttagttcgtgagaatgagtattacaacgcgtggtgtgcatat祖tt讽tccc1440150015601620168017401800I860192019802040210021602220228023402400246025202S8026402700276028202880294030003060312031803240330033G0342034803S40360036S037203*7803840390039604020408041404200426043204380"4045004560468047404800486049204980504051005160522052805340S4005460attcttttttaatgataatcacttgttttaatttctccatatttatttaccgttgttgtgggtaatggtttcaatgcagccttatcatcatcataatgtaggcgtttctgstcttctattgaaaa_3ctctccata_a_tcggagcggttgtacgttcaacgttccccttttcatgcccgctataatgcatttgaaattgtaaaaatgcatccgtgactacagcggcagataagttatcaatttgaaataatcgttttaagccttctaaaaagcgtaaaacaaaacgttcttgatccactgtg5520tccattgagaagaccgttaggtcttccaaa5S80ttccaaaggtcttcaatcatgacttccttcS640gcttgtacttctaaccattgtgcgagttga5700aaccaattgtttcgtgtataactgactttc5760taaggattacaaggttgcacttcaaagtta5820gtataagttcgattatcaccttttcctact5880cgaagatgcgttggtacaccaccagcttga5940ccttctgtat5980<210>13<211>2082<212>DNAdlh苏云金芽孢杆菌<220><221>CDS(1)-.(2082)<400>13gtgaaaaagLysUysMet;atgagtMetSer5CC3Prot3tc站GinsateL33Lys10331AsngaaGlutatTyargasGlu3t3lie15ttgLeu48gaaGlutecteaSerSerteg站t20AsnTtiraicgThr2Scca_ProtatTyrcctPro30tttPheAla96aataategggatatgtctactatgtcttggaatgattgtcagggaateAsnAsnArgAspMetSerThrMetSerTrpAsnAspCysGin(Slylie354045144teatgggatgaaatttgggaateagtcgaaacgataacaagtattgggSerTrpAspGlulieTrpGluSerValGluThrlieThrSerlieGly50S560"1S2lie65aatcttAsnlieuatagaglieGlutttPhe70ValataliegaaGlucctProagtSer75ttgLeuggtGlyGlyattlie貼t80240ThretattaIjeulieutC3Serlie85lieGly333cta_Leuattlie卯ccgProactThraatcgtArgc幼Gin95actThr288gtgteagcactttctatatgtgatttattettctataattcgtaaageigValSerAlalieuSerlieCysAsplieuLeuSerlielieArgLysGlu10010511033GgtagecgatagtgttttaagtgatgcgattgcagattttgacggtaaaValAlaAspSerValLeuSerAspAlalieAlaAspPheAspGlyLys115120125384ttgaaaaattatagagagtattatctttcttatcttggggettggcttLeuLysAsnTyrArgGluTyrTyrLeuSerTyrlieuGlyAlaTrpLeu130135140432SLSL5Lgateggtaaaccacttcaaaagaca幼taattctgatateg诉caaLysAspGlyLysProLeuGinLysThrAsnAsnSerAsplieGlyGin145ISO155160480ttagtttattatttta站cttteagaaagagatttcaatgaaattetaLeuValTyrTyrPheLy曰LeuSerGluArgAspPheAsnGlulieLeu17017552899a999teattgteaa诉aacaatgetcaagtattgttattacctact<31yGlySerLeuSerArgAsnAsnAlaGinVallieulieuIeuProThr57618018S190tttPhegca^c的AlaGin195getAlagcaaatgtgAlaAsnVaicagtta_GinLeu200ttalieuCt3ljeutt3lieu鄉Arg205gatAspgcaAlagttVal624GintataaaTyrIjys210gcaAlacmtggttcGinTrpPhe215ccatttProPhettgLeuagtSerAla220gagGlu貼tAsngtaValArg672tegSer225gaattaGluLeulieteacctaacSerProAsn230a9tggtSerGlytgtCysgatAsp235tttPheThrggtGlygatAspt3CTyr240720tatTyrgagcgaGluArgttaLeu333tgC333IiysCysLys245ThrAlaGlu2S0t3tTyrThr33tAsntatTyrtgtCys2S5tta7"匕attggta匕caggtaggtttaaatcaga_taaaacagggggggeicaggtTyrTrpTyrGinValGlyLeuAsnGin工leLysGinGlyGlyThrGly260265270getAlagacactAspThr275tggTrptegaaatttSerIjysPhe站tAsnLys280tttPhecgtArg3g3Arg933Glu28Sa±gMet3cgThrttg8"9c3AlagtattgVallieu290gatAspattategetlielieAla295atatttliePheCC3ProsetThrTyr300AsptttPhe9&933a>912tstTyr30SccattgProLeuCC3ProacacatgtaThxHisVal310g3gttgGlulieuactThr鄉Arg315Sf貼GluattlieTyrThrgatA印320%0gcagtgggatatteategggaacttatagttggttaeggaattggcctAlaValGlyTyrSerSerGlyThrTyrSerTrpLeuArgAsnTrpPro3253303351008站tacttttThrPhe站tAsn340gggttagagGlyIjeuGlugetaatAI3Asn345Gly3C3ThreggArg9g3Glycc仁Pro350ggtGlyttaLeu1056gttacttggcttageaaaataggtatatataatgagtatgtttegagaValThrTrpUeuSerLyslieGlylieTyrAsnGluTyrValSerArg35S3603651104tattttgccggctgggtaggaactcgtcattatgaagactacacaaagTyrPheAlaGlyTrpValGlyThrArgHisTyrGluAspTyrThrLys3703753801152ggtaacggtatttttcaacgtatgtctggaactacgagtaatgatetaGlyAsnGlyliePheGinArgMetSerGlyThrThrSerAsnAspLeu3853903954001200cgtaatattgattttcagaatgccgatgtatataaaattactteattaArgAsnlieAspPheGinAsnAlaAspValLyslieThrSerLeu405"041S1248getateatgaacetagtagga诉gactaceget的accagagtatcgtAlalieMetAsnlieuValGlyGluThrThrAlaArgProGluTyrArg4204254301236gttteaaaggca讽ttttcgtagggtagggggacctgatttaaattatValSerIiysAlaAspPheArgArgValGlyGlyProAspIeuAsnTyr435440"513"gatgcaggtaataatg的etaageaggatgacaattgaatctacgttcAspAlaGlyAsnAsnGly*LeuSerArgMetThrlie虹uSerThrPhe4S0455460CC3cttProLeu465gtaValttgcacIjeuHistctSet470aatAsnggtGlygttagaggaValArgGly475ccctctcatagattaProSerHisArgLeu4801440teaaatSe:rAs:nAlagcatgtAlsCys485gttValgtatatTyrggaaactecGlyAsnSer490agagttaacgtatatArgValAsnValTyr4951488ggttggGlyTrpThrC3t3C3HisThr500agtSertt3Leu333IjyscgtgaaaatArgGluAsnS05ataattgaagecaatlielieGluAlaAsn5101536caaattGinlieThr515GinlieccgProAlaVal520aagagttatLysSerTyrtaccttcaa33ttstTyrLeuGinAsnTyr5251584cttgetLeuAla530AsngectatAla_Tyr3CCThrt3tTyr535gtaValataaaaggclieLysGlyactcatacaggtgggThrHisThrGlyGly5401632gatttaAspLeu5453tCliecgttttArgPhettaLeu550agaArgThraaateagagIjysSerGlu555tataacgcagtttatTyrAsnAlaValTyr5601680gca_ggtAlaGlyggcGlyggaattGlylieS65agattglieuattlieattaataaclieAsnAsnS70aaaactgcaggacaaLysThrAlaGlyGin5751728agttaccgtattcgttttcgttatgetgcagataaagetgetttctttSerTyrArglieArgPheArgTyrAlaAlaAspLysAlaAlaPhePhe5S0S855901776agtgtatatctttatccagga^gttggggtteaaatcgttttgtategSerValTyrLeuTyrProGlyGlyTrpGlySerAsnArgPheValSer595GOO6051824cttgaalieuGlu610333IjystcttactctSerGly615astAsnt3tgsicgatTyrAspAsptta站atatagtgatLeuIiysTyrSerAsp6201872ttt3EiaPheIiys625ttcPhegetgaaAlaGluattlie6303tClieThrcctccattaProProIjeu635cctagtteaaacattProSerSerAsnlie6401920cag3tgGinMetgatAspgtggagValGlu"5atgMetC33GinAlaaatagttttAsnSerPhe650caateagatgtaaacGinSerAspValAsn1968gtggttetcgacaaaattgaattcetcccaagtaatacaacaactttaValValLeuAspLyslieGluPheLeuProSerAsnThrThrThrLeu"06656702016gaatatgagggagaac的gacetagaaaaaacaaagaacgcggtgaacGluTyrGluGlyGluArgAspI*euGluLyeThrLysAsnAlaValAsn67S68068SgatctgtttaccaattaaAspIeuPheThrAsn*6902082<210>14<211>633<212>PRT<213>苏云金芽孢杆菌<400>14MetLysMetSerProTyrGinAsnAsnGluTyrGlulieLeu151015GluSerSerSerAsnAsixThrAsnThrProAsnArgTyrProPheAla202530AsnAsnArgAspMetSerThrMetSerTrpAsnAspCysGinGlylie354045SerTrpAspGlulieTrpGluSerValGluThrlieThrSerlieGly505560lieAsnLeulieGluPheVallieGluProSerLieuGlyGlylieAsn707580ThrIjeuLeuSerlielieGlyLyslieulieProThrAsnArgGinThr859095ValSerAlaL>euSerlieCysAspLieuLieuSer工le工leArgGlu100105110ValAlaAspSerValIjeuSerAspAla工leAlaAspPheAspGlyIjys115120125LeuLysTyrArgGluTyrTyrLeuSerTyrLeuGlyAlaTrpL/eu130135140LysAspGlyLysProLeuGinLysThrAsnAsnSerAsplieGlyGin145150155160UeuValTyrTyrPheLyslieuSerGluArgA叩PheAsnGlulielieu165170175GlyGlySerUeuSerArgAsnAsnAlaGinValUeuLeuUeuProThr18018S190PheAlaGinAlaAlaAsnValGinLieuLeuLeuUeuArgAspAlaVal195200205GluTyr!>ysAlaGinTrpPheProPheSerAlaGluAsnValArg210215220SerGluUeulieSerProAsnSerGlyCysAspPheThrGlyAspTyr225230235240TyrGluArglieuijysCysLysThrAlaGluTyrThrAsnTyrCysIjeu245250255TyrTrpTyrGinValGlyLeuAsnGlulieLysGinGlyGlyThrGly265270AlaAspThrTrpSerLysPheAsnIjysPheArgArgGluMetThrIjeu275280285AlaVallieuAsplielieAlaliePheProThrTyrAspPheGluLys290295300TyrProLeuProThrHisValGluIjeuThrArgGlulieTyrThrAsp305310315320AlaValGlyTyxSerSerGlyThrTyrSerTrpIjeuArgAsnTrpPro32533033SAsnThrPheAsnGlyIjeuGluAlaAsnGlyThrArgGlyProGlyUeu34034S350ValThrTrpIjeulieGlylieTyrGluTyrValSerArg3553603G5AlaGlyTrpValGlyThrArgHisiyrGluAspTyrThrLys37037S380GlyAsnGlyliePheGinArgMetSerGlyThrThrSerAsnAspLeu385390395400ArgAsnlieAspPheGillAsnAlaA8pValTyrLyslieThrSerlieu405410415AlalieMetAsnlieuValGlyGluThrThrAlaArgProGluTyrArg420425430ValSerLysAlaAspPheArgArgValGlyGlyProAsplieuAsnTyr435440"5AspAla<31yAsnAsnGlylieuSerArgMetThrlieGluSerThrPhe45045S460ProLeuVallieuHisSerAsnGlyValArgGlyProSerHisArgIjeu465470475480Sea:AsnAlaAlaCysVal"ValTyrGlyAsnSerArgValAsnValTyr485<31yTrpThrHisThrSerlieuLysArg<31uAsnlielieGluAlaAsnSOOS05GinlieThrGinlieProAlaValIiysSerTyrTyrlieuGinAsaTyrS15520525IjeuAlaAsnAlaTyrThrTyrVallieLys<31yThrHisThrGlyGlyS3053SS40AsplieulieArgPheIjeuArgThrLysSerGluTyrAsnAlaValTyr545550555560AlaGlyGlyGlylie565ArgLeulielieAsn570AsnIiysThrAlaGinSerTyrArglie580ArgPheArgTyrAla585AlaAspliysAlaAla590PhePheSerValTyr595lieuTyrProGlyGlyGOOTrpGlySerAsnArg605PheValSerLeuGlu610ljysSerTyrSerGly615AsnTyrAspAspLeu620LysTyrSerAspPheLysPheAlaGlulielieThrProProlieuProSerSerAsnlie625630635640GinMetAspValGlu645MetGinAlaAsnSer650PheGinSerAspVal655AsnValValLieuAspliyslieGluPheIjeuProSerAsnThrThr670ThrUeuGluTyrGlu675GlyGluArgAspLeu680GluIjysThrLysAsn685AlaValAsnAspLeuPheThrAsn690<210>15<211>2073<212>DNA<213>苏云金芽孢杆菌<220><221>CDS<222><1)…(2073)<400>15atgagtccaMetSerProtatcaaaataaaaatgaatatgaaatattggaatecteaTyrGinAsnLysAsnGluTyrGlulielieuGluSerSer5101548tegaataacacaaatacgccaaacagatatccttttgcaaataateggSerAsnAsnThxAsnThrProAsnArgTyrProPheAlaAsnAsnArg20253096gatatgtctactatgtcttggaatgattgtcagggaateteatgggatAspMetSerThrMetSerTrpAsnAspCysGinGlylieSerTrpAsp354045144gaaatttgggaateagtcgaaacgataacaagtattgggataaatcttGlulieTrpGluSerValGluThrlieThrSerlieGlylieAsnLeu505560192atagagtttgtgatagaacctagtttgggtggaattaatacaetattalieGluPheVallieGluProSerLeuGlyGlylieAsnThrLeuLeu65707580240teaataataggaaaaetaattccgactaatcgtcaaactgtgteagcaSerlielieGiyLysLeulieProThrAsnArgGinThrVaiSerAla8S9095288ctttctatatgtgatttattatctataattcgtaaagaggtagecgatUeuSerlieCysAspLeulieuSerlielieArgLysGluValMaAsp10010S110336agtgttttaagtgatgcgattgcagattttgacggtaaattgaaaaatSerVallieuSerAspAlalieAlaAspPheAspGlyliysLeuItysAsn11512012S384tataga诉gtattatctttcttatcttg的gettggcttaaa诉cggtTyrArgGluTyrTyrLeuSerTyrLeuGlyAlaTrpLeuLysAspGly130135140432acaaataattctgatateLysProLeuGinLysThrAsnAsnSerAsplie145150155ggacaattagttGlyGinLeuValTyr160480tattttaaacttteagaaagagatttcaatgaaTyrPheLysLeuSerGluArgAspPheAsnGlu165170attetaggagggtealieDeuGlyGlySer175528ttgteaagaaacaatgetcaagtattgttattaLeuSerArgAsnAsnAlaGinValLeuLeuLeu180185cctacttttgcacaaProThrPheAlaGin190576getgcaaatgtgcagttattaetattaagggatAlaAlaAsnValGinLeuLeuLeuLeuArgAsp195200gcagttcaatataaaAlaValGinTyrLys205624gcacaatggttcccatttttgagtgcagagaatAlaGinTrpPheProPheLeuSerAlaGluAsn210215gtaagateggaattaValArgSerGluLeu220672atateacctaacagtggttgtgattttaccggtlieSerProAsnSerGlyCysAspPheTht:Gly225230235gattactatgagAspTyrTyrGlucgaArg240720ttaaaatgcaaaacggcagagtataccaattatLeuLysCysLysThrAlaGluTyrThrAsnTyr2452S0tgtttatattggtatCyslieuTyrTrpTyr255768caggtaggtttaaatcagataaaacagggggggseaggtgetgacactGltiValGlyLeuAsnGinlieLysGinGlyGlyThrGlyAlaAspThr2602S5270816t的tegaaatttaataaatttcgtagagaaatgacgttggcggtattgTrpSerLysPheAsnLysPheArgArgGluMetThrlieuAlaVallieu27S280285gatattettcgetatatttccaacttatgattttgagaaatatccattgAsplielieAlaliePheProThrTyrAspPheGluLysTyrProLeu2902953008S4912ccaacac&tgtagagttgactagggaaatttatProThrHisValGluLeuThrArgGlulieTyr305310315acagatgcagtgThrA叩AlaVal993Gly320960tatteategggaacttatagttggttaeggaattggcctaatacttttTyrSerSerGiyThrTyrSerTrpLeuArgAsnTrpProAsnThrPhe32S3303351008aatgggttagaggetaatggaacac的ggacctggtttagttacttggAsnGlyLeuGluAlaAanGlyThrArgGlyProGlyLeuValThrTrp3403453501056cttageaaaata的tatatataatgagtatgtttegagatattttgecLeuSerLyslieGiylieTyrAsnGluTyrValSerArgTyrPheAla35S3603651104ggctgggtaggaactcgtcattat诉agactacacaaagggtaacggtGlyTrpValGlyThrArgHisTyrGluAspTyrThrliysGlyAsnGly37037530011S2atttttcaacgtatgtctggaactacgagtaatgatetacgtaatattliePheGinArgMetSerGlyThrThrSerAsnAspLeuArgAsnlie3853903954001200gattttcag站tgecgatgtatataaaattactteattagetateatgAepPheGinAsnAlaAspValTyrLyslieTbrSerlieuAlalieMet40S4104151248aacctagtaggagagactaccgetagaccagagtatcgtgttteaaagAsnLeuValGlyGluThrThrAlaArgProGluTyrArgValSerLys4204254301296gesigattttcgtagggtagggggacctgatttaaattatgatgcaggtAlaAspPheArgArgValGlyGlyProAspLeuAsnTyrAspAlaGly4354404451344aettaatgggetaageaggatgacaattgaatctacgttcccacttgtaAsnAsnGlyLeuSesrArgMetThrlieGluSerThrPheProLeuVal4504554601392ttgcactctaatggtgttagaggaccctctcatagattateaaatgcgUeuHisSerAsnGlyValArgGlyProSerHisArgLeuSer*AsnAla4654704754801440gcatgtgttgtatatggaaactecagagttaacgtatatggttggacaAlaCysValValTyrGlyAsnSerArgValAsnValTyrGlyTrpThr4854904951488catacaagtttaaaacgtgaaaatataattgaagecaatcaaattacaHisThrSerLeuLysArgGluAsnlielieGluAlaAsnGinlieThr5005055101536caaataccggcggtgaagagttattaccttcaaaattatcttgetaatGinlieProAlaValLysSerTyrTyrLeuGinAsnTyrLeuAlaAsn515S205251584gectatacctatgtaataaaaggcactcatacaggtggggatttaateAlaTyrThrTyrVallieIysGlyThrHisThrGlyGlyAspUeulie530535540cgttttttaagaacaaaateagagtsttaacgcagtttatgcaggtggcArgPheLeuArgThrLysSerGluTyrAsnAlaValTyrAlaGlyGly5455505555"ggaattagattgattattaataacaaaactgcaGiylieArgLeulielieAsnAsnLysThrAla565S70ggacaaagttaccgtGlyGinSerTyxArg5751728attcgttttcgttatgetgcagataaagetgetttctttagtgtatatlieArgPheArgTyrAlaAlaAspLysAlaAlaPhePheSerValTyr580S8S5901776ctttatccaggaggttggggtteaaatcgttttgtategcttgaaaaaIjeuTyrProGlyGlyTrpGlySea:AsnArgPheValperLeuGluIiys59560060S1824tcttactctggaaattatgacgatttaaaatatagtgattttaaattcSerTyrSerGlyAsnTyrAspAspLeuLysTyrSerAspPheLysPhe"06201872getgaaattateacacctccattacctAlaGlulielieThrProProLeuPro625630agtteaaacattcagatggatSerSerAsnlieGinMetAsp63S64013203tggagatgcaagcgaatagttttcaateagatgtaaacgtggttetcValGluMetGinAlaAsnSerPheGinSerAspValAsnValVallieu"S65065S1968gacaaaattgaattcetcccaagtaatacaacaactttagaatatgagAspLyslieGluKieLeuProSerAsnThrThrThrLeuGluTyrGlu€606656702016ggagaaegggaceta诉aaaaacaaagaacgcggtgaac诉tctgtttGlyGluArgAspLeuGluLysThrLysAsnAlaValAsnAspLeu跑e67S68068520"accaatta_a20*73ThrAsn*690<210>16<211>690<212>PRT<2lh苏云金芽孢杆菌<400>16MetSerProTyrGinAsnLysAsn15SerAsnAsnThrAsnThrProAsn20AspMetSerThrMetSerTrpAsn3540GlulieTrpGluSerValGluThr5055lieGluPheVallieGluProSer6570SerlielieGlyLyslieuliePro85LieuSerlieCysAspIjeuLeuSer100SerValLeuSerAspAlalieAla115120TyrArgGluTyrTyrIjeuSerTyr130135LysProIjeuGinThrAsnAsn145150TyrPheLysIjeuSerGluArgAspLeuSerArgAsnAsnAlaGinVal180AlaAlaAsnValGinLeuLeuLeu195200AlaGinTrpPheProPheLeuSer210215lieSerProAsnSerGlyCysAsp225230IjeuLysCysLysThrAlaGluTyr245GinValGlyLeuAsnGinlieLysTrpSerLysPheAsnLysPheArg275280AsplielieAlaliePheProThr2902S5ProThrHisValGlulieuThrArg305310TyrSerSerGlyThrTyrSerTrp325AsnGlylieuGluAlaAsnGlyThr340lieuSerLyslieGlylieTyrAsn355360GlyTrpValGlyThrArgHisTyr370375liePheGinArgMetSerGlyThr385390AspPheGinAsnAlaAspValTyr405AsnLeuValGlyGluThrThrAla420AlaAspPheArgArgValGlyGly43S440GluTyrGlulie!LeuGluSerSer1015ArgTyrProPheAlaAsnAsnArg2530AspCysGinGlylieSerTrpAsp45lieThrSer工leGlylieAsnLeu60LeuGlyGlylieAsnTharLeuLieu7580ThrAsnArgGinThrValSerAla9095lielieArgLysGluValAlaAsp105110AspPheAspGlyLysIjeuUysAsn125IjeuGlyAlaTrpLeuIjysAspGly140SerAsplieGlyGinUeuValTyr155160PheAsnGlulieLeuGlyGlySer170175LeuLeuUeuProThrPheAlaGin185190IjeuArgAspAlaValGinTyrLys205AlaGluAsnValArgSerGluLeu220PheThrGlyAspTyrTyrGluArg235240ThrAsnTyrCysIjeuTyrTrpTyr2S0255GinGlyGlyThrGlyAlaAspThr265270ArgGluMetThrLeuAlaVallieu285TyrAspPheGluLysTyrProLieu300GlulieTyrThrAspAlaValGly315320LeuArgAsn.TrpProAsnThrPhe330335ArgGlyProGlylieuValThrTrp345350GluTyrValSerArgPheAla3S5GluAapTyrThrLysGlyAsnGly380ThrSerAsnAspLeuArgAsnlie335400LyslieThrSerLeuAlalieMet"0415ArgProGluTyrArgValSerLys42S430ProAspLeuAsnTyrAspAlaGly"5AsnAsnGlyLeuSerArgMetThrlieGluSerThrPheProUeuVal450455460LeuHisSerAsnGlyValArgGlyProSerHisArgLeuSerAsnAla465470475480AlaCysValValTyr485GlyAsnSerArgVal490AsnValTyrGlyTrp495ThrHisThrSerLeu500LysArgGluAsnlie505lieGluAlaAsnGin510lieThrGinliePro515AlaValLysSerTyr520TyrLieuGinAsnTyr525LieuAlaAsnAlaTyrThrTyrVallieLysGlyThrHisThrGlyGlyAspLeulie530535540ArgPheLeuArgThrIjysSerGluTyrAsnAlaValTyrAlaGlyGly545550555GlylieArgLeulielieAsnAsnLysThrAlaGlyGinSerTyrArg565570575lieArgPheArg580TyrAidAlaAspLys585AlaAlaPhePheSer590ValTyrLeuTyrPro595GlyGlyTrpGlySer600AsnArgPheValSer605LeuGluLysSerTyrSerGlyAsnTyrAspAspIjeuLysTyrSerA印PheLysPhe620AlaGlulielieThrProPro!LeuProSerAsnlieGinMetAsp625630ValGluMetGinAlaAsnSerPheGinSerAspValAsnValValLeu645650SS5AspLyslieGluPheLeuProSerAsnThrThrThrlieuGluTyrGlu660665670GlyGluArgG75AspIjeuGluLysThr680LysAsnAlaValAsn685AsplieuPhe690<210>17<211>1686<212>DNA<213>苏云金芽孢杆菌<220><221>CDS<222>(1)…(1686)<400>17gtgagtcctatgtttacaagtagtacgaaaaatacgttaaaaatagaa48MetSerProMetPheThrSerSerThrLysAsnThrLeuLyslieGlu151015atcgacagattsitgaaatagatcaagcggccattThrThrAspTyrGlulieAspGinAlaAlalie2025tcteitELgaatgtatgSerlieGluCysMet3096teagatgaacaaaatcctcaggaaaaaataatgttatgggatgaaataSerAspGluGinAsnProGinGluLyslieMetlieuTrpAspGlulie3S4045144ctgLeuSOgcaAlaa站c站GincttagtcaglieuSerGin55tctcgtaatSerArgAsnetaetccaaaatggaLeuIjeuGinAsnGly60192gacttttctgggaatgattggacattcggtaatgatattateatagga240AspPheSerGlyAsnAspTrpThrPhe<3lyAsnAsplielielieGly6S707580tecaataatcctatttttaaag诉aaatttetacagatgcgt诉agcaSerAsnAsnProliePheLysGlyLysPheLeuGinMetArgGlyAla859095288<table>tableseeoriginaldocumentpage98</column></row><table>ttacagggatggcacgcaacaggaaatgccgcggtacaacaaatggatLeuGinGlyTrpHisAlaThrGlyAsnAlaAlaValGinGinMetAsp3703753801152Gly385getAlatC3SergtattagttValLeuVal390etateaaattggageLeuSerAsnTrpSer39SAlagggGlygtaValtctSerGin4001200aacttgcatgetcaagatcatcatggatatgtgttacgtgtgattAsnLeuHisAlaGinAspHisHisGlyTyrValLeuArgVal工le405410415gccAla1248aaaaaagaaggacctggaaaagggta_tgtaacgatgatggattgtaatLysLysGluGlyProGlyIjysGlyTyrValThrMetMetAspCysAsn4204254301296ggaaagcaggaaacacttaagttcacttcttgcgaagaaggatatatgGlyLysGinGluThrLeuLysPheThrSerCysGluGluGlyTyrMet435"04451344seaaaaacagtagaggtattcccagaaagtgatcgtgtaeggattThrLysThrValGluValPheProGluSerAspArgValArglie4504554S0Glu13923taggagaaeiccgaaggtacattttatatagatageategagttgcttlieGlyGluThrGluGlyThfPheTyrlieAspSerlieGluLeuLeu46547047S4801440tgtatgcaaggatatgataacaata_ataacctgcacacgggtaatatgCysMetGinGlyTyrAspAsnAsnAsnAsnLeuHisThrGlyAsnMet4854S04951488tatgagcaaagttataatggaaattataatcaaaatactagegatgtgTyrGluGinSerTyrAanGlyAsnTyrAsnGinAsnThrSerAspVal500S055101536tattacca_a_gggtata_ca_etacaactataaccaagactctagtaatatgTyrTyrGinGlyTyxThrAsnAsnTyrAsnGinA印SerSerAsnMetS155205251584tataatcaaaattatactaacaatgatgacctgcattecggttgcacaTyrAsnGinAsnTyxT)irAsnAsnAspAspLeuHisSerGlyCysThr53053S5401632tgtaaccaagggcataactctggctgtacatgtaatcaaggatataacCysAsnGinGlyHisAsnSerGlyCysThrCysAsnGinGlyTyrAsn54S550S555S01680cgttaaiArg*<210>18<211>561<212>PRT<S13>苏云金芽孢杆菌<40()>18MetSerProMetPheThrSerSer15ThrThrAspTyrGlulieAspGin20SerAspGluGinAsnProGinGlu3540LysUeuAlaLysGltiLeuSerGin50S5AspPileSetGlyAsnAfipTrpThrThrLyeAsnThrLeuI*yslieGlu1015AlaAlalieSerlieGluCysMet2530liyslieMetLeuTrpA8pGlulie45SerArgAsnlieulieuGinAsnGly60PheGlyAsnAsplielielieGly65707580SerAsnAsnProliePheGlyLyaPheLeuGinMetArgGlyAla8595ArgAsplieTyrGlyThrLeuPheProThrTyrlieCysGinlie100105110AspGluSertjysProTyrThrArgTyrArgValArgGlyPhe115120125ValGlySerSerLysAspLeuliysLeuMetValThrArgTyrGlyLys130135140GlulieAspAlalieMetAsnValProAsnAspLeuAlaTyrMetGin1451501S5ISOProAsnProSerCysGlyAspTyrArgCysGluSerSerGinTyr165170175ValSerGinGlyTyrProThrProThrAspGlyTyrAlaProAspMet180185190TyrAlaCysProGinAsnlieAspArgLysHisValIjysCysHisAsp195200205ArgHisProPheAspPheHislieAspThxGlyGluValAspThrAsn210215220ThrAsnValGlylieAspValLeuLeuLyslieSerAsnProAspGly22S230235240TyrAlaThrValGlyAsnLeuGluVallieGluGluGlyProLeuThr24S25025SGlyGluAlaLeuAlaHisValIjysGinIjysGluLysLysTrpLysGin265270HisMetGluUysUysArgTrpGluThrGinGinAlaTyrAspProAla275280285LysGinAlaValAspAlaIjeuPheThrAsnGluGinGlulieuHisTyr290295300HislieThrLeuAspHislieGinAsnAlaAspArgIeuValGinSer30531031S320lieProTyrValTyrHisAsnTrpLeuProAsnAlaProGlyMetAsn325330335TyrAspValTyrGinGlulieuAsnAlaArglieMetGinGlyTyrAsn340345350LeuTyxAspAlaArgAsnVallieThrAsnGlyAspPheThrGinGly35536SLeuGinGlyTrpHisAlaThrGlyAsnAlaAlaValGinGinMetAsp370375380GlyAlaSerLeuValLieuSerAsnTrpSerAlaGlyValSerGin385390400asx1IiSUHisAlaGinAspHisHisGlyTyrValLeuArgVallieAla405410415GluGlyProGlyUysGlyTyrValThrMetMetAspCysAsn420425430GlyLysGinGluThrLeuLysPheThrSerCysGluGluGlyTyrMet435"0"5ThrLysThrValGluValPheProGluSerAspArgValArglieGlu4S0455460lieGlyGluThrGluGlyThrPheTyrlieAspSerlieGluLeuUeu470475480CysMetGinGlyTyrAspAsnAsnAsxiAsnlieuHisThrGlyAsnMet48543043STyrGlu<3ltlSerTyrAsnGlyAsnTyrAsnGinAbxiThrSerAspVal500S05S10TyrTyrGinGlyTyrThrAsnAsnTyrAsnGinAspSerSerAsnMet515520S25TyrAsnGinAsnTyrThrAsnAsnAspAspLeuHisSerGlyCysThr530S3S540CysAsn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satAsnatgccaiMetProaaattcLysPhe170GluagaArg貼cAsiltttPhe175Glu528attliettalieuttglieuttalieu180CC3ProValtatgcaLTyrAlacaagecGinAls185gcgAlaAsnttgLeucsitHis190tt￡llieulie576ttattaagagatgetgattattttggagcacagtggcaattaggtgatlieuLeuArgAspAlaAspTyrPheGlyAlaGinTrpGinLeuGlyAsp19520020S624gatgaaattcgtgataattatateagaetacaaggactgattagagaaAspGlulieArgAspAsnTyrlieArgLeuGinGlyLeulieArgGlu210215220tataaagatcattgtataacattctataaccagggtttaaatcaatttTyrLysAspHisCyslieThrPheTyrAsnGinGlyLeuAsnGinPhe225230235240720aatcgcteaaatgetcsagattgggtgagetttaataggtttcgtacaAsnArgSerAsnAlaGinAspTrpValSerPheAsnArgPheArgThr245250255768gatatgacattaacagtattagatetcgcaatattatttccaaactatAspMetThrLeuThrValLeuAspLeuAlalieLeuPheProAsnTyr26026.5270816gatccacgtaggtatccattagcagtaaaaacggaattgactagggaaAspProArgArgTyrProLeuAlaValLysThrGluLeuThrArgGlu275280285864gtttatacagatccagtagggtttactggggtattagaaagtggaggtValTyrThrAspProValGlyPheThrGlyValLeuGluSerGlyGly2S029S300912aggacttacccttggtataatcctaataatacaacctttactgetatgArgThrTyrProTrpTyrAsnProAsnAsnThrThrPheThrAlaMet305310315320960gaaaataacgcaagacgacgtccttcttataccacttggcttaatcgtGluAsnAsnAlaArgArgArgProSerTyrThrThrTrpLeuAsnArg3253303351008atttttgtatatacaaggactetaggtaatatgtctgatgtgagaaatliePheValTyrThrArgThrLeuGlyAsnMetSerAspValArgAsn3403453501056atttggggagggcatacattagttgaaaatggaaatgatggttctgaalieTrpGlyGlyHisThrLeuValGluAsnGlyAsnABpGlySerGlu3553603651104ataacccataactttggtaaaactgattctattactcctattcaatatlieThrHisAsnPheGlyLysThrAspSerlieThrProlieGinTyr3703753801152tttaatttcgcgaacctttctgttttcagtattgagteacttgetcgtPheAsnPheAlaAsnLeuSerValPheSerlieGluSerLeuAlaArg3853903954001200atatatttaggaggaacagaggetaataattatattactagtcagtatlieTyrlieuGlyGlyThrGluAlaAenAsnTyrlieThrSerGinTyx40S4104151248ggagtctegagagttatttttaatacateaaatataaataatgtacctG^yValSerArgValliePheAsnThrSerAsnlieAsnAsnValPro4204254301296ggatctttaagatacgaagtgcctgetaatcttccateccaaactataGlySerLeuArgTyrGluValProAlaAsnLeuProSerGinThrlie43544044S1344ttateagaattaccaggaaaggataagccaagaccaaacgcaggagatLeuSet*GluLeuProGl女LysAspLysProArgProAsnAlaGlyAsp4504554601392ttcagecatagattatcttatatatea貼ttttgatgcac的cgaagtPheSerHisArgI*euSerTyrlieSerAsnPheAspAlaArgArgSer46547047S4801440agtteaggcggtattgttagtcttttaacgtttggttgggcacataccSerSer<31yGlylieValSerLeuLeuThrPheGlyTxpAlaHisThr4854904951488agtatggatcgtaataatcgtcttgaaccagataaaattactcaaataSerMetAspArgAsnAsnArgLeuGluProAspLyslieThrGinlie5005055101536gatgcagttaaaggttggggggggaatategggtttgtcateccaggaAspAlaValLysGlyTrpGlyGlyAsnlieGlyPheVallieProGly51552052S1S84CCtProactThr530鹏GlygggGly貼tttgAsnIjsugtaVal535gtcValSergatagtAspSer540TrpC3tHisteaSercttlieu1632Lys545gttValc站GingcaAlaCC3ProGin550ArgGinseaThrSertatcgtTyrArg555liecgtArgttgLeucgt5601680tatTyrgetAlatgtttaLeugttaccValThr565catHisgggGlygatAspgetAla570attttt工lePheValgasGluHis575age1728GlySeragtSercatHis580BitsgttlieValteaSertttPhetttPhe585gatAsptgcteaCysSsraattea590tC3ggtGly1776cgtArgccaProteaSer595AsnactcttThrlieueta弼Glu600SergatAsptttcgcPheArgTyr605attliegatAspgttVal1824CC3Pro的tGly610attlietttPheThrProteaSei:615lie33tAstlCCCProttaataLeulie620tatTyraga1872C3ELGill625agetttPheggtGly￡LCCC3CThrHis630Ala3t3liegacAsp貼3tttgaaPheGlu635tttPhe.attlieCC3ProcttLeu64019203CtThrtttPheccgProAsnGin€45SerttaLeug貼Glu貼s650的sgaaArgGluGing33GlugtaVal65533tAsnAspLeutttPheatelie660aattaa1986'<210>24<211:>661<212>ERT<213>苏云金芽孢杆菌<400>24MetSerAsnCysTyrProArgTyrProLeuAlaLysAspProGinMet151015ThrMet;ArgAsnThrAsnTyrLysGluTrplieuAsnMetCysAspSer202S30AsnThrGinPhelieGlyAsplieSerThrTyrSerSerProGluAla35404SAlalieuSerValArgAspAlaValIeuThrGlylieAsnSerValGly505560ThrlielieuSerAsnlieuGlyValProLeuAlaSerGinSetPheGly65707S80lieSerArglieulieGlylieLeuTrpAlaGlyProAspProPhe859095GluAlaUeuMetValIi的ValGluGluIjeulieLysLysSerlieAsp10010S110GinArgValArgGluAsnAlaLeuArgGluIjeuGluGlylieuGinGly115120125lieMetArgLeuTyrGinThrArglieuGinAlaTrplieuValAsn130135AsilAspAspAsnArgArgAlaLeu14S150AsnPhePheGluAsnMetProlieIjeuLeulieuProValTyrAla180LeuIjeuArgAspAlaA印TyrPhe19S200AspGlulieArgAspAsnTyrUe210215TyrLysAspHisCyslieThrPhe225230AsnArgSerAsnAlaGinAspTrp245AspMetThrLeuThrValLieuAsp260AspProArgArgTyrProLeuAla275280ValTyrThrAspProValGlyPhe290295ArgThrTyrProTrpTyrAsnPro305310GluAsnAsrxAlaArgArgArgPro325liePheValTyrThrArgThrIjeu340lieTrpGlyGlyHisThrUeuVal355lieThrHisAsnPheGlyLysThr370375PheAsnAlaAsnLeuSerVal385lieTyrLeuGlyGlyThrGluAlaGlyValSerArgValliePheAsnGlySerLeuArgTyrGluValPro435440IjeuSerGlulieuProGlyUysAsp450455PheSerHisArgIjeuSerTyrlie465470SerSerGlyGlylieValSerLeu485SerMetAspArgAsnAsnArgLeu500AspAlaValLysGlyTrpGlyGlySIS520ProThrGlyGlyAsnLeuValLys530535LysValGinAlaProGinArgGinS45550TyrAlaCysheuValThrHis<31y565GlySerSerHislieValSer*Phe580ArgProSerAsnThrLeulieuGluS95600ProGlylie处eThrProSeirlieGinSerPheGlyThrHisAlaUeAsnThrPheProAsnGinSerlieu"5AspUeulieAsn660140ValThrGinTyrAlalieValAsp155160LysPheLysGluArgAsnpheGlu170175GinAlaAlaAsnLeuHisLeulie185190GlyAlaGinTrpGinLeuGlyAsp205ArglieuGinGlyIjeulieArgGlu220TyrAsnGinGlyLeuAsnGinPhe235ValSerPheAsnArgPheArgThr250255LeuAlalieLeuPheProAsnTyr265270ValLysThrGluLeuThrArgGlu285ThrGlyValLeuGluSerGlyGly300AsnAsnThrThrPheThrAlaMet315320SerTyrThrThrTrpLeuAsnArg33033SGlyAsnMetSerAspValArgAsn345350GluAsnGlyAsnAspGlySerGluAspSer工leThxPi:olieGinTyr380PheSerlieGluSerLeuAlaArg395400AsnAsnTyrlieThrSerGinTyr41041SThrSerAsnlieAsnAsnValPro425430AlaAsnLeuProSerGinThrlie445LysProArgProAsnAlaGlyAspSerAsnPheAspAlaArgArgSer475480LeuThrPheGlyTrpJUaHisThrGluProAspLyslieThrGinlie505510AsnlieGlyPheVallieProGly525ValSerAspSerTrpHisSerLeu540ThrSerTyrArglieArgLeuArg555AspMaliePheValGluHisSerS70S7SPheAspCysSerAsnSerSerGly585SerAspPheArgTyrlieAspVal605AsnProLeulieArgTyrArgThrAspLysPheGluPhelieProLeuLysArgGluGltiGluValAsn650655<table>tableseeoriginaldocumentpage113</column></row><table>5055603C3Thr65TyrcttLieu3t3lieC33GinttcPhe70ttgLeuctaLeugaaGlucccProggtGly753t3lieGlyGly3ttliecctPro80240gtaVal3t3lietttPheteaSerlie85lie33C333LysetcLeu3ttlie90ccgProtctSertctSerGlyC33Gin95tct288gtgAlagcaAlactt100tctSerlietgtCysgatAspttaLeu105gtatctSerlieattliecgt110Iiys弼Glu336ValgacAspgagGlu115SerValttaLeuSergacAsp120鹏GlygttVal9C6LAlagatAsptttPhe125gagGlu的tGlyGlu384atgactgettatcaagattattatcttcattatct匕gaggattggcttMetThrAlaTyrGinAspTyrTyrLeuHisTyrLeuGluAspTrpLeu13013S140432SC3Thr145gatAsp貼3IiysteaSerastAsncctPro150LysLyscttgetAlaAsp155gtaValgttVal乙ysGinttcPhe160480c站GingcaAlaeggArggaaGlug站Glu165gatAspttcPhesetThr333UyscttLeu170ttaLeuAla鹏GlySertt3Leu175teaSer528ArgcagGin333LysgetAla180GluliettaLeuttalieuttgUeu185cctProacgThrtatTyrValGin190getAlagcaAla576aatValC3tHis195tt3ttaLeuctattalieuArg200AspAlagttValtatTyr205Ly日LysGlu624tggGly210cta!Leu9tgValtgtCyscca_ProccgPro215ttgTyrCC3ProgggGlyteaSer220999GlyagaactThrgatAspt9tCys225Asn弼GlueggttaLieu站aiUys230AlaLysatalie貼333gGlu235tatTyrElCtThrsettAsiitettTyrtgtCys240720gtagggtggtataacaagggtttagatcagataagacaggcgggtacaValGlyTrpTyrAsnLysGlyLeuAspGinlieArgGinAlaGlyThr24S250255768agtgetgaagtttggtegataatttaataaatttcgtagagaaatgacgSerAlaGluValTrpSerLysPheAsnLysPheArgArgdluMetThi270816ttggcggtattggatattattgetatatttccaacttatgattttgaaLeuAlaVallieuAsplielieAlaliePheProThrTyrAspPheGlu2752802858"aaatatccattagcaacaagtgtagagttaactagggaaatttatacaLysTyrProlieuAlaThrSerValGluLeuThrArgGlulieTyrThr290300S12gatccagtgggatatteaggg的aaattatggttgggaaeggttttttAspProValGlyTyrSerGlyGlyAsnTyrGlyTrpGluArgPhePhe305310315320agetttaatteggtagaagcaaatg诉acaeggggacctggtttagttSerPheAsnSerValGluAlaAsnGlyThrArgGlyProGlyLeuVal10083303353CttggTrpcttLeuGin340getAla3t3liegatAsp3talieTyr345agtSerestHistctatt工lesatAsii350CttLeucagGin1056cttggttatcttagtggctgggggggaactcgtcattatgaagacttcLeuGlyTyrLeuSerGlyTrpGlyGlyThrArgHisTyrGJLuAspPhe3553603651104ThraagLiys370Gly33CGlygetAlatttPhe375C33GincgtArgatgMettctSer9gaGly380actThraegThragtSeraatAsn115233tAsn385CC3Procgt33t3ttlie3ttlie390tttPhe的cGly33tAsn3CCThrgatAsp39SlietttPheatt工lelie4001200tC3tt3lieugetAlat3tTyr405AlaMetGinccgProtttPhe410gttValgggGlytatTyrteaSeratelie415CC3Pro1248c的ArgC3tHiscttlieugttval420tC3SercgtgcaAlaGlutttPhe425tttPheccgProThrseaThretaLeu430Asnact1296ttcPhectgIjeut3tTyr435gagGlugtaVal3gt440鹏Glyt3CTyrteaSerGinTiu:44Satt工leGlutctSer1344gtgValttaLeu450ProggtGlyatt工leAsnLy日455gatAspIjeuCC3ProcctProagtSer460cgtAT9ThrAsntacTyr1392tctSer465estHissg3ArgttateaSerAsn470AlaAlatgtCysgttValca_3Gin47S犯tAsn9站GluThrtecSeragaArg4801440gttValAsngtaValtttPhe9gtGly485tggTrpseaThrestHisThrSer490MetLiysgatAsp的tAsn495Arg1488attlietatTyrCC3ProAsp500LysattlieacgThrGinlieSOScctProgcaAlaValLysgetAla510tttPhegecAlaetaccagcaggtacaggatatgcaggaggttacgtcacagetgggcctLieuProAlaGlyThrGlyTyrAlaGlyGlyTyxValThrAlaGlyPro5155205251584ggttatacaggaggagatgtagtaacgttaccttatcaagcaagtttaGlyTyrThrGlyGlyAspValValThrLeuProTyrGinAlaSerLeu530S35S401632aaaatacgtttaacttctgcacccacgaataaaaattaccgtgttagaliyslieArglieuThrSerAlaProThrAsnLysAsnTyrArgValArg5455505555601680cttcgctacgcgagtggaggacctggtccgttccgagtagaaagatggLeuArgTyrAlaSerGlyGlyProGlyProPheArgValGluArgTrp5655705751728tegccaagttctgttteaaatgetaatttttctcgtccagetacaggtSerProSerSerValSerAenAlaAsnPheSerArgProAlaThrGly5805855901776ggctatagtteatttgattatgtggacaccttagttactacatttaatGlyTyrSerSerPheAspTyrValAspThrLeuValThrThrPheAsn1824595600605caateaggtgttgaaataattatacaaaatctatctggttaccacctt1872GinSerGlyValGlulielielieGinAsnLeuSerGlyTyrHisLeu610615620attgttgacaaagtcgaatttateccaattgacatecaaattgaaaaa1920lieValAspLysValGluPhelieProlieAsplieGinlieGluLysG30635640tgtacgaaatgtcaattcgaaggagacatatgtagatgtgaaggagta1968CysThrLysCysGinPheGluGlyAsplieCysArgCysGluGlyVal645650655caatec匕tggaaacaaaaaaagagattgtaaatagtttatttateGinSerLeuGluThrLysLysGlulieValAsnSerLeuPhelie660670taaaat2016Asn2019<210>27<211>672<212>PRT<2">苏云金芽孢杆菌<400>27MetAsnMetAsiaSerTyrGinAsnThrASIlGluTyrGlu工lelieu151015AspGlySerPro20AsnAsnThrAsnMet25SerAsnArgTyrPro30PheAlaLysAspPro35AsnliePheProUe40AsnLeuAspAlaCys4SGinGlyArgProTrp50GinAspThrTrpGlu55SerValSerAsplieGOValThi:lieGlyThrTyrIjeulieGinPheLeuIjeuGluProGlylieGlyGlyliePro707S80ValliePheSerlie85lieAsnLyeLeulie90ProSerSerGlyGinSerValAlaAlaLeu100SerlieCysAspLeu105ValSerlielieArg110LysGluValAspGluSerValLeuSerAspGlyValAlaAspPheGluGlyGlu115120125MetThr130AlaTyrGinAspTyr13STyrlieuHisTyrLeu140GluAspTrplieuThrAspIjysSerAsnProLysLyslieuAlaAspValValLysGinPhe145150155160GinAlaArgGluGlu165AspPheThrLysLeu170LeuAlaGlySerLeu175SerArgGinLysAla180GlulieUeulieuLeu185ProThrTyrValGlu190AlaAlaAsnValHisUeuLeuI/euLeuArgAspAlaValLysTyrLysGlu195200205TrpGlyLeuValCysProProLeuTyrProGlySerGlyArgThrAsp210215220CysAsnGluArgLeuLysAl8LyslieLysGluTyrThrAsnTyrCys22S230235240ValGlyTrpTyxAsnLysGlyLeuAspGinlieArgGinAlaGlyThr24S250255SerAlaGluValTrpPheAsnLysPheArgArgGluMetThr260265270lieuAlaVal275LeuAsplielieAla280liePheProThrTyr285AspPheGluLysTyr290ProlieuAlaThrSer29SValGluLeuThrArg300GlulieTyrThrAspProValGlyTyrSerGlyGlyAsnTyrGlyTrpGluArgPhePhe305310315320SerPheAsnSerVal325GluAlaAsnGlyThr330ArgGlyProGlyIjeu335ValThrTrpIjeuGin340AlalieAsplieTyr345SerHisSerlieAsn350IjeuGinLeuGlyTyrLeuSerGlyTrpGlyGlyThrArgHisTyrGluAspPhe3553603G5ThrLjysGlyAsnGlyAlaPheGinArgMetSerGlyThrThrSerAsn370375380AsnProArgAsn工leliePheGlyAstiThrAspliePheIiyslielie3853S0395400SerLeuAlaArgTyr405AlaMetGinProPhe410ValGlyTyrSerlie415ProArgHisLeuValSerArgAlaGluPhePheProThrThrLeuAsnThr420425430PheLieuTyr435GluValAsnSerSer440GlyTyrSerGinThr445UeGluSerValLieu450ProGlylieAsnUys45SAspLeuProProSer460ArgThrAsnTyrSerHisArgUeuSerAsnAlaAlaCysValGinAsnGluThrSerArg470475480ValAsnValPheGly485TrpThrH丄sThrSer490MetljysljysAspAsn495ArglieTyrProAsp500LyslieThrGinlie505ProAlaVallaysAla510PheAlaLieuProAlaGlyThrGlyTyrAlaGlyGlyTyrValThrAlaGlyPro515520525GlyTyrThrGlyGlyAspValValThrLeuProTyrGinAlaSerLeu530535540UyslieArgLeuThrSerAlaProThrAsnLysTyrArgValArg545SSO555560lieuArgTyrAlaSerGlyGlyProGlyProPheArgValGluArgTrpS6S570575SerProSerSerValSerAsnAlaAsnPheSerArgProAlaThrGly580585590GlyTyrSerSerPheAspTyrVal600AspThrLeuValThr605ThrPheAsnGinSer610GlyValGlulielielieGinAsnLeuSer620GlyTyrHisIjeulieValAspLysValGluPhelieProlieAsplieGin工leGluUys625630635CysThrljysCysGinPheGluGlyAsplieCysArgCysGluGlyValS45650655GinSerIjeuGlu660ThrLysGlulie"5ValAsnSerlieuPhe670lieAsn<210><211><212><213>282010DNA苏云金芽孢杆菌<220><221>CDS<222>(1)(2010)<400>28atgaattctMetAsnSertatTyrCS3GluAsnThraatgaaAsnGlutatTyr10gaaatt(31uliectglieugatAspggttecGlySerIS48ccg站tProAsa站cAbuThr2033tAsnatgteaMetSeraacag这AsnArg25tatTyrccttttProPhegcaAlaaagLys30诉tccaAspPro96站tatatttcctattaacctggacgettgtcagggaaggccat的caaAsnliePheProlieAsnLeuAspAlaCysGinGlyArgProTrpGin354045gat.AspacgThrSOTrpgaaGlutCSLSergtcValtcggatSerAsp553t3liegt￡iVal3Ct5LttThrlie60gggGlyThrTyrcttLeu192atalie6SGinttcPhettgLeuetaIjeug貼Glu70cccggtProGlyatalieggtGlyggaa_ttGlylie75cctProgta_VallietttPhe80240tC3SerlielieAsn3a_3Uys85etcLeuattccglieProtctSertct90ggtcaaGlyGintctSerValgcaAla95Ala288ctttctatatgtgatttagtatctataattcgtaaagaggtagacgagLeuSerlieCysAspLeuValSerlielieArgLysGluValAspGlu100105110336agegtgttaagtgacggggttgcagattttgagggtgaaatgactgetSerValLeuSerAspGlyValAlaAspPheGluGlyGluMetThrAla115120125384tatcaagattattatcttcattatcttgaggattggcttacagataaaTyrGinAspTyrTyrLeuHisTyxLeuGluAspTrpLeuThrAspLys130135140432Ser145貼tAsncctProLyscttLeu150getgacgtagttAlaAspValVal155cagGinttcPheGingcaAlaeggArg160480gaagaagatttcactaaacttttagcagggteattateaagacagaaaGluGluAspPheThrLysLeuLeuAlaGlySerLeuSerArgGinLys16517017S528getgaaatattattattgcctacgtatgtgcaagetgcaaatgtgcatAlaGlulieLeuLeuLeuProThrTyrValGinAlaAlaAsnValHis180185190576ttattaetattaagggacgcagttaaatataaaaaagaatggggaetaIjeuLeuLeuLeuArgAspAlaValLysTyrLysLysGluTrpGlyLeu"5200205624gt'gtgtccaccgttgtatccagggteagggagaactgattgtaacgagValCysProProLeuTyrProGlySerGlyArgThrAspCysAsnGlu21021S220eggArg225ttaIjeu3站LysAla333Lys3t3lie230aaagagtatactLysGluTyrThr235tatTyrtgtCysgtaValgggGly240720tataacaagggtttagatcagEttaagacaggcgggtacaagtgetgaaTyrAsnLysGlyLeuAspGinlieArgGinAlaGlyThrSerAlaGlu24S250255gtttggtcgaaatttaat幼atttcgtagagaaatgacgttggcggtaValTrpSerLysPheAsnLysPheArgArgGluMetThrlieuAlaVal2S5270816ttggatattattgetatatttccaacttatgattttgaaaaatatccaLeuAsplielieAlaliePheProThrTyrAspPheGluLysTyrPro27S280285864ttagcaacaagtgtagagttaactagggaaatttatacagatccagtgLeuAlaThrSerValGlulieuThrArgGlulieTyrThrAspProVal290235300912ggatatteagggggaaattatggttgggaaeggttttttagetttaatGlyTyrSerGlyGlyAsnTyrGlyTrpGluArgPhePheSerPheAsn30S310315320360tcggtagaagcaaatggaacseggggacctggtttagttacttggcttSerValGluAlaAsnGlyThrArgGlyProGlyLeuValThrTrpLeu3253303351008caagetatagatatatatagcattctattaatcttcagcttggttatGinAlalieAsplieTyrSerHisSerlieAsnLeuGinLeuGlyTyr3403453501056cttagtggctgggggggaactcgtcattatgaagacttcacasagggtLeuSerGlyTrpGlyGlyThrArgHisTyrGluAspPheThrLysGly3553603651104aacggtgettttcaacgtatgtctggaactacgagtaeitaatccacgtAsnGiyAlaPheGinArgMetSerGlyThrThrSerAsnAsnProArg3703753801152aatattatttttggcaataccgatatatttaaaattattteattagetAsnlieliePtieGlyAsnThrAspliePheLyslie工leSerLeuAla3853903954001200agatatArgTyrAla3tgC33MetGin405ccgtttgttgggProPheValGlytatTyr410teaSeratelieccaeggProArgcatHis415cttLeu1248gttteacgtgcagaattttttccgacaacaetaaatactttcctgtatValSerArgAlaGluPhePheProThrThrLeuAsnThrPheLeuTyr4204254301296gaggtaaacagttctgggtacteacagacaattgaatctgtgttaccaGluVSlAsnSerSerGlyTyrSerGinThrlieGluSerValLeuPro4354404451344ggtattaataaggatetaccacctagtcgtacaaattactctcatagaGlylieAsnLysAspLeuProProSerArgThrAsnTyrSerHisArg4S04554601392tt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s170175LieuThrValTyrAlaGinAlaAla185190AspSerThrLeuTyrGlyAspIjys205GluGluAsnTyrAsnArgGinIjys220HisCysThrLysTrpTyrAsnSer235240ThrTyrGluGinTrplieAsnTyr250255UeuMetAlalieuAspLeuValAla265270ArgTyrSerMetGluThrSerThr28SAspProValSerLeuSerlieSer300SerGinMetGluAsnThrAlalie315320lieuAspGluUeuTyrlieTyrThr330335GlulieGinProAspLeuPheTyx345350LysLysSerGluGinSerAsnLeu36SThrSerGlyTyrlieSerSerGly380lieTyrArgThrLeuAlaAlaPro395400GinAsnTyrGlyValGluGinVal410415ValHisTyrArgGlyAspLys425430AspGinlieuProProAspGlyGlu44SAr9LeuCysHisAlaThrAlalie460AsnAlaThrlieProliePheSer475480TyrAsnArglieTyrProAsiiLysMetTyrLysIjeuAspAspProSer50S510ThrGlyGlyAspLeuValLysArg525lieLysAlaThrValAsnSerProS40ValArgTyrAlaThrAsnValSerSS5AspLyslieThrlieuGillThrLysS70S75GlyGluLysAspLeuThrTyr580585590GlySerPheGlyTyrlieGluTyrSerThrThrlieGinPheProAsp605GluHisProLyslieThrIjeuHisIjeuSerAspIjeuSerAsnAsnSer610620SerPheTyxValAspSerlieGluPhelieProValAspValAsnTyr630635AlaGluLysGluLysLeuGluLysAlaGinLysAlaValAsnThr650655PheThrGluGlyArgAsnAlaIjeuGinLysAspValThrAspTyrLysValAspGinValSerlieIjeuValAspCyslieSerGlyAspLeuTyr675680ProAsnGluLysArgGluLeuGinAsnLeuValLysTyrAlaLysArg690700LieuSerTyxSerArgAsnLeuLeuLeuAspProThrPheAspSerlie705710715720AsnSerSerGluGluAsnGlyTrpTyrGlySerAsnGlylieVallie725730735GlyAsnGlyAspPheValPheLysGlyAsnTyrLeuliePheSerGly740745750ThrAsnAspThrGinTyrProThrTyrIjeuTyrGinLyslieAspGlu755760765SerLyslieuLy，GluTyrThrArgTyrIjysLeuLysGlyPhelieGlu770775780SerSerGinAsplieuGluAlaTyrVallieArgTyrAspAlaLysHis785790795800ArgThrLieuAspValSerAspAsnLeuIjeuProAsplielieuProGlu805810815AsnThrCysGlyGluProAsnArgCysAlaAlaGinGinT^xLeuAsp820825830GluAsnProSerProGluCysSerSerMetGinAspGlylieLeuSer835840845AspSerHisSerPheSerIjeuAsnlieAspThrGlySerlieAsnHis850855860AsnGluAsnUeuGlylieTrpValLeuPheLyslieSerThrLeuGlu.865870875880GlyTyrAlaIiysPheGlyAsnLeuGluVallieGluAspGlyProVal885890lieGlyGluAlaLeuAlaArgValLysArgGinGluThrLysTrpArg900905910AsnLysIjeuAlaGinUeuThrThrGluThrGinAlalieTyrThrArg91SS25AlaIysGinAlaLeuAspAsnIjeuPheAlaAsnAlaGinAspSerHis935LeuLysArgAspValThrPheAlaGlulieAlaAlaAlaArgLyslie945350355S60ValGinSerlieArgGluAlaTyrMetSerTrpIjeuSerValValPro965370GlyValAsnHisProliePheThrGluLeuSerGlyArgValGinArg980S8S990AlaPheGinUeuTyrAspValArgAsnValValArgAsnGlyArgPhe99510001005lieuAsnGlyLeuSerAspTrplievalThrSerAspValLysValGin101010151020GluGluAsnGlyAsnAenVallieuVallieuA8nAsnTrpAspAlaGin10251030103S1040ValLeuGinAsnValLyslieuTyrGinAspArgGlyTyrlieLeuHis104510501055ValThrAlaArgLyslieGlylieGlyGluGlyTyrlieThrlieThr10S010651070AspGluGluGlyHisThrAspGinLeuArgPheThrAlaCysGluGlu107510801085lieAspAlaSerAsnAlaPhelieSerGlyTyrlieThrLysGluLeu10901100GluPhePheProAspThrGluLysValHislieGlulieGlyGluThr1105111011151120GluGlyliePheLeuValGluSerlieGluLeuLeuMetGluGlu112511301135LieuCys<210>42<211>1157<212>PRT<213>苏云金芽孢杆菌<400>42MetSerProAsnAsnGluAsnGluTyrGlulielieAspAlaThrPro1510SerThrSerValSerSerAspSerAsiaArgTyrProPheAlaAsnGlu202530ProThrAspAlaLeuGinAsnMetAsnTyrLysAspTyrLeuLysMet354045SerGlyGlyGluAsnProGlu:LeuPheGlyAsnProGluThrPhelie5055GOSerSerSerThrlieGinThrGly工leGlylieValGlyArglieLeu65707580GlyAlalieuGlyValProPheAlaSerGinlieAlaSerPheTyrSer8590PhelieValGlyGinLeuTrpProSerLysSerValAsplieTrpGly100105110Glu工leMetGluArgValGluGluLeuValAspGinLyslieGluLys115120125Tyrval]jysAspLysAlaLeuAlaGluLeuLysGlyLeuGlyAsnAla130135140LeuAspValTyrGinGinSerLeuGluAspTrpUeuGluAsnArgAsn14515015S160AepAlaArgThrArgSerValValSerAsnGinPhelieAlaLeuAsp1G5170175IjeuAstlPheValSerSerlieProSerPheAlaValSerGlyHisGlu180185ValLeuLeuLeuAlaValTyrAl3GinAlaValAsnLeuHislieuLeu200205'LieuLeuArgAspAlaSerliePheGlyGluGluTrpGlyPheThrPro210215220GlyGlulieSerArgPheTyrAsnArgGinValGinLeuThrAlaGlu225230235240TyrSerAspTyrCysValLiysTrpTyrLyslieGlylieuAspIiysLeu245250255LysGlyThrThrSerLysSerTrpLieuAsnTyrHisGinPheArgArg260265270GluMetThrIjeuValIjeuAsp3JeuValAlaLeuPheProAsnTyr275280285AspThrHisMetTyrProlieGluThrThrAlaGinLeuThrArgAsp290300ValTyrThrAspProlieAlaPheAsnlieValThrSerThrGlyPhe305310315320CysAsnProTrpSerThrHisSerGlylieLeuPheTyrGluValGlu325330335AsnAsnVallieArgProProHisLeuPheAsplielieuSerSerVal34034S350GlulieAsnThrSerArgGlyGlylieThrlieuAsnAsnAspAlaTyx355365lieAsnTyrTrpSerGlyHisThrlieuLysTyrArgArgThrAlaAsp370375380SerThrValThrTyrThrAlaAsnTyrGlyArglieThrSerGluLys385390395400AsnSerPheAlalieuGluAspArgAspliePheGlulieAstiSerThr405410ValAlaAsnlieuAlaAsnTyrTyrGinLysAlaTyrC51yValProGly42042S430SerTrpPheHisMetValLysArgGlyThrSerSerThrThrAlaTyr43S"5lieuTyrSerLysThrHisThrAlaLeuGinGlyCysThr*GinValTyr455460GluSerSerAspGlulieProLeuAspArgThrValProValAlaGlu470475480SerTyrSerHisArgLeuSerHis工leThrSerHisSerPheSerLys485490495AsnGlySerAlaTyrTyrGlySerPheProValPheValTrpThrHis500505S10ThrSerAlaAspLeuAsnAsnThrlieTyrSerAspLyslieThrGin515520525工leProAlaValLysGlyAspMetLeuTyrlieuGlyGlySerValVal530535540GinGlyProGlyPheThrGlyGlyAsplieIjeuLysArgThrAsnPro545550555560SerlieLeuGlyThrPheAlaValThi:V汪lAsnGlySerLeuSerGin570575ArgTyrArgValArglieArgTyrAlaSerThrThrAspPheGluPhe580585590ThrLeuTyrLeuGlyAspThrlieGluLysAsnArgPheAsnLysThr595600605MetAspAsnGlyAlaSerLeuThrTyrGluThrPheLysPheAlaSer620PhelieThrAspPheGinPheArgGluThrGinAspLyslieIjeuLieu630。5640SerMetGlyA印PheSerSerGlyGinGluValTyrlieAspArglie€50655GluPhelieProValAspGluThrTyrGluAlaGluGinGlu670AlaAlaLysLysAlaValAsnAlaLeuPheThrAsnThrLysAspGly6806B5LeuArgProGlyValThrAspTyrGluValAsnGinAlaAlaAsnLeu。0700ValGluCysUeuSerAspAspUeuTyrProAsnGluLysArglieu705710715720PheAspAlaValArgGluAlaLysArgLeuSerGlyAlaArgAsnLeu725730735LeaGinAspProAspPheGinGlu工leAsnGlyGluAsnGlyTrpAla740745750AlaSerThrGlylieGlulieValGluGlyAspAlaValPheIiysGly755765ArgTyrLeuArgUeuProGlyAlaArgGlulieAspThrGluThrTyr770775780ProThrTyrUeuTyrGliiIiysValGluGluGlyValUeuIiysProTyr785790795800Thi:ArgTyrArgLeuArgGlyPheValGlySerSerGinGlyLeuGlu80581081S工leTyrThrlieArgHisGinThrAsnArglieValLysAsnValPro820825830AspAsplieuJjeuProAspValSerProValAsnSerAspGlySerlie835840845AsnArgCysSerGluGinLysTyrValAsnSerArgIjeuGluGlyGlu850855AsnArgSerGlyAspAlaHisGluPheSerProlieAsplieGly865870875880GluIj€UAspTyrAsnGluAsnAlaGlylieTrpVeQGlyPheLyslie885895ThrAspProG工uGlyTyrAlaThrI>euGlyAsnlieuGluUeuValGlu900905910GluGlyProLeuSerGlyAspAlalieuGluArglieuGinArgGluGlu920S2SGinGinTrpLyslieGlaMetThrArgArgArgGluGluThrAspArg930335ArgTyrMetAlaSerLysGinAlaValAspArgLeuTyrAlaAspTyr94S950SS5GinAspGinGinI/euAsnProAspValGlulieThrA印LeuThrAla$70AlaGinAeplieulieGinSerlieProTyrValTyrAsnGluMetPhe380S85ProGlulieProGlyMetAsnTyrThrLysThrGluLeuThrAsp99510001005ArgLeuGinGinAlaTrpAsnLeuTyrAspGinArgAsnAlaliePro101010151020AsnGlyAspPheArgAsnGlyLeuSerAsnTrpAsnAlaThrProGly10251030103S1040ValGluValGinGinlieAsnHisThrSerValLeuVallieProAsn104510501055TrpAspGluGinValSerGinGinPheThrGinProAsnGinArg106010651070TyrVal:LeiiArgValThrAlaArgLysGluGlyValGlyAsrtGlyTyr107510801085ValSerlieArgAspGlyGlyAsnGinSerGluThrUeuThrPheSer109010951100AlaSerAspTyrAspThrAsnGlyValTyrAsnAspGinThrGlyTyr1105111011151120工leThrIjysThrValThrPhelieProTyrThrAspGinMetTrplie112511301135GluUeSerGluThrGluGlyThrPheTyrlieGluSerValGlulieu114011451150工leValAspValGlu1155<210>43<211>675<212>PRT苏云金芽孢杆菌<400>43MetAsnProTyrGluAsnIjysAsnGluTyrGluliePheAsnAlaPro151015SerAsiiGlyPheSerLysSerAsnAsnTyrSerArgTyrProlieuAla202530AsnLysProAsnGinProLeuLysAsnThrAsnTyrLysAspTrpUeu354045AsnValCysGinAspAsnGinGinTyrGlyAsnAsnAlaGlyAsnPhe5055AlaSerSerGluThrlieValGlyValS6rAIelGlylielieValVal65707580GlyThrMetLeuGlyAlaPheAlaAlaProValLeuAlaAlaGlylie859095lieSerPheGlyThrLeuLeuProliePheTrpGinGlySerAspPro100105110AlaAsnValTrpGinAspUeuAsn工leGlyGlyArgProlieGin115120125GlulieAspAsnlielieAsnValIjSUThrSerlieValThrP:ro130135140UeLysAsnGinlieuAspLysTyrGlxiGluPhePheAspLysTrpGlu145150155ProAlaArgThrHisAlaAsnAlaLysAlaValHisAspLeuPheThr165170175TlirLeuGluProHelieAspLysAspIjeuAspMetLeuLy日AsnAsn180185ISOAlaSerTyrArglieProThrLeuProAl3TyrAlaGinlieAlaThr200205TrpHisAen￡jeuLeuIiysHisAlaAlsThrTyrTyrAsnlieTrp210215220lieuGinAsn<31nGlylieAenProSerThrPheAsnSerSerAsnTyr225230235240TyrGinGlyTyrLeuLysArgLyslieGluGluTyrThrAspTyrCys24S2S02SSUeGinThrTyrAsnAlaGlyLeuThrMetlieArgThrAsnThrAsn260265270AlaThrTrpMetTyrAsnThrTyrArgLeuGluMetThrI*euThr27528028SValLeuAsplieulieAlaliePheProAbjiTyrAspProGluLysI*yr29S300ProlieValSerGlulieulieArgGluValTyrThrAsnVal30S310AsnSerAspThrPheArgThrlie325ArgProThr340LieuPheThrTrpArgAsnSer355ArgAsplieIjeuGlyAsnGinMetAlaPheThrHis370375TrpGlyAlaValHisGlyAsnlie385390PheProPheTyrArgAsnLysPro405HisArgGluTyr420SerAsp工lelieSerSerGlu435PheArgTyxSer440TyrLysArgThrAspSerTyrMet450455GluGluTyrGlyHisThrLieuSer470PheSerValValArgGluArgArg485ThrSerValAspsooPheGinAsnThrlieHisAla515IjeuLysAlaLeuLys520LysGlyProGlyHisThrGlyGly530535MetAspPheArgValPheLeuS45550ValArgUeArgTyrAlaThrAsn565ThrGlylieAsp580ThrlieSerValGinAsnProAsnAlaThrAspLeuGOOThrPheProArgThrValPro615AsnLeuLeuMetThrLeuTyrGlyThr625630lieAspLyslieGluPhelieProThrGluLysGin660lieGluLysValAsn675<210>44<211>648<212>PRT<213>苏云金芽孢杆菌<400>44MetHisTyrTyrGlyA6txArgAsn15SerAsnAspSerAsnMetSerAsn20AsnProGinGluAspLeuMetGlu3540AsnValCysGluGlyTyrHislieSO5SArgAlaGlyLeuGlyLysGlylieu6570PhePheGlyGlySerlielieLeu85lieSerGluLeuTrpProGlu315320ThrGluLeu<31uAsnGlyThr33033S工leAsnGinGlyArgPheTyrThr345350ProTyrAspliePteSerPheThrThrAsnA印AspArgAsnlielie380工leSerGinAspThrSerLysVal395400lieASpLysValGlulieValArg410415Tyr'GluMetliePhePheSerAsn425430SerAsnSerThrlieGluAsnAsn445工leProLysGinThrTrpLysAsnTyrlieLysThrAspAsnTyr工le475480ArgValAlaPheSerTrpThrHis49049S工leAspLeuAspAsnlieThrGin505S10ValSerSerAspSerLyslieVal525AspLeuVallieUeuLysAspSer540Ly日AsnValSerArgGinTyrGin555560AlaProLysThrThrValPheIjeu570575GluLeuProSerThxThrSearArg5855S0ThrTyrAlaAspPheGlyTyrValSOSLysThrPheGluGlyGluAspThr620ProAsnHisSerTyrAsnlieTyr635lieThrGinSerValUeuAspTyrThrGinLyslieValAsnAsplieuMSGluTyrAsplieLeuAsnAlaSer10ThrTyrProArgTyrProlieuAla2530AsaThrAsnTyxLysAspTrpLeu45GluAsnProArgGluAlaSerVal60GlylieValSerThrlieVal<31y7580AspThrlieGlyLeuPheTyrGin9095AspAspThrGin<31nTyxThrTrp100GinAsplieMet115GluVallieArg130LysValAspAsp145LiysSerThrGlyAspSerAspPhe180ProGlyTyrGlu195IjeuHisLeuLeu210TrpSerAlaArg225GluLysThrLyBGlyljeuAsnAsp260TyrArgArgGlu275Pro工leTyrAsp290ThrArgGlulie305MetThrProTyrAlaProH丄slieuSerlieSerTyr355TyrThrAsnAsn370ThrAspTyrGly385TyrValTyrAsnGlyAspProVal420AsnSerSerLys435V3lVaiArgThr450lieuAlaLiysTyr465AsnGlyGluThrThrHisSerSer500ThrGin工lePro51SlieGlulysGly530AlaAspSerGly54SLysTyrArgVallieI*euArgLys580AsnlieSerPro595lieThrThrThr610ProTyrGlyArg625LyslieGluPheH丄sValGlu120GlyAsnAla工le135TyrAsnAsnTrpISOAsnLeuSerThrAsnGlyAla工leLeuLeuLeuLeu200lieuLieuArgAsp215AlaAsnAlaArg230GluTyrThrGlu245PheArgThrAlaMetThrUeuThr280AlaArgLeuTyr295TyrSerAspVal310PheSerPheGlu325PheThrTrpJjeuTrpThrPhelieuSerSerlieAsn37SGlyThrSerSerLeuTrpThrGlu405AsnlieThrijysGluLeulieTyr440AspPheAspPhe455AsnAspTyrAsn470PheGlyGinLys485ValAspProAsnValValLysAla520ProGlyPheThr535LeuThrMetArgS50ArglieArgTyrTrpLysGlyGluThrTyrGlyAla600GluAsnliePhe615GinPheValGlu630LeuProThrAsn105AsplieAspArgThrLeuAla140LeuLysLysTrp155LeuValThr170ArgThrValAsn185ProValTyrAlaAla(Unlieoyr220AspAsnTyrTyr235TyrCyslieAsn250GlyGinTrpVal265VallieuAsplieProThrGluVal300lieAsnGlyGlu315LysAlaGlu330■LysGlyPheArg345SerGlyGlyGinGluGlySerPhe380ThrlieAsnlie395AsnTyrGluTyrMetAsnPheSer425GlyAlaHisArgLeuThrAsn460HislieLeuSer475ArgHisGlyTyr4S0AsnThrlieAlaSOSSerSerlieAsnplyGlyAspLeu540PheLysAlaGluS55LysCysAenTyrS70GlyTVrlieGin58SPheSerTyrLeuAspLeuThrMetASplieProSer635110ArglieThr125AspLeuGinGlyLysAspAspProPileThi:AlaIjeu175AsnGinGlySerGinlieAlaAsn205GlyAepLysTrpGinlieGinLeu240TrpTyrAsnLys255AsnPheAsnArg270lieSerMetPhe285LysThrGluLieulieTyrGlylieu320lieu*TyrThrArg335PheValThrAsn350AsixLysTyrSer365ArgGlyGinAspProSerAsnSer400lieTyrProTrp415ValThrAspAsn430ThrAsnLysPro445GluGlyThrGluTyrMetLeulie480SerPheAlaPheAlaAsnLyslie510GlySerlieSer525ValLysMetArgLeulieuAspLysSerSerLysLeuS75饥nGinlieHis5S0GluSerPheThr605GluValThrTyrlieulielieuAsp"5<210>45<211>682<212>PRT<2">苏云金芽孢杆菌<400>45MetAsnSerTyrGinAsnLysAsnGluTyrg:lulieLeuAspAlaLys151015ArgAsnThrCysHisMetSerAsnCysTyrProLysTyrProIjeuAla202530AsnAspProGinMetTyrIjeuArgAsnThrHisTyxIiysAspTrplie354045AsnMetCysGluGluAlaSerTyrAlaSerSerGlyProSerGinLeu505560PheValGlyGlySerlieValAlaLyslielieuGlyMetliePro65707580GluValGlyProLieuLieuSerTrpMetSerlieuPheTrpProThr859095lieGluGluAsnThrValTrpGluAspMet工leLysTyrV3lAla100105110AsnLeuljeuLysGinGluLeuThrAsnAspThxlieuAsnArgAlaThr115120125SerAsnUeuSerGlylieuAsnGluSerlieuAsnlieTyrAsnArgAla130135140LeuAlaAlaTrpLtysGinAsnLyaAsnAsnPheAlaSerGlyGluIjeu14S150155160lieArgSerTyr工leAsnAspIjeuHislieLeuJPheThrArgAsplie170175GinSerAspPheSerLeuGlyGlyTyrGluThrValIjeuIjeuProSer180185190TyrAlaSerAlaAlaAsnlieuHisIjeuLeuLeuLeuArgAspValAla195200205lieTyrGlyLysGlulieuGlyTyrProSerThrAspValGluPheTyr210215220TyrAsnGluGinLysTyrTyrThrGluJjysTyrSerAsnTyrCysVal225230235240AsnThrTyrLysSerGlyLeuGluSerLysLysGin工leGlyTrpSer245250A印PheAsnArgTyrArgArgGluMetThrLeuSerValLieuAsplie270ValAlaLeuPheProLeuTyrAspThrGlyLeuTyrProSerLysAsp275280285GlyLyslieHisValIysAlaGluLieuThrArgGlulieTyrSerAsp290295300VallieAsnAspHisValTyrGlyLeuMetValProTyrlieSerPhe305310315320GluHisAlaGluSerLeuTyrThrArgArgProH丄sAlaPheThrTrp325330335LeuIiysGlyPheArgPheValThrAsnSerlieAsnSerTrpThrPhe340345350LeuSerGlyGlyGluAsnArgTyrPheIjeuThrHisGlyGluGlyThr355lieTyrAsnGlyProPheLeuGlyGinAspThrGluTyrGlyGlyThr370375380SerSerTyrlieAsplieSerAsnAsnSerSerlieTyrAsnLeuTrp38S3S0395400ThrLysASHTyrGluTrplieTyrProTrpThrAspProValAsnlie405410415ThrLyslieAsnPheSerlieThrAspAsnSerAsnSerSerGluSer42042S430lieTyrGlyAlaGluArgMetAsnLysProThrValArgThrAspPhe435445AsnPheLeuLeuAsnArgAlaGlyAsnGlyProThrThrTyrAsnAsp4S0TyrAsnHislieLeuSerTyrMetlieulieAsn<3lyGluThrPheGly465470475480GinLysArgHisGlyTyrSerPheAlaPheThrHisSerSerValAsp485490495ArgTyrAsnThrlieValProAspIiyslieValGinlieProAlaVal500505510LysThrAsnLeuValGlyAlaAsn工lelieLysGlyProGlyHisThr515520525GlyGlyAspLeuIjeuLyslieuGluTyrGluArgPHeLeuSerLeuArg530535540lieLysLeulieAlaSerMetThrPheArglieArglieArgTyrAla545550555SerAsnlieSerGlyGinMetMet工leAsnlieGlyTyrGinAsnPro565570575ThrTyrPheAsnlielieProThrThrSerArgAspTyrThrGlulieu580585590laysPheGluAspPheGinLeuValAspThrSerTyr工leTyrSerGly595600605GlyProSerlieSerSerAsnThrUeuTrpUeuAspAsnPheSerAsn610615GlyProVallielieAspLyslieGluPhe工leProGlylieThr625630635640LeuAsnGinAlaGinGlyTyrAspThrTyrAspGinAsnAlaAsnGly655MetTyrHisGinAsnTyrSerAsnSerGlyTyrAsnTyrAsnGinGlu665670TyrAsnThrTyrTyrGinSerTyrAsnAsn680<210>46<211>529<212>PRT苏云金芽孢杆菌<400>46AsnPheMetlieuThrSerGlyAlaAsnMetLeuLysIeuGlu151015ThrThrAspTyrGlulieAspGinMetAlaAsnAlalieGluAsnMet202530SerGlyGluGluTyrSerGinGluLysMetMetGinTrpHisAsplie354045LysTyrAlaLysGinLeuSerGinAlaArgAstiLeuLeuGinAsnGly5055AspPheGluAspIseuPheSerGlyTrpThrThrSerAsnGinMetSer707580lieGinAlaAspAsnAlaThrPheLysGlyAsnTyrLeuHisMetSer8595GlyAlaArgAsplieTyrGlyThrliePheProThrTyrlieTyrGin100105110LyslieAspGluSerLyslieuLysProTyrThrArgTyrlieuValArg115120125GlyPheValGlySerSsrLysAspIjeuGluLeuMetValMetArgTyr130135140GlyLysGlulieAspThrValMetAsnValProAsnAsplieProTyr150155ValProSerMetValCysAsnGluIjeuTyrAspGlyGinGinPro165170175TyrProAsnArgHisValGlyTyrTyrAsnProMetProValSerGin180185ISOProSerTyrThrSerAspThrCysGinCysThrProGlyLysLysHis20020SValValCysHisAspSerHisGinPheLysPheHislieAspThrGly210220GluValAspTyrAsnThrAsnLeu<33LylieTrpValLeuPheLyslie22523023S240SerSerProAspGlyTyrAlaTfarLeuAspAsnlieuGluVallieGlu2452S02S5GluGlyProValArgGlyGluAlaValThrHisValLysGinLysGlu260265270LysTrp275AsnGinGinMetGlu280LysLysArgMetGlu285ThrLysArgValTyr290AspArgAlaIjysGinAlaValAspAlaIjeu300PheThrGlyGluGluLieuAsnTyrAspValThrLeuSerHislieIjysAsnAlaAspAsp305310315320lieuValGinSerHe325ProTyrValHisAsn330GluTrpIjeuProAsp335PheProGlyMetAsn340TyrAsplieTyrGin345GluLeuAsnAlaArg350lieMetGinAlaArg355TyrLeuTyrAspAla360ArgAsnVal工leThrAsnGlyAspPheAlaGinGlyLeuGinGlyTrpHisAlaGluGlyIjysValGluVal370375380GinGinMetAsnGlyThrSerValLeuValLeuSerAsnTrpSerSer385390395400GlyValSerGinAsn405]jeuHisValGinHis410ProHisGlyTyrLeu415lieuArgValSerAlaijysIjysGluGlySerGlyLiysGlyTyrValThrArg420425430MetSerCys435AsnGlyLysGinGlu440ThrtieuThrPheThr445SerCysAspGlyGly450TyrMetThrIjysThr4S5ValGluValPheProGluSerAspArgValArglieGlulieGlyGluThrGluGlySerPheTyrlieGluSer470475480lieGluLeu工leCys485MetAsnGlyTyrThr490SerAsnAsnAsnGlu495AsnMetSer■AsnMetTyrAspGinSerTyrGlyAsnTyrSerGinAsn500S05510ThrSerAspMetTyrAspGinGlyGlySerValAlaLysPheGluIjys51S520525Glu<210>47<211>674<212>PRT<2">苏云金芽孢杆菌<400>47MetAsnGinTyrGinAbuLys15GinAsnAsnMet20AsnMetProAsnAsnAlaVal35MetLysAsnGlyAsn40GluGlySOSerAsnlieSerPro55SerlieValSerlieVallieulaysThr6570LeuAlaAspSerlie85LysSerSerGluAsnAsnVal100SerGinValSerAsnArgArg115lieGinGluThrlie120AsnGly130LeuValAlalieTyr135AsnAlaTrpAsnAsnAsnAsn150GluAlaPtieLysThrValGluArglieTyrArgThr180SerSerSerGinGinAlaAlaAsnIi€UHislieuSerGluTyr10ArgTyr2STyrLysProAl3LeuAlaLeuGly90MetVal105lieuAspArgAsplieAsnGlulie185Met170ThrLeuGlu工leProPheAspTrpAlaAl360LysAlei7SlieSerGinVallieuGlyTyrlieuTyrGin1S5PheThrlieulieuArgAspLeuAlaVal45lievalLysHisGlu125GlyThrLysProAlaGluAsp30AsnThrAlaThrGin110SerAlaAsnLeuThr190ValSerSer15AspProGluCysSerSer80lieThrlielieSerLeulieuGluValAla160LysGly175PheThrMetTyr195200205GinGluGlyTrpAsnlieuGinSerHislieAsnTyrSerLysGluIjeu210215220AspAspAlaLeuGluAspTyrThrAsnTyrCysValGluValTyrThr22S230235240I/ysGlylieuAsnAlaLeuArgGlyThrAlalieAspTrpLeuGlu24525025SPheAsnSerPheArgArgAspMetThrIjeuMetValLeuAspIjeuVal260265270AlaliePheProAsnTyrAsnProValArgTyrProLeuSerThrIiys275280285lieSerLieuSerArglieTyrThrAspProValGlyArgThrAsp29029S300SerProSerPheGlyAspTrpThrAsnThrGlyArgThrLeuAlaAsn305310315320PheAsnAspIjeuGluArgGluValThrAspSerProSerIjeuValIjys325330335TrpLeuGlyAspMetThrlieTyrThrGlyAlalieAspSerTyrArg340345350ProThrSerProGlyAspArglieGlyValTrpTyrGlyAsn工leAsn355360365AlaPheTyrHisThrGlyArgThrAspValValMetPheArgGinThr370375380GlyAspThrAlaTyrGluAspProSerThrPhelieSerAsnlieLeu385390395TyrAspAsplieTyrLysLeuAspLeuArgAlaAlaAlaValSerThr405410415lieGinGlyAlaMetA印ThrThrPheGlyValSerSerSerArgPhe420425PheAsplieArgGlyArgAsnGin'LieuTyrGluSerAsnLysProTyr43S440445ProSerLeuProlieThrlieThrPheProGlyGluGluSerSerGlu4S045SGlyAsnAlaAsnAspTyrSerHisIj6ULeuCysAspValLyslieLeu465470475.480GinGluAspSerSerASlllieCysGluGlyArgSerSerIjeuLeuSer485490495HisAlaTrpThrHisAlaSerLeuAspArgAsnAsnThrlieLeuPro500505510AspGlulieThrGinlieProAlaValThrAlaTyrGluIjeuArgGly515520525AsnSerSerValValAlaGlyProGlySerThrGlyGlyAspLeuVal530535540LysMetSerTyrHisSerValTrpSerPheLysValTyrCysSerGlu545550555560LeuLysAsnTyrArgValArg工leArgTyrAlaSerHisGlyAsnCys565570S75GinPheLeuMetLysArgTrpProSerThrGlyValAlaProArgGin580585590TrpAlaHisAsnValGinGlyThrPheSerA扫nSerMetArgTyr595600GluAlaPheljysTyrlieuAspliePheThrlieThrProGluGluAsn€10615620AsnPheAlaPheThrlieAspLeuGluSerGlyGlyAspLeuPhelie€2563063SAspLyslieGluPhelieProValSerGlyPheGlutTyrGlu"5GlyLysGinAsnlieGluLysThrGinLysAlaValAsnAspLeuPhe660665670lieAsn<210>48<211>。5<212>PRT<2">苏云金芽孢杆菌MetAsnProTyxGinAsnLysSerGluCysGlulieIeuAsnAlaPro151015LeuAsnAsnlieAsnMetProAsnArgTyi:ProPheAlaAsnAspPro202530AlaValMetLysAsnGlyAsnTyi:LiysAspTrpLeuAsnGluCys354045AspGly工leThrProSer工lePheGlyThrIjeuGlyValLeuAlaSer505560lieVallieSerThrlieAsnLieuAlaThrSerProSerlieGlyAsp707580AlaPheAlaLeuValSerSerlieGlyGluTyrTrpProGluThrLys859095ThrSerPheProIjeuSerValAlaAspValAsnArgLeulieArgGlu100105110AlaLeuAspGinAsnAlalieAsnArgAlaThrGlyLysPheAsnGly115120125IjeuMetAspThrTyrAsnThrValTyrLeuLysAsnLeuGinAspTrp130135140TyrAspThrArg工leProAlaAsnProGinGlyAspSerGinIjeuArg145150155160GluAlaAlaArgArgSerLeuGluGlu工leGluArgAspPheArgLys170175AlaLeuAlaGlyGluPheAlaGluAlaGlySerGinlieValIjeulieu180185190ProlieTyrAlaGinAlaAlaAsnlieHisIjeulieulieUeuLysAsp200205AlaMetGinPheArgThrAspGlyUeulieArgProValGlyVal210215220Pxo工leThrThrSerAlaGluAspProPheGluSerGluPheIjeuIjeu225230235240ArglieLysLysTyrThrAspHisCyslieSerTyrTyrAspAspGly245250255lieuAlaLyslieArgSerArgGlySerAspGlyGluThrTrpTrpGlu2602S5270PheAsntiysPheArgArgGluMetThrLeuThrValLeuAspI/euVal275280285AlaLeuTyrProThrHisAsnlieLysLeuTyrProlieProThrGin290295300ThrGluLeuSerArgValValTyr*ThrAspProValGlyCysPheGly305310315320AsnArgLysSerAspliePheSerArgLieuAsnPheAspTyrlieuGlu325330335AsnArglieuThrArgProArgGluProPheAsnTyrIjeuAsnSerVal3403453S0GinI/euPheAlaSerThrValSerAsnSerAsnAsnGlyGluValLeu35536036SArgGlyAsnI^euAsiiLyslieMetPheGluGlyGlyTrpThrAlaSer370375380ArgSerGlyAspGlyValThrThrGlyThrProPheSerThrMetAsp385390395400TrpSerTyrGlyTrpGlyTyrProArgLysHisTyrAlaGlulieThr405410415SerArgSerGinAlalieuProGlylieuAsnAsnSerUeHisVallie420425430ValGlylieAspSerPheArgAlalieGlyProGlyGlyGinGlyAsp435440"5HisThrPheSerlieuProGlyGlyAspMetTyrAspCysGlyLysVal4S04SS460GinlieAsaProLeuGluAspTyrArgAsnSerAspHisTrplieSer465470475480AspMetMetThrlieAsnGinSerValGinlieuAlaSerAsnProThr4&S490GinThrPheAlaPheSerAlaLeuSerlieuGlyTrpHisHisSerSerSOOS05S10AlaGlyAsnArgAsnValTyrValTyrAspLyslieThrGinliePro515520S2SAlaThrIiysThrValArgGluHisProMetlieLysGlyPro530535540ThrGlyGlyAspLeuAlaAspLeuSerSerAsnSerAsplieLeuGinS45S50555TyrAsplieuArgSerAspTyrAspAspArgLeuThrGluAspValPro565570575PheArglieArglieArgCysAlaSerlieGlyValSerThrlieSer580585590ValAspAsnTrpGlySerSerSerProGinValThrValAlaSerThr595605AlaAlaSerAspThrLeuLysTyxGluSerPheGinTyrValSer620lieProGlyAsnTyrTyrPheAspSerAlaProArglieArgLeuLeu630640ArgGinProGlyArglieuLeuValAspArglieGlulielieProVal"5655AsnPhePheProSerGluGinGluAsnLysSerValAspSerLeu670PhelieAsn675<210>49<211>6S9<212>PRT<213>苏云金芽孢杆菌<400>49AsnSerTyrGluAsnLysAsnGluTyrGlulieLeuAsnAspSerLys151015LysSerAsnMetSerAsnProTyrlieuArgTyrProLeuAlaAsnAspAla2025、，30SerLeuSerMetGinAsnThrAsnTyrLysAspTrpLeuThrMet3S4045CysAspArgThrAspThrAspValLeuSerSerArgGlyAlaValSer5055GOThrGlyValGlyMetLeuSerThrlielieuSerUeuPlieGlyliePro65707580LeulieGlyGluGlylieAspLeuLeuUeuGlyAlaAlaAspPheLeu8S9095TrpProGluSerAspGlyGlyHisGinTyrThrTrpGluAspUeuMet100105110AsnHislieGluGluIjeuMetAspGluArgLeuGluThrGluIiysArg115120125ThrThxAlalieuAspAspLeuArgGlyLeuLysAlaLeuIjeuGlyIjeu130135140PheArgAspAlaPheA印SerTrpGluLysAsnGinAsnAspProlieISO155160AlaLysAsnArgValGlyGlyTyrPheGluAspValHisThrHisPhe170175ValLysAspMetAlaSerliePheSerAlaThrAsnTyrGluVallieu180185ISOUeuIjeuProValTyrAlaGinAIelAlaAsnUeuHisLeuLeulieuLeu19520020SArgGluGlyVallieTyrGlySerArgTrpGlylieAlaProAlaAla210215220AspPheTyrHisAspGinLeuLeuLysTyrThrAlalieTyrAlaAsn225230235240HisCysValThrTrpTyrAsnAsnGlyLeuAlaGinGluLysGlulieu2452502S5PheAlaIiysSerProAsaTrpAsnArgPheAsnAlsiTyrArgArgAsp270MetThrlieThrValLeuAsplielieAlaLeuPheProThrTyrAsp275280285AlaArglieuTyrThrLysProlieLysThrGluLeuThrArg<31ulie2卯295300TyrSerAspVallieuAsnLeuAspValTyrGlyValGinGinThrAsp30531031S320LeuAsnLysAsnGluAl3AlaPheThrArgSerProHisLeuValThr325330335ArgLeuArgGlyPheAspPheTyrThrArgThrLysTyrAlaTyrTrp340345350ArgTyrLeuAlaGlyHisThrAsnTyrPheSerPheThrGlyAsnGly355360Thr工leTyrSerSerSerPheAsnAsnTrpTyrAspThrAspMetThr370375380LysSerThrlieAsnlieProAspTyrAlaAsnlieTyrLysLeuTrp385395400ThrLysSerTyrThrAsnlieSerProTyrThrAspPi:oValGlylie405410415SerGinMetGinPheSerLeuThrAsnAsnGinGinLeuThrTyrThrf420425430GlyThrSerAlaProLysTyrProVal■ArgGluThrPhePheGlulie435440445ProProThrAspGluLysProIjeuThrTyrGluAsnTyrSerHislie4504S5460IjeuSerTyrMetThrSerAlaGinH丄sPheGlyAspLysLyslieGly465470475480TyirThrPheAlaTrpMetHisGluSerValAspPheAspAsn485435AspProAspLyslieThrGinlieProAlaValLysGlyAspTyrIjeu500505510GinTyrGlyTyrValGinGlyProGlyHisThrGlyGlyAspLeu515520525ValSerMetlieArgThrAspArgLeuGlylieAsiiValTyrPhePro530535540GinProLieuAspTyrArglieArglieArgTyrSerThrSerSerAsn545550555560GlyTyrUeuTyr工JLeTyrSerProAsnThrLyslieValTyrIjeuPro570575ProThrThrLeuValAspGlyGinProThrPheAspProMetAspPhe58058SS90SerAlaPheArgValValGluValProAlaSerPheArgAlaSerVal595600605AlaGlyTyrThrAsnPheThrlieGluAlaGlyPheGlyProValTyr€15工leAsplieGluPhelieProAspAsnThrThrThrlieuGluTyr625630635640GluGlyGlyArgAspLeuGluUysThrLysAsnAlaValAsnAsplieu650655PheThrAsn<210>50<211>55B<212>PRT<213>苏云金芽孢杆菌<400>50MetPhelieSerAsnlieLysAsnThr3beuLyslieGluThrThrAsp1S1015GlulieAspGinAlaAlalieSerlieGluCysMetSerAsnGlu202530HisSerSerI*ysGluGluMetMetLeuTrpAspGluGinAla354045LysGinIjeuSerTrpSerArgAsnIjeuLeuTyrAsnGlyAspPheGlu505560AspValSerAsnGlyTrpLysThrSerAsnThrlieGlulieArgGlu65707580AsnSearProValPheLysGlyHisTyrlieuHisMetPheGlyAlaArg8530AsplieAspGlyThrLeuPheProThrTyrlieTyr<3lnliyslieGlu10S110GluSerLysLeuLysProTyrThrArgTyrArgValArg<31yPheValUS12012SGlySerSerLysAspLeuLysLeuMetValThrArgTyr(SlyGlu<table>tableseeoriginaldocumentpage155</column></row><table>SOS560GluPheLieuMetGluProSerLeuGlyGlylieAsnThrLeuPheSer65707580lielieGlyLyslieulieProThrAsnHisGinSerValSerAlaLeu859095SerlieCysAspLeuLeuSerlielieArgLiysGluValAlaAspSer100105110ValLeuSerAspAlalieCysArgPhelieuAspGlyLysUeuLysAsn115120125TyrArgGluTyrTyrLieuProTyrL;euGluAlaTrpLeuIjysAspGly130135140LysProGinliysThrAsnAsnSerAsplieGlyGinLieuValLiys145150155160TyrPheGluLeuSerGluArgAspPheAsnGlu工leLeuGlyGlySer165170175LieuAlaArgAsnAsnAlaGinlieLeuUeuIjeuProTyrPheCysAla180IBS190SerCysLysCysGinLeuIjeuLeuArgAspAlaValGinTyrGlu195200205GluGinTrpPheProPheLeuSerAlaGluAsnValArgSerGluLeu210215220lieSerProAsnSerGlyCysAspPheThrGlyAspTyrTyrGluArg225230235240LeuLysCysLyslieAlaGluTyrTtirAspTyrCysGluTyrTrpTyr245250255GinAlaGlyLeuAsnGilllieLysGinAlaGlyThrGlyAlaAspThr260265270TrpAlaIjysPheAsnLysPheArgArgGluMetThrLeuThrValUeu27528028SAsplielieAla工leGinThrTyrAspPheLysLysTyrProIjeu290295300Pro丁hrHisValGluLeuThrArgGlu工leTyrThrAspProValGly305310315320TyrSerSerGlyThrTyrSerTrpLieuLiysTyrTrpThrGlyAlaPhe325330335AsnThrLeuGluAlaAsnGlyThrArgGlyProGlyIjeuValThrTrp34034S350IjeuArgSerlieGlylieTyrAsnGluTyrValSerArgTyrPheSer355360365GlyTrpValGlyThrArgHisTyrGluAspTyrThrThrGlyAsnGly370375380AsnPheGinArgMetSerGlyThrThrSerAsnAspLeuArgAsplie38539039S400Sei:PheProAsnSerAspliePheLyslieGluSerLysAlalieMet405410415AsnLeuValGlyGlulieAsnAlaArgProGluTyrArgValSerArg420425430AlaGluPheSerGluSerThrAlaPhelieTyrLeuTyrAspAlaGly435440445AsnSerGlyLeuSerSerMetThrlieThrSerLysLeuProGlylie450455"0LysAsnProGluProSerTyrArgAspTyrSerHisArgLeuSerAsn470475480AlaAlaCysValGlyAlaGlyAsnSerArglieAsnValTyrGlyTrp4854904S5ThrHisThrSerMetSerIiysTyrAsnLeulieTyrProAspLyslie500505510Thr<51nneProAlaValLysAlaPheAsplieSerAspThrPro51SS20525GlyGinVallieAlaGlyProGlyHisThrGlyGlyAfinValValSerS30S35540lieuProTyrTyrSerArglieuUyslieArglieulieProAlaSerThr54555055S560AsnLysAsnTyrIjeuValArgValArgT^rThrSerThrSerAeaGlyS6557057SArgIjeuLeuValGluArgTrpSerProSerSerlielieAsnSerTyr580S8SS90PhePheLeuProSerThrGlyProAspPheGlyTyrValAsp59S600605ThrLeuVaiThrThrPheAsnGinProGlyValGlulielielieGin610615620AsnLeuAspThrProlieAsnValAspUysValGluPhelieProVal625630635640AsnSerThrAlaLieuGluTyrGluGlyLrysGinSerLeuGluLysAlaGinAspValValAsnAspIteuPheValLys660665<210;>52<211>558<212>PRT<21>苏云金芽孢杆菌<400>52MetPheThrAsnGlyThrLysAsnThrLeuIiyslieGluThrThrAsp151015GlulieAspGinAla'AlalieSer工leGluCysMetSerAspGlu202530HisSerProIiysGluLysMetMetIjeuTrpAspGluValLysArgAla354045LysIjeuIjeuSerGinSerArgAsnLieuLeuGinAsnGlyAspPheGly50S560AspPheTyrGlyAsnAspTrpLysPheGlyAanAsnlielielieGly65707580SerAanAsnSerliePheLysGlyAsnPhelieuGinMetSerGlyAla859095ArgAspUeTyrGlyThrliePheProThrTyrlieTyrGinLys工le100105110AspGluSerLysUeuLysProTyrThrArgTyrArgValGlyPhe11S120125ValSerSerIjysAspLeuArgUeuMetValThrArgTyrGlyLys130135140GlulieAspAlaMetMetAsnValProAsnAsplieuAlaTyrMetGin145150155160ProAsnProSerCysGlyAspSerArgCysGluSerSerSerGinTyr165170175"ValSerGinGlyTyrProThrProThrAspGlyTyrAlaProAspArg180185190TyrAlaCysProSerSerSerAspLysLysHisValMetCysHisAsp200205ArgHisProPheAspPheHislieAspThrGlyGluLeuAspThrAsn210215220ThrAsnValGlylieAspValLeuPheLyslieSerAsnProAspGly22S230235240TyrAlaThrLeuGlyAsnLeuGluVallieGluGluGlyProLeuThr2452502S5GlyGluAlaIjeuThrHisValLys(31nLysGluLysTrpLysGin260270HisMetGluLiysLysArgTrpGluThrGinGinAlaTyrAspProAla27S280285IiysGinAlaValAspThrPheThrAsnGluGinGluLeuHisTyr295300HislieThr乙euAspTyrlieGltiThrLeulieAspTrpTyrSerArg305310315320PheProlieTyrThrMetThrGlyTyrArgAspAlaProGlyMetAsn32S33033STyrAsp<31yTyrGin<31yLeuAsnAlaArglieMetGinAlaTyrAsn34034S350LeuTyrAspAlaArgAsnVallieThrAenGlyAspPheThrLys<3iy3SS360365LeuGinGlyTrpHisAlaAlaGlyLysAlaAlaValGinGlulieAsp370375380GlyAlaSerVallieuValIjeuSerAsnTrpSerAlaGlyValSerGin38S330400AsnlieuHisAlaGinAspHisHisGlyTyrMetlieuArgVallieAla405LysGluGlyProGlyLysGly420GlyAsnGinGluThrLeuLysPhe435440ThrliysThrValGluValPhePi:o4S0455lieGlyGluThrGluGlyThrPhe465470CysMetGinGlyTyrAlaSerAsn485TyrGlyGinSerTyrAsnGlyAsn500TyrHisGinGlyTyrThrAsnAsn515520TyrAsnGinAsnTyrThrHisAsn530535CysAsnGinGlyHisAsnSerGly545550410415TyrValThrMetMetAspCysAsn425430ThrSerCysGluGlu445GlyTyrMetGluSerAspArgValAxglieGluTyrValAsp475SerlieGluLeutjeu480AsnAsnProHisThrGly495MetTyrAsnGinAsnTh;rSerAspVal505510TyrAsnGinAsnSer525SerAsnMetAspAspLeuHis540SerGlyCysThrCysThrCysSerGinGly55权利要求1.选自下组的分离核酸分子a)含有SEQIDNO1、2、4、6、8、10、12、13、15、17、19、21、23、25、26、或28所示核苷酸序列的核酸分子；b)含有与SEQIDNO1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列具有至少95％序列同一性的核苷酸序列的核酸分子，其中所述核苷酸序列编码具有杀虫活性的多肽；c)编码含有SEQIDNO3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列的多肽的核酸分子；d)含有编码具有与SEQIDNO3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列有至少95％氨基酸序列同一性的多肽的核苷酸序列的核酸分子，其中所述多肽具有杀虫活性；和e)a)-d)中任一项的互补物。2.权利要求1的分离核酸分子，其中所述核苷酸序列是设计成在植物中表达的合成序列。3.权利要求2的核酸分子，其中所述合成序列具有升高的GC含量。4.含有权利要求1的核酸分子的载体。5.权利要求4的载体，其中还含有编码异源多肽的核酸分子。6.含有权利要求4的载体的宿主细胞。7.权利要求6的宿主细胞，它是细菌宿主细胞。8.权利要求6的宿主细胞，它是植物细胞。9.含有权利要求8的宿主细胞的转基因植物。10.权利要求9的转基因植物，其中所述植物选自下组玉米、高粱、小麦、向日葵、番茄、十字花科植物、胡椒属植物、马铃薯、棉花、稻、大豆、甜菜、甘蔗、烟草、大麦、和油籽油菜。,11.权利要求9的植物的转基因种子。12.选自下组的分离多肽a)含有SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列的多肽；b)由SEQIDNO:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列编码的多肽，其中所述多肽具有杀虫活性；c)含有与SEQIDNO:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列具有至少95%序列同一性的氨基酸序列的多肽，其中所述多肽具有杀虫活性；和d)由与SEQIDNO:l、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列至少95%相同的核苷酸序列编码的多肽。13.权利要求12的多肽，其中还含有异源氨基酸序列。14.选择性结合权利要求12的多肽的抗体。15.含有权利要求12的多肽的组合物。16.权利要求15的组合物，其中所述组合物选自下组粉剂、尘剂、丸剂、颗粒剂、喷雾剂、乳液、胶体和溶液。17.权利要求15的组合物，其中所述组合物是通过对苏云金芽孢杆菌细胞进行干燥、冻干、匀浆、抽提、过滤、离心、沉降、或浓缩而制备的。18.权利要求15的组合物，其中含有约1重量％至约99重量％的所述多肽。19，用于生产具有杀虫活性的多肽的方法，其中包括在编码所述多肽的核酸分子得到表达的条件下培养权利要求6的宿主细胞，所述多肽选自下组a)含有SEQIDN0:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列的多肽；b)由SEQIDN0:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列编码的多肽，其中所述多肽具有杀虫活性；c)含有与SEQIDN0:3、5、7、9、11、14、16、18、20、22、24、27或29所示氨基酸序列具有至少95%序列同一性的氨基酸序列的多肽，其中所述多肽具有杀虫活性；和d)由与SEQIDN0:1、2、4、6、8、10、12、13、15、17、19、21、23、25、26或28所示核苷酸序列至少95%相同的核苷酸序列编码的多肽。20.用于控制鳞翅类或鞘翅类害虫种群的方法，其中包括使所述种群接触杀虫有效量的权利要求12的多肽。21.用于杀死鳞翅类或鞘翅类害虫的方法，其中包括使所述害虫接触或给其喂食杀虫有效量的权利要求12的多肽。22.在其基因组中稳定掺入了DNA构建体的植物，其中所述DNA构建体含有编码具有杀虫活性的蛋白质的核苷酸序列，该核苷酸序列选自下组a)SEQIDN0:1、2、4、6、8、10、12、13、15、19、21、23、25、26、或28所示核苷酸序列；b)与SEQIDN0:1、2、4、6、8、10、12、13、15、19、21、23、25、26或28所示核苷酸序列具有至少95%序列同一性的核苷酸序列，其中所述核苷酸序列编码具有杀虫活性的多肽；c)编码含有SEQIDN0:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列的多肽的核苷酸序列；和d)编码与SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列具有至少95%氨基酸序列同一性的多肽的核苷酸序列，其中所述多肽具有杀虫活性；表达的启动子。23,权利要求22的植物，其中所述植物还含有SEQIDNO:17所示核苷酸序列。24.在其基因组中稳定掺入了DNA构建体的植物细胞，其中所述DNA构建体含有编码具有杀虫活性的蛋白质的核苷酸序列，该核苷酸序列选自下组a)SEQIDN0:1、2、4、6、8、10、12、13、15、19、21、23、25、26、或28所示核苷酸序列；b)与SEQIDNO:l、2、4、6、8、10、12、13、15、19、21、23、25、26或28所示核苷酸序列具有至少95%序列同一性的核苷酸序列，其中所述核苷酸序列编码具有杀虫活性的多肽；c)编码含有SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列的多肽的核苷酸序列；和d)编码与SEQIDNO:3、5、7、9、11、14、16、20、22、24、27或29所示氨基酸序列具有至少95%氨基酸序列同一性的多肽的核苷酸序列，其中所述多肽具有杀虫活性；中表达的启动4。"''—一25.权利要求24的植物细胞，其中所述植物细胞还含有SEQIDNO:17所示核苷酸序列。全文摘要本发明提供了赋予细菌、植物、植物细胞、组织和种子以杀虫活性的组合物和方法。本发明提供了含有δ-内毒素和δ-内毒素相关多肽的编码序列的组合物。所述编码序列可用于DNA构建体或表达盒中，以便用于植物和细菌中的转化和表达。组合物还包括被转化的细菌、植物、植物细胞、组织和种子。具体而言，本发明提供了分离的δ-内毒素和δ-内毒素相关核酸分子。另外，本发明还涵盖与多核苷酸对应的氨基酸序列。具体而言，本发明提供了含有编码SEQIDNO3、5、7、9、11、14、16、18、20、22、24、27和29中所示氨基酸序列的核苷酸序列和SEQIDNO1、2、4、6、8、10、12、13、15、17、19、21、23、25、26和28中所示核苷酸序列的分离的核酸分子及其变体和片段。文档编号C07K16/12GK101328484SQ20081013609公开日2008年12月24日申请日期2004年2月20日优先权日2003年2月20日发明者B·卡尔,M·G·科奇埃尔,N·B·达克,N·卡罗奇,T·哈吉斯申请人:阿则耐克斯公司