杀虫基因和使用方法与流程

相关申请的交叉引用本申请要求美国临时申请系列号62/095,524(提交于2014年12月22日)的权益，该申请的内容通过引用整体并入本文。发明领域本发明涉及用于控制害虫，特别是植物害虫的方法和组合物。对经由efs-web以文本文件提交的序列表的引用经由efs-web以ascii格式的序列表以电子方式提交序列表的正式副本，并使用名为agb006.pct_seqlist.txt的文件，其创建于2015年12月14日，大小为956kb，与本说明书同时提交。包含在该ascii格式文档中的序列表是说明书的一部分，其全部内容通过引用并入本文。发明背景害虫、植物病害和杂草是对作物的严重威胁。由于病虫害造成的损失估计为全球农业生产的37％，其中13％是由昆虫、细菌和其它生物导致的。毒素是毒力决定因素，其在微生物致病性和/或逃避宿主免疫应答中发挥重要作用。来自革兰氏阳性菌芽孢杆菌，特别是苏云金芽孢杆菌(bacillusthuringensis)的毒素已被用作杀虫蛋白(通常称为bt毒素)。目前的策略是使用表达这些毒素的基因来生产转基因作物。表达杀虫蛋白毒素的转基因作物用于对抗昆虫造成的作物损害。虽然使用芽孢杆菌毒素已经能成功地控制昆虫，但是在世界许多已经密集使用了这些毒素的区域的某些目标害虫中已经发展出对bt毒素的抗性。解决这个问题的一个方法是将bt作物和常规非bt作物(庇护)交替排列播种。避免或减缓发展出昆虫抗性的另一种方法是在转基因植物中堆叠对昆虫有不同作用模式的杀虫基因。使用表达杀虫蛋白毒素的转基因作物的策略目前越来越强调新型毒素的发现，超出从苏云金芽孢杆菌细菌中产生的毒素之外。新型毒素可能被证明是可有效替代那些衍生自苏云金芽孢杆菌的那些毒素，用于开发昆虫和害虫抗性转基因植物。因此，需要新型毒素蛋白。附图说明图1提供seqidno：8、9、123和124的氨基酸比对。突出显示的区域表示在四种多肽之间氨基酸不同的区域。图2提供seqidno：25、26、125和126的氨基酸比对。突出显示的区域表示在四种多肽之间氨基酸不同的区域。图3提供seqidno：72、94、127和128的氨基酸比对。突出显示的区域表示在四种多肽之间氨基酸不同的区域。图4提供seqidno：70、71、129、130和131的氨基酸比对。突出显示的区域表示在五种多肽之间氨基酸不同的区域。图5a-5h提供seqidno：6、7、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和159的氨基酸比对。突出显示的区域表示每个多肽保守的区域。存在于该比对中的保守区域列于表4中。图6提供seqidno：6和132-159各自的序列同一性百分比关系。图7提供了对西方玉米根虫的生物测定中使用的分析评分指标(大小x死亡率矩阵)。发明概述提供了具有杀虫活性的组合物及其使用方法。组合物包括具有杀虫活性的分离的和重组的多肽序列、编码所述杀虫多肽的重组的和合成的核酸分子、包含所述核酸分子的dna构建体、包含所述核酸分子的载体、包含所述载体的宿主细胞，以及针对所述杀虫多肽的抗体。编码本文提供的多肽的多核苷酸序列可用于dna构建体或表达盒中，用于在目的生物(包括微生物和植物)中转化和表达。本文提供的组合物和方法可用于生产具有增强的害虫抗性或耐受性的生物。这些生物和包含这些生物的组合物理想地用于农业目的。还提供了包含编码本发明的杀虫蛋白的核苷酸序列的转基因植物和种子。这些植物对昆虫和其他害虫具有抗性。提供了用于产生本文公开的多种多肽的方法，以及使用那些多肽来控制或杀灭害虫的方法。还包括用于检测样品中本发明多肽的方法和试剂盒。发明详述现在将参考附图更全面地描述本发明，其中示出了本发明的一些但不是全部的实施方式。实际上，这些发明可以以许多不同的形式实施，并且不应被解释为限于本文所阐述的实施方式；相反地，提供这些实施方式，使得本公开满足适用的法律要求。通篇中相同的数字都指代相同元素。本发明相关
技术领域：
的技术人员将会认识到本文提供的发明的许多修饰和其它实施方式，这些发明具有在前文描述和相关附图教导中得到益处。因此，应当理解，本发明不限于所公开的特定实施方式，且修饰和其它实施方式旨在被包括在所附权利要求的范围内。尽管这里采用了具体的术语，但它们仅在通用和描述性意义上使用，而不是为了限制的目的。i.多核苷酸和多肽提供了赋予生物以杀虫活性的组合物和方法。经修饰的生物显示对害虫的抗性或耐受性。提供了保留杀虫活性的重组杀虫蛋白或多肽及其片段和变体，包括序列seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的那些。杀虫蛋白对包括昆虫、真菌、线虫等的害虫具有生物学活性(例如，是杀虫的)。可以使用编码杀虫多肽包括例如seqidno：1-159的多核苷酸，或者其活性片段或变体，以产生转基因生物，如植物和微生物。转化的生物的特征在于基因组包含至少一种稳定掺入的dna构建体，其包含本文公开的杀虫蛋白的编码序列。在一些实施方案中，编码序列可操作地连接至驱动所编码的杀虫多肽表达的启动子。因此，提供了转化的微生物、植物细胞、植物组织、植物、种子和植物部分。各种多肽、其活性变体和片段及编码其的多核苷酸的总结于下文表1。如表1所示，提供了各种形式的多肽。提供全长杀虫多肽，以及原始全长序列的修饰版本(称为变体)。表1还表示“crybp1”序列。这些序列(seqidno：24、67和73)包含可与一些毒素基因相关的辅助多肽。在这种情况下，crybp1序列可以单独使用或与本文提供的任何杀虫多肽组合使用。表1还提供了分裂-cryc'-末端多肽(seqidno：3、74、77、80和91)。这些序列包括与cry类型毒素基因的c'-末端具有同源性的下游蛋白的序列，这些序列通常在非全长并缺少预期的c'-末端区域的cry基因后面。i.杀虫蛋白分类本文提供的杀虫蛋白和编码其的核苷酸序列可用于影响害虫的方法。换言之，本发明的组合物和方法可用于农业以控制或杀灭害虫，包括许多作物植物的害虫。本文提供的杀虫蛋白是来自细菌的毒素蛋白，并且显示针对某些害虫的活性。杀虫蛋白来自几类毒素，包括cry、cyt、bin、mtx毒素。本文提供的各种seqidno的特定蛋白质分类参见例如表1。此外，在本公开内容中还参考pfam数据库条目。pfam数据库是蛋白质家族的数据库，每个家族都由多个序列比对和特征隐马尔科夫模型表示。finn等人(2014)nucl.acidres.数据库第42期:d222-d230.苏云金芽孢杆菌(bt)是一种革兰氏阳性菌，其在生长的孢子生长阶段产生晶体夹杂物形式的杀虫蛋白。bt的蛋白质性质的内含物称为晶体蛋白或δ-内毒素(或cry蛋白)，对昆虫纲和其他无脊椎动物的成员有毒。类似地，cyt蛋白是来自bt的伴胞包涵体蛋白，其表现出溶血性(细胞溶解)活性或与已知的cyt蛋白具有明显的序列相似性。这些毒素对其靶生物具有高度的特异性，对人类、脊椎动物和植物无害。cry毒素的结构显示有五个保守的氨基酸嵌段，主要集中在结构域的中心或结构域之间的连接处。cry毒素由三个结构域组成，每个结构域具有特定的功能。结构域i是七个α螺旋的束，其中中心螺旋被六个外螺旋完全包围。该结构域涉及在膜中的通道形成。结构域ii表现为三个反平行β-折叠片的三角柱，其与免疫球蛋白的抗原结合区域相似。结构域iii含有β-夹心形式的反平行β-链。毒素蛋白的n-末端部分负责其毒性和特异性，并含有五个保守区域。c末端部分通常是高度保守的，可能负责晶体形成。参见，例如，美国专利号8,878,007。苏云金芽孢杆菌菌株对不同的昆虫目(鳞翅目、双翅目、鞘翅目、膜翅目、同翅目、虱目或食毛目和螨)及其他无脊椎动物(线形动物门、扁形动物门和sarocomastebrates)具有广泛的特异性。基于对各种昆虫和无脊椎动物群体的毒性，cry蛋白质已被分类成不同的组。通常，cryi蛋白显示对鳞翅目的毒性，cryii蛋白显示对鳞翅目和双翅目的毒性，cryiii蛋白显示对鞘翅目的毒性、cryiv蛋白显示对双翅目的毒性，并且cryv和cryvi蛋白显示对线虫的毒性。基于氨基酸同一性，可以鉴定新的cry蛋白并将其分配到某个cry组。参见例如bravo,a.(1997)j.ofbacteriol.179:2793-2801；bravo等人(2013)microb.biotechnol.6:17-26，通过引用并入本文。超过750种不同的cry基因序列被分类为73组(cry1-cry73)，同时该基因家族的新成员持续被发现(crickmore等人(2014)www.btnomenclature.info/)。cry基因家族由几种可能具有不同作用模式的系统发育非相关蛋白质家族组成：三结构域cry毒素家族、灭蚊cry毒素家族、二元样毒素家族和cyt毒素家族(bravo等，2005)。一些bt菌株产生称为vip毒素的其他杀虫毒素。也可参见cohen等人(2011)j.mol.biol.413:4-814；crickmore等人(2014)bacillusthuringiensistoxinnomenclature，可在万维网上找到，网址为lifesci.sussex.ac.uk/home/neil_crickmore/bt/；crickmore等人(1988)microbiol.mol.biol.rev.62:807-813；gill等人(1992)ann.rev.entomol.37:807-636；goldbert等人(1997)appl.environ.microbiol.63:2716-2712；knowles等人(1992)proc.r.soc.ser.b.248:1-7；koni等人(1994)microbiology140:1869-1880；lailak等人(2013)biochem.biophys.res.commun.435:216-221；lopez-diaz等人(2013)environ.microbiol.15:3030-3039；perez等人(2007)cell.microbiol.9:2931-2937；promdonkoy等人(2003)biochem.j.374:255-259；rigden(2009)febslett.583:1555-1560；schnepf等人(1998)microbiol.mol.biol.rev.62:775-806；soberon等人(2013)peptides41:87-93；thiery等人(1998)j.am.mosq.controlassoc.14:472-476；thomas等人(1983)febslett.154:362-368；wirth等人(1997)proc.natl.acad.sci.u.s.a.94:10536-10540；wirth等人(2005)appl.environ.microbiol.71:185-189；以及zhang等人(2006)biosci.biotechnol.biochem.70:2199-2204；其各自全部内容通过引用并入本文。cyt表示来自苏云金芽孢杆菌的具有溶细胞活性的伴胞结晶内涵体蛋白，或与已知cyt蛋白具有序列相似性的蛋白质。(crickmore等人(1998)microbiol.mol.biol.rev.62：807-813)。该基因记为cyt。这些蛋白质与cry蛋白质的结构和活性不同(gill等(1992)annu.rev.entomol.37：615-636)。cyt毒素首先在苏云金芽孢杆菌以色列亚种中发现(goldberg等人(1977年)，mosq.news.37：355-358)。目前的命名法有3种cyt毒素家族，包括11种全型毒素(crickmore等人(2014)bacillusthuringiensistoxinnomenclature，可在万维网上找到，网址为lifesci.sussex.ac.uk/home/neil_crickmore/bt/)。大多数具有cyt基因的苏云金芽孢杆菌分离株表现出对双翅目昆虫(特别是蚊子和黑蝇)的活性，但也有苏云金芽孢杆菌菌株中描述的cyt基因针对的是鳞翅目昆虫或鞘翅目昆虫(guerchicoff等人(1997)appl.environ.microbiol.33：2716-2721)。通过x射线晶体学解析的cyt2a的结构显示了一个单结构域，其中两个外层α-螺旋缠绕在混合的β-折叠上。cyt毒素的其他可得到的晶体结构支持保守的α-β结构模型，其中两个α-螺旋发夹在含有七至八个β-链的β-折叠核心的两侧。(cohen等人(2011)。j.mol.biol.413:804-814)。诱变研究鉴定β折叠残基对于毒性是关键的，而在螺旋结构域中的突变不影响毒性(adang等人；diversityofbacillusthuringiensiscrystaltoxinsandmechanismofaction.在t.s.dhadialla和s.s.gill,eds,advancesininsectphysiology,vol.47,oxford:academicpress,2014,pp.39-87中)。cyt毒素的代表性结构域是δ-内毒素，bac_thur_toxin(pfampf01338)。对cyt毒素的作用模式提出了多种模型，这仍然是一个研究活跃的领域。已经显示一些cyt蛋白(cyt1a)需要存在辅助蛋白质用于结晶。cyt1a和cyt2a原毒素在n和c末端的相同位点受到消化蛋白酶处理形成稳定的毒素核心。然后cyt毒素与非饱和膜脂质如磷脂酰胆碱、磷脂酰乙醇胺和鞘磷脂相互作用。对于cyt毒素，已经提出了孔形成和去污剂样膜破坏作用作为非排他性机制；并且通常认为二者都依赖于毒素浓度而发生，较低浓度有利于低聚化的孔，而和较高的浓度导致膜破裂。(butko(2003)appl.environ.microbiol.69:2415-2422)。在孔形成模型中，cyt毒素与细胞膜结合，诱导在膜囊泡中形成阳离子选择性通道，导致细胞的胶体渗透裂解。(knowles等人(1989)febslett.244:259-262；knowles等人(1992)proc.r.soc.ser.b.248:1-7andpromdonkoy等人(2003)biochem.j.374:255-259)。在去污剂模型中，脂质双分子层表面会有毒素的非特异性聚集，导致膜解体和细胞死亡。(butko(2003)见上文；manceva等人(2005)biochem.44:589-597)。多重研究显示cyt毒素和其他苏云金芽孢杆菌毒素，特别是cry、bin和mtx毒素之间有协同的活性。这种协同作用甚至被证明可以克服昆虫对所述其他毒素的抵抗力(wirth1997、wirth2005、thiery1998、zhang2006)。研究者提出cyt对cry毒素的协同效应涉及cyt1a在溶液中或膜表面上结合cry毒素的结构域ii，以促进cry毒素前体-孔寡聚体的形成。该寡聚体的形成不依赖于与cyt的寡聚化、结合或插入(lailak2013、perez2007、lopez-diaz2013)。一些苏云金芽孢杆菌和蜡状芽孢杆菌(b.cereus)菌株质在营养生长期会产生大量与cry蛋白不相关的杀虫蛋白(estruch等人(1996)procnatlacadsciusa93：5389-5394；warren等人(1994)wo94/21795)。这些营养性杀虫蛋白或vip不形成伴胞晶体蛋白，并且显然会从细胞中分泌出来。vip目前被排除在cry蛋白质命名之外，因为它们不是晶体形成蛋白质。术语vip是一种误称，因为一些苏云金芽孢杆菌cry蛋白质也会在营养生长期以及在静止期和孢子形成期中产生，最著名的是cry3aa。据报道，苏云金芽孢杆菌基因组中的vip基因定位在也编码cry基因的大质粒上(mesrati等人(2005)femsmicrobiol.lett.244(2)：353-8)。可以在万维网上找到bt毒素命名法的网站，网址为lifesci.sussex.ac.uk，路径为“/home/neil_crickmore/bt/”，也可在“btnomenclature.info/”这一网站获得。另见schnepf等人(1998)microbiol.mol.biol.rev.62(3):775-806。这些参考文献通过引用并入本文。迄今为止，已经确定了四个vip分类。一些vip基因形成二元双组分蛋白质复合物；其中“a”组分通常是“活性”部分，“b”组分通常是“结合”部分(pfam_pfam.xfam.org/family/pf03495)。vip1和vip4蛋白通常含有二元毒素b蛋白质结构域。vip2蛋白通常含有二元毒素a蛋白质结构域。vip1和vip2蛋白是显示对鞘翅目有毒性的二元毒素的两个组分。vip1aa1和vip2aa1针对玉米根虫，尤其是玉米根叶甲(diabroticavirgifera)和叶甲蜱(diabroticalongicornis)有非常强的活性(han等人(1999)nat.struct.biol.6:932–936；warrengw(1997)“vegetativeinsecticidalproteins:novelproteinsforcontrolofcornpests”，carozzinb,kozielm(eds)advancesininsectcontrol,theroleoftransgenicplants；taylor&francisltd,london,pp109–21)。膜结合的95kdavip1多聚体为52kda的vip2adp-核糖基化酶进入靶标西方玉米根虫细胞的细胞质提供了途径(warren(1997)，见上文)。所述nad依赖的adp-核糖基化酶vip2很可能在arg177处修饰单体肌动蛋白而阻断多聚化，导致肌动蛋白细胞骨架的丢失并且最终由于体内肌动蛋白纤维内的快速亚单位交换而造成细胞死亡(carlierm.f.(1990)adv.biophys.26:51-73)。像cry毒素一样，活化的vip3a毒素是能够在膜中形成稳定离子通道的成孔蛋白(lee等人(2003)appl.environ.microbiol.69：4648-4657)。vip3蛋白对几种主要的鳞翅目害虫具有活性(rang等人(2005)appl.environ.microbiol.71(10)：6276-6281；bhalla等(2005)femsmicrobiol.lett.243：467-472；estruch等人(1998)wo9844137；estruch等人(1996)procnatlacadsciusa93：5389-5394；selvapandiyan等人(2001)appl.environmicrobiol.67：5855-5858；yu等人(1997)appl.environmicrobiol.63：532-536)。vip3a对小地老虎(agrotisipsilon)、草地贪夜蛾(spodopterafrugiperda)、甜菜夜蛾(spodopteraexigua)、烟芽夜蛾(heliothisvirescens)和玉米穗虫(helicoverpazea)有活性(warren等人(1996)wo96/10083；estruch等人(1996)procnatlacadsciusa93：5389-5394)。像cry毒素一样，vip3a蛋白质必须先被蛋白酶活化，才能在中肠上皮表面识别cry毒素不能识别的特定膜蛋白。毒素蛋白质的mtx家族的特征在于存在保守结构域，etx_mtx2(pfam03318)。该家族的成员与来自球形芽孢杆菌(bacillussphaericus)的灭蚊毒素mtx2和mtx3，以及与来自产气荚膜梭菌(clostridiumperfringens)的ε毒素etx(cole等人(2004)nat.struct.mol.biol.11:797-8；thanabalu等人(1996)gene170:85-9)具有序列相似性。mtx样蛋白在结构上不同于三结构域的cry毒素，因为它们具有细长且占主要的基于β-折叠的结构。然而，类似于三结构域的毒素，mtx样蛋白被认为在靶细胞的膜中形成孔(adang等人(2014)，见上文)。与三结构域的cry蛋白不同，mtx样蛋白长度上短很多，在267个氨基酸(cry23)到340个氨基酸(cry15a)之间。迄今为止，只对15种属于mtx样毒素家族的蛋白质指了cry名字，使其与三结构的cry家族相比成为相对小的一类蛋白(crickmore等人(2014)，见上文，adang等(2014)，见上文)。mtx样毒素家族的成员包括cry15、cry23、cry33、cry38、cry45、cry46、cry51、cry60a、cry60b和cry64。该家族具有一系列杀虫活性，包括对鳞翅目和鞘翅目昆虫害虫的活性。该家族的一些成员可能与其它蛋白质形成二元伙伴关系，杀虫活性可能需要或不需要这种关系。cry15是苏云金芽孢杆菌血清型thompsonihd542中鉴定的34kda的蛋白质。cry15天然地与一种约40kda的不相关蛋白一起存在于晶体中。编码cry15及其伙伴蛋白的基因被一同排列在操纵子中。已经证明单独的cry15具有针对鳞翅目昆虫害虫的活性，包括烟草天蛾(manducasexta)、苹果蠹蛾(cydiapomonella)和菜粉蝶(pierisrapae)，所述40kda蛋白质的存在已被证明仅增加cry15对苹果蠹蛾的活性(brownk.和whiteleyh.(1992)j.bacteriol.174：549-557；naimov等人(2008)appl.environ.microbiol.74：7145-7151)。需要进一步的研究来阐明cry15的伙伴蛋白的功能。类似地，cry23是一种29kda的蛋白质，已被证明与其伙伴蛋白cry37一起时具有针对鞘翅目害虫赤拟谷盗(triboliumcastaneum)和日本金龟子(popilliajaponica)的活性(donovan等人(2000)美国专利号6,063,756)。mtx样家族的新成员正在持续被鉴定出来。最近在苏云金芽孢杆菌血清型索奈氏菌株na205-3基因组中鉴定出etx_mtx毒素基因。发现该菌株对鳞翅目害虫棉铃虫(helicoverpaarmigera)具有毒性，并且还含有cry1、cry11、vip1、vip2和vip3的同源物(palma等(2014)genomeannounc.2(2)：e00187-14。在线发表于2014年3月13日，doi：10.1128/genomea.00187-14；pmcid：pmc3953196)。由于mtx样蛋白具有相对于三结构域cry蛋白的独特结构域结构，因此被认为具有独特的作用模式，从而使它们成为昆虫控制和对抗昆虫抗药性的有价值的工具。细菌细胞产生大量对宿主和非宿主生物具有多样特异性的毒素。已经在许多细菌家族中鉴定出大的二元毒素家族，包括具有抗昆虫害虫活性的毒素。(poopathi和abidha(2010)j.physiol.path.1(3):22-38)。球形赖氨酸芽孢杆菌(lysinibacillussphaericus，ls)，即以前的球形芽孢杆菌(bacillussphaericus，ls)，(ahmed等人(2007)int.j.syst.evol.microbiol.57：1117-1125)是众所周知的用于昆虫生物防治的菌株。ls产生数种杀虫蛋白，包括高效二元复合物bina/binb。这种二元复合物在ls细胞中形成伴胞晶体，并且具有针对双翅目昆虫的强且特异的活性，特别针对蚊子。在某些地区，已经报道了昆虫对现有的ls灭蚊菌株的抗性。具有不同靶特异性或克服昆虫抗性的能力的新二元毒素的发现是非常有意义的。ls二元杀虫蛋白复合物含有两个主要多肽，即42kda多肽和51kda多肽，分别命名为bina和binb(ahmed等(2007)，见上文)。这两种多肽协同作用对其靶标产生毒性。作用方式涉及所述蛋白质与幼虫中肠的受体结合。在一些情况下，所述蛋白质在幼虫肠中经过蛋白酶的消化得到修饰而产生活化形式。认为binb组分参与结合，而bina组分赋予毒性(nielsen-leroux等人(2001)appl.environ.microbiol.67(11)：5049-5054)。当单独克隆和表达时，bina组分对蚊子幼虫有毒，而binb组分不具有毒性。然而，两种蛋白质的共施用显著增加了毒性(nielsen-leroux等人(2001)，见上文)。也描述了细菌来源的少数bin蛋白质同源物。priest等人(1997)appl.environ.microbiol.63(4):1195-1198描述尝试使用杂交方法鉴定新的ls菌株，尽管他们鉴定的大多数基因都编码的是与已知的bina/binb蛋白质相同的蛋白质。bina蛋白含有被称为toxin10超家族结构域的已经确定的保守结构域。这种毒素结构域最初由其在bina和binb中的存在而得到确定。所述两种蛋白质都具有这种结构域，尽管bina和binb之间的序列相似性在该区域是有限的(<40％)。cry49aa蛋白也具有杀虫活性，其具有该结构域(如下所述)。ls的cry48aa/cry49aa二元毒素具有杀灭致倦库蚊(culexquinquefasciatus)蚊子幼虫的能力。这些蛋白质是在结构上与苏云金芽孢杆菌(bt)cry蛋白复合物有一定相似性。已知bt的cry34/cry35二元毒素也能杀灭昆虫，包括西方玉米根虫——一种重要的玉米害虫。cry34是一种小(14kda)多肽，已经鉴定了其的几种变体；而cry35(也由若干变体编码)是44kda的多肽。这些蛋白质与bina/binb蛋白质组具有一些序列同源性，并被认为是进化相关的(ellis等人(2002)appl.environ.microbiol.68(3)：1137-1145)。本文提供了来自这些毒素类别的杀虫蛋白。根据其结构、与已知毒素的同源性和/或其杀虫特异性对这些杀虫蛋白其进行分类。ii.杀虫蛋白和编码其的多核苷酸的变体和片段本发明的杀虫蛋白或多肽包括seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159及其片段和变体所示的那些。“杀虫毒素”或“杀虫蛋白”或“杀虫多肽”是指这样的毒素或蛋白质或多肽，其具有针对一种或多种害虫(包括昆虫、真菌，线虫等)的活性，致使害虫被杀灭或控制。“分离的”或“纯化的”多肽或蛋白质，或其生物学活性部分大体上或基本上不含在其天然存在的环境中发现时通常与所述多肽或蛋白质相伴随或相互作用的组分。因此，当通过重组技术产生时，分离的或纯化的多肽或蛋白质大体上不含其它细胞材料或培养基，或者当其为化学合成时，大体上不含化学前体或其它化学物质。大体上不含细胞材料的蛋白质包括这样的蛋白质制备物，其具有小于约30％、20％、10％、5％或1％(以干重计)的污染性蛋白质。当本发明的蛋白质或其生物学活性部分是通过重组产生时，最佳的情况下，培养基中有小于约30％、20％、10％、5％或1％(以干重计)的化学前体或非感兴趣蛋白质的化学物。术语“片段”指代本发明的多肽序列的部分。“片段”或“生物学活性部分”包括包含足够数量的连续氨基酸残基以保持生物学活性(具有杀虫活性)的多肽。杀虫蛋白的片段包括短于全长序列的那些，可以是由于使用了替代的下游起始位点而形成，或由于加工过程产生具有杀虫活性的较短蛋白质而形成。加工过程可以发生在表达蛋白质的生物中或在该蛋白质被摄入后的害虫体内。蛋白质的片段的实例可以在表1中找到。杀虫蛋白的生物学活性部分可以是多肽，例如，seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的任一的长度为10、25、50、100、150、200、250或更多个氨基酸的多肽。可以通过重组技术制备这些生物学活性部分并评估其杀虫活性。如本文所用，片段包含seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的至少8个连续的氨基酸。细菌基因，包括编码本文公开的杀虫蛋白的那些基因经常在开放阅读框的起始附近具有多个甲硫氨酸起始密码子。通常，在这些起始密码子中的一个或多个处的翻译启动将会产生功能性蛋白质。这些起始密码子可以包括atg密码子。然而，细菌如芽孢杆菌属也将密码子gtg识别为起始密码子，在gtg密码子处启动翻译的蛋白质包含在第一个氨基酸处的甲硫氨酸。在极少数情况下，在ttg密码子处启动细菌系统的翻译，而在这种情况下ttg编码甲硫氨酸。此外，先前并不会常常确定哪些密码子在细菌中是天然使用的。从而，应当理解，使用一个替代甲硫氨酸密码子也可以产生杀虫蛋白。这些杀虫蛋白包括在本发明中并且可以用于本文公开的方法。应当理解，当在植物中表达时，有必要将替代的起始密码子改变为atg以进行适当的翻译。在多种实施方案中，本文提供的杀虫蛋白包括从全长核苷酸序列推导出的氨基酸序列以及由于使用替代的下游起始位点生成的短于全长序列的氨基酸序列。因此，本发明的核苷酸序列和/或包含本发明的核苷酸序列的载体、宿主细胞和植物(以及制备和使用本发明的核苷酸序列的方法)可以包含编码替代起始位点的核苷酸序列。认为可以对本文提供的杀虫多肽进行修饰而产生变体蛋白质。可以通过应用定点诱变技术引入人为设计的改变。备选地，也可以鉴定出处于本发明范围内的天然的、尚未知的或尚未鉴定的与本文公开的序列在结构上和/或在功能上相关的多核苷酸和/或多肽。可以在非保守区域中进行保守氨基酸置换而不改变杀虫蛋白功能。备选地，可以进行修饰以改进毒素活性。例如，多种cry蛋白变体也在预期之内。通过结构域iii互换(swapping)对cry毒素的修饰已经在一些情况下产生出一些杂合毒素，其对某些昆虫物种的毒性有所改进。因此，结构域iii互换可以是改进cry毒素的毒性或产生针对那些对亲本cry毒素不敏感的害虫有毒性的新型杂合毒素的有效策略。结构域ii环序列的定点诱变可以产生具有增加的杀虫活性的新毒素。结构域ii环区是初始cry毒素的关键结合区，其是用于诱变和选择具有改进的杀虫性质的cry毒素的合适靶标。可以修饰cry毒素的结构域i以引入蛋白酶裂解位点而改进针对某些害虫的活性。在大量cry基因之间交换(shuffling)这三个不同的结构域的策略和高通量生物测定筛选方法可提供具有改进的或新型毒性的新cry毒素。如所指出的，本文公开的多肽的片段和变体将保留杀虫活性。杀虫活性包括组合物达到减少目标害虫的发生或活性的可观察的效应的能力，包括例如导致至少一种有害生物的死亡，或有害生物生长、饲养或正常生理发育的显著减少。所述数量、有害生物生长、饲养或正常发育的降低可以包括任何统计学显著的降低，包括例如降低约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、85％、90％、95％或以上。本文提供的对一种或多种不同害虫的杀虫活性，包括例如对鞘翅目、双翅目、膜翅目、鳞翅目、羽虱目、同翅目、半翅目、直翅目、线虫、缨尾目、革翅目、等翅目、虱目、蚤目、毛翅目等，或本文所述的任何其他害虫的杀虫活性。人们认识到，其杀虫活性相对于天然蛋白质的活性可能不同或改进，或者可以不变，只要保留杀虫活性就行。杀虫活性测定方法在本文另有提供。另参见czapla和lang(1990)j.econ.entomol.83:2480-2485；andrews等人(1988)biochem.j.252:199-206；marrone等人(1985)j.ofeconomicentomology78:290-293；和u.s.pat.no.5,743,477，其全部全部内容通过引用并入本文。本公开的变体包括具有与seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的任一氨基酸序列具有至少约60％、约65％、约70％、约75％、约80％、约85％、约86％、约87％、约88％、约89％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的氨基酸序列并保留杀虫活性的多肽。表1提供了seqidno：1-159中每一个的变体多肽(及编码其的多核苷酸)的非限制性实例。本文提供的杀虫多肽的生物学活性变体有低至约1-15个氨基酸残基、低至约1-10个，例如约6-10个、低至5个、低至4个，如低至3个、低至2个，或低至1个氨基酸残基的差异。在具体实施方案中，多肽可以包含n’末端或c’末端截短，其可以包含在多肽的n’末端或c’末端至少10、15、20、25、30、35、40、45、50或更多个氨基酸的删除。表2提供了基于pfam数据在seqidno：1-159中发现的蛋白质结构域。表2中提供了结构域描述和在给定seqidno中的位置。在具体实施方案中，包含seqidno：1-159的任一的活性变体可以包含与seqidno：1-159的任一至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、94％、95％、96％、97％、98％或99％的序列同一性，并且还包含表2所示的至少一个保守结构域。例如在一个实施方案中，活性变体包含与seqidno：15至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且还包含78-329位的天然氨基酸、554-708位的天然氨基酸，或包括78-329位及554-708位的天然氨基酸。表2.seqidno：1-159各自序列中的pfam结构域的概述seqidno：8的变体包含seqidno：9、123和124。图1提供了seqidno：8、9、123和124的氨基酸序列比对，表3提供了seqidno：9、123和124的修饰类型及其与seqidno：8共有的序列同一性百分比。seqidno：25的变体包含seqidno：26、125和126。图2提供了seqidno：25、26、125和126的氨基酸序列比对，表3提供了seqidno：26、125和126的修饰类型及其与seqidno：25共有的序列同一性百分比。seqidno：72的变体包含seqidno：94、127和128。图3提供了seqidno：72、94、127和128的氨基酸序列比对，表3提供了seqidno：94、127和128的修饰类型及其与seqidno：72共有的序列同一性百分比。seqidno：70的变体包含seqidno：71、129、130和131。图4提供了seqidno：70、71、129、130和131的氨基酸序列比对。表3提供了seqidno：71、129、130和131的修饰类型及其与seqidno：70共有的序列同一性百分比。表3.seqidno:8、25、72和70的变体概述如图5a-5h所述，seqidno：6、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和159各自都共有共同基序，如下表4所示。图5a-5h表示每个保守序列在seqidno：6和132-159序列中的位置。表4.seqidno：6和132-159中的保守区域保守序列seqidno:icsingsakfdpntn161nsqagaiagkta162igsatgaann163plnyepiglkatd164vpviddgwengdp165edeenalngkwvf166dkhvaiykqve167在具体实施方案中，包含seqidno：6或132-159的活性变体包含与seqidno：6和132-159的任一至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且还包含表4所示的保守结构域中的至少一个。例如，在一个实施方案中，活性变体可包含与seqidno：6和132-159至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且还包含表4所示的一个或多个氨基酸结构域(即seqidno：161、162、163、164、165、166和/或167的至少一个)。在一个非限制性实施方案中，活性变体包含与seqidno：6和132-159至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，并且还包含seqidno:166。在另一个实施方案中，seqidno：6和132-159的活性变体包含与seqidno：6或132-159至少70％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的序列同一性，其中所述活性变体不是seqidno:160。图6提供了seqidno：6、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和159之间的序列同一性百分比关系。还提供了编码本文公开的杀虫多肽的重组或合成核酸。特别目的是已被设计用于在目的植物中表达的核酸序列。例如，可以优化核酸序列以在宿主植物中增加表达。本文提供的杀虫蛋白可以反向翻译以产生包含优化用于在特定宿主例如作物植物中表达的密码子的核酸。在另一个实施方案中，可以优化编码本文提供的多肽的多核苷酸，以增加在转化植物中的表达。例如，可以使用植物优选的密码子来合成多核苷酸以改进表达。参见，例如，campbell和gowri(1990)plantphysiol.92：1-11讨论宿主优选密码子使用情况。用于合成植物优选基因的方法是本领域已知的。参见，例如，美国专利号5,380,831和5,436,391，及murray等人(1989)nucleicacidsres.17：477-498，其各自通过引用并入本文。由转化的植物(例如双子叶植物或单子叶植物)表达这样的编码序列将产生杀虫多肽并赋予植物对害虫增加的抗性。编码本发明的杀虫蛋白的重组和合成核酸分子不包括编码所述蛋白质的天然存在的细菌序列。“重组多核苷酸”或“重组核酸”包含两个或多个化学连接的核酸片段的组合，天然状态下不存在这些核酸片段的直接连接。“直接连接”是指两个核酸片段通过化学键直接相邻并且彼此连接。在具体实施方案中，重组多核苷酸包含目的多核苷酸或者其变体或片段，从而另外的化学连接的核酸片段位于目的多核苷酸的5'、3'端或内部。备选地，可以通过序列的删除而形成重组多核苷酸的化学连接的核酸片段。所述另外的化学连接的核酸片段或所述被删除以将连接的核酸片段相连的序列可以是任何长度，包括例如1、2、3、4、5、6、7、8、9、10、15、20个核苷酸或更多。用于制备这种重组多核苷酸的多种方法包括化学合成，以及通过基因工程技术操作分离的多核苷酸片段。在具体实施方案中，重组多核苷酸可以包含重组dna序列或重组rna序列。“重组多核苷酸或核酸片段”包含两个或多个化学连接的氨基酸片段的组合中的至少一种，其在自然界中不直接连接。多核苷酸(rna或dna)的片段可以编码保留活性的蛋白质片段。在具体实施方案中，重组多核苷酸或重组多核苷酸构建体的片段包含两个或多个化学连接的或可操作地连接的核酸片段的至少一种连接形式，天然状态下这些核酸片段不直接连接。编码保留杀虫活性的多肽的生物学活性部分的多核苷酸的片段将编码seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的全长多肽中存在的至少25、30、40、50、60、70、75、80、90、100、110、120、125、130、130、140、150、160、170、175或180个连续氨基酸，或多至这些多肽的氨基酸总数。在具体实施方案中，这些多肽片段是活性片段。在一些实施方案中，多肽片段包含重组多肽片段。如本文所用，重组多肽的片段包含两个或多个化学连接的氨基酸片段的至少一种组合，天然状态下这些核酸片段不直接连接。本文所用的术语“变体”旨在表示大体相似的序列。对于多核苷酸，变体包含在天然多核苷酸内一个或多个内部位点处的一个或多个核苷酸的缺失和/或添加，和/或在天然多核苷酸内一个或多个位点处的一个或多个核苷酸的置换。如本文所用，“天然”多核苷酸或多肽分别包含天然存在的核苷酸序列或氨基酸序列。也可以通过比较由变体多核苷酸编码的多肽和由参比多核苷酸编码的多肽来评估本发明的特定多核苷酸(即参比多核苷酸)的变体，以确定二者之间的序列同一性百分比。因此，公开了例如分离的多核苷酸，其编码的多肽具有与seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的多肽给定的序列同一性百分比。可以使用本文别处描述的序列比对程序和参数来计算任何两个多肽之间的序列同一性百分比。当通过比较其编码的两个多肽共有的序列同一性百分比来评估本发明的任何给定的一对多核苷酸时，两个编码的多肽之间的序列同一性百分比为与seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159至少约40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高的序列同一性。在其他实施方案中，本文提供的多核苷酸的变体与天然序列相比差异为至少1、2、3、4、5、6、7、8、9、10个或更多个核苷酸。iii.序列比较本文所用的术语“同一性”或“同一性百分比”当用于特定一对的比对氨基酸序列时，是指通过计数比对中相同匹配的数目，将相同匹配的数目除以对齐序列的长度，获得的氨基酸序列同一性百分比。本文所用的术语“相似性”或“相似性百分比”当用于特定一对的比对氨基酸序列时，是指从比对中的每个氨基酸对的评分矩阵除以比对序列的长度获得的评分的总和。除非另有说明，同一性和相似性是通过needleman-wunsch全局比对和计分算法而计算的(needleman和wunsch(1970)j.mol.biol.48(3):443-453)，其由称为“needle”的程序来实现，该程序作为emboss软件包的一部分(rice,p.longden,i.andbleasby,a.,emboss:theeuropeanmolecularbiologyopensoftwaresuite,2000,trendsingenetics16,(6)pp.276-277,6.3.1版可从embnet网站(网址为embnet.org/resource/emboss和emboss.sourceforge.net)和其他来源获得，其中包括使用默认的缺口罚分和评分矩阵(eblosum62用于蛋白质，ednafull用于dna)。也可以使用等效的程序。通过“等效程序”旨在表示任何序列比较程序，在用于任意两个所讨论的序列时，生成的相同核苷酸残基匹配的比对及序列同一性百分比相比于由emboss的6.3.1版获得的对应比对是完全相同的。其他的数学算法在本领域中是已知的，可以用于两个序列的比较。参见，例如，karlin和altschul(1990)proc.natl.acad.sci.usa87:2264的算法，在karlin和altschul(1993)proc.natl.acad.sci.usa90:5873-5877中得到改进。该算法被并入altschul等人(1990)j.mol.biol.215:403的blast程序中。可以使用blastn程序进行blast核苷酸搜索，以获得与本发明的杀虫样核酸分子同源的核苷酸序列。可以使用blastp程序进行blast蛋白质检索，以获得与本发明的杀虫蛋白质分子同源的氨基酸序列。为了获得缺口比对进行比较，可以如altschul等人(1997)nucleicacidsres.25：3389所述方法使用gappedblast(在blast2.0中)。备选地，可使用psi-blast执行检测分子之间的远距离关系的迭代搜索。参见altschul等人(1997)，见上文。当使用blast、gappedblast和psi-blast程序时，可以使用相应程序的默认参数(例如，blastx和blastn)。比对也可以手动地通过目视检查进行。当使用限定的氨基酸置换矩阵(例如blosum62)、缺口存在罚分和缺口延伸罚分对两条序列比对进行相似性评分以达到这对序列可能得到的最高分时，这两条序列是“最佳比对的”。氨基酸置换矩阵及其在量化两条序列之间相似性上的用途在本领域是公知的，描述于例如dayhoff等人(1978)“amodelofevolutionarychangeinproteins”。还描述于“atlasofproteinsequenceandstructure,”vol.5,suppl.3(ed.m.o.dayhoff),pp.345-352.natl.biomed.res.found.,washington,d.c.及henikoff等人(1992)proc.natl.acad.sci.usa89:10915-10919。blosum62矩阵通常用作序列比对协议中的默认评分置换矩阵。在一个比对的序列中引入单个氨基酸缺口会有缺口存在罚分，在插入到已经打开的缺口的每个附加的空氨基酸位置会有缺口延伸罚分。比对被限定为每条序列在比对开始和结束时的氨基酸位置，其中一条或两条序列中会插入一个缺口或多个缺口，以达到最高可能得分。虽然可以手动完成最佳比对和评分，但是通过使用计算机实现的比对算法可有助于该过程的进行，例如gappedblast2.0，描述于altschul等人(1997)nucleicacidsres.25:3389-3402，该算法在国家生物技术信息网站(www.ncbi.nlm.nih.gov)向公众提供。可以使用例如psi-blast(可通过www.ncbi.nlm.nih.gov获得，由altschul等人(1997)nucleicacidsres.25：3389-3402描述)来得到包括多重比对在内的最佳比对。对于与参考序列最佳比对的氨基酸序列，氨基酸残基“对应于”在比对中该残基配对的参考序列中的位置。“位置”由基于其相对于n-末端的位置顺序地确定在参考序列中每个氨基酸的数字表示。例如，在seqidno：1中，1位为m、2位为a、3位为n等。当测试序列与seqidno：1得到最佳比对时，测试序列中与3位的n对齐的残基被称为“对应于seqidno：1的3位”。由于在确定最佳比对时必须考虑到删除、插入、截短、融合等等，一般来说，通过从n-末端简单计数确定的测试序列中的氨基酸残基数不一定与参考序列中其相应位置的数字相同。例如，在进行比对的测试序列中存在删除的情况下，参考序列在删除位点的位置将不存在相对应的氨基酸。在进行比对的参考序列中有插入的情况下，该插入将不对应于参考序列中的任何氨基酸位置。在截短或融合的情况下，在参考或比对序列中可能有氨基酸的延伸片段不对应于相应序列中的任何氨基酸。iv.抗体本发明还包括的是针对本发明的多肽或者其变体或片段的抗体。生产抗体的方法是本领域公知的(参见例如harlow和lane(1988)antibodies：alaboratorymanual，coldspringharborlaboratory，coldspringharbor，ny；和美国专利号4,196,265)。这些抗体可用于试剂盒中以检测和分离毒素多肽。因此，本公开提供了包含与本文所述的多肽特异性结合的抗体的试剂盒，所述多肽包括例如具有seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、1143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的序列的多肽。ii.害虫本文提供的组合物和方法可用于针对各种害虫。所述“害虫”包括但不限于昆虫、真菌、细菌、线虫、螨虫、原生动物病原体、动物寄生肝吸虫等。特别目的害虫是昆虫害虫，特别是对农业植物造成重大损害的昆虫害虫。昆虫害虫包括选自鞘翅目、双翅目、膜翅目、鳞翅目、羽虱目、同翅目、半翅目、直翅目、缨尾目、革翅目、等翅目、虱目、蚤目、毛翅目或线虫的昆虫。在非限制性实施方案中，昆虫害虫包括西方玉米根虫，玉米根叶甲(diabroticavirgifera)；秋夜蛾，草地贪夜蛾(spodopterafrugiperda)；科罗拉多马铃薯甲虫，马铃薯甲虫(leptinotarsadecemlineata)；玉米穗虫，玉米穗虫(helicoverpazea)(北美同类物种攻击棉花，称为棉铃虫)；欧洲玉米螟，玉米螟虫(ostrinianubilalis)；黑色夜蛾，小地老虎(agrotisipsilon)；小钻石背蛾，钻石背蛾(plutellaxylostella)；天鹅绒毛虫，大豆夜蛾(anticarsiagemmatalis)；西南玉米螟，西南玉米杆草螟(diatraeagrandiosella)；棉铃虫，玉米穗蛾(helicoverpaarmigera)(除美国以外的世界其他地区)；南部绿蝽，稻绿蝽(nezaraviridula)；绿蝽，绿蝽(chinaviahalaris)；棕纹蝽，茶翅蝽(halyomorphahalys)；和褐臭蝽，褐臭蝽(euschistusservus)。在其它实施方案中，所述害虫包括线虫，包括但不限于根结线虫(meloidogynehapla)(北部根结线虫)；子叶根结线虫(meloidogyneenterolobii)；花生根结线虫(meloidogynearenaria)；和爪哇根结线虫(meloidogynejavanica)。本文所用的术语“昆虫害虫”是指昆虫和其他类似的害虫，例如螨，包括但不限于螨虫和蜱虫。本发明的害虫包括但不限于鳞翅目昆虫，例如蜡蛾(achoroiagrisella)、西方黑头虫蚜虫(aclerisgloverana)、黑头长翅卷蛾(aclerisvariana)、夏季果实卷夜蛾(adoxophyesorana)、小地老虎(agrotisipsilon)、棉叶虫(alabamaargillacea)、秋尺蠖(alsophilapometaria)、脐橙蛾(amyeloistransitella)、地中海粉蛾(anagastakuehniella)、桃树枝钻心虫(anarsialineatella)、橙色条纹橡树虫(anisotasenatoria)、中国橡树丝蛾(antheraeapernyi)、黎豆毛虫(anticarsiagemmatalis)、黄卷蛾属(archipssp.)、条卷蛾属(argyrotaeniasp)、粗皮地香(athetismindara)、家蚕(bombyxmori)、棉叶潜蛾(bucculatrixthurberiella)、粉斑螟蛾(cadracautella)、色卷蛾属(choristoneurasp)、向日葵细卷蛾(cochyllshospes)、纹黄豆粉蝶(coliaseurytheme)、米螟(corcyracephalonica)、棒树虫(cydialatiferreanus)、苹果蠹蛾(cydiapomonella)、datanaintegerrima、西伯利亚丝蛾(dendrolimussibericus)、desmiafeneralis、甜瓜虫(diaphaniahyalinata)、泡菜虫(diaphanianitidalis)、西南玉米钻心虫(diatraeagrandiosella)、甘鹿钻心虫(diatraeasaccharalis)、偷树尺蠖(ennomossubsignaria)、墨西哥水稻钻心虫(eoreumaloftini)、烟草蛾(esphestiaelutella)、erannistilaria、盐泽灯蛾(estigmeneacrea)、圆纹卷叶蛾(euliasalubricola)、葡萄缀穗蛾(eupocoelliaambiguella)、女贞细卷蛾(eupoeciliaambiguella)、黄毒蛾(euproctischrysorrhoea)、黑边地香(euxoamessoria)、大蜡螟(galleriamellonella)、梨小食心虫(grapholitamolesta)、雕叶虫(harrisinaamericana)、棉花暝蛉(helicoverpasubflexa)、玉米穗虫(helicoverpazea)、烟草夜蛾(heliothisvirescens)、三色大蚕蛾(hemileucaoliviae)、向日葵斑螟(homoeosomaelectellum)、美国白蛾(hyphantiacunea)、番茄茎麦蛾(keiferialycopersicella)、铁杉尺蠖(lambdinafiscellaria)、西方铁杉尺蠖(lambdinafiscellarialugubrosa)、缎蛾(leucomasalicis)、葡萄卷叶蛾(lobesiabotrana)、草地螟(loxostegesticticalis)、舞毒蛾(lymantriadispar)、锄须丛螟(macallathyrisalis)、天幕毛虫属(malacosomasp.)、甘蓝夜蛾(mamestrabrassicae)、蓓带夜蛾(mamestraconfigurata)、番茄天蛾(manducaquinquemaculata)、烟草天蛾(manducasexta)、豆荚钻心虫(marucatestulalis)、斑马纹夜蛾(melanchrapicta)、冬尺蠖蛾(operophterabrumata)、毒蛾属(orgyiasp.)、欧洲玉米螟(ostrinianubilalis)、春尺蠖(paleacritavernata)、美洲大芷凤蝶(papiliocresphontes)、棉红铃虫(pectinophoragossypiella)、加州橡树蛾(phryganidiacalifornica)、潜叶虫(phyllonorycterblancardella)、暗脉粉蝶(pierisnapi)、菜粉蝶(pierisrapae)、苜蓿绿夜蛾(plathypenascabra)、杂色卷叶蛾(platynotaflouendana)、厚背卷叶蛾(platynotastultana)、洋蓟羽蛾(platyptiliacarduidactyla)、印度谷螟(plodiainterpunctella)、小钻石背蛾(plutellaxylostella)、多形云粉蝶(pontiaprotodice)、美洲粘虫(pseudaletiaunipuncta)、大豆尺蠖(pseudoplasiaincludens)、杂食尺蠖(sabulodesaegrotata)、花生红灯蛾(schizuraconcinna)、麦蛾(sitotrogacerealella)、苹白小卷蛾(spilontaocellana)、灰翅夜蛾属(spodopterasp.)、松行列蛾(thaurnstopoeapityocampa)、幕谷蛾(tinsolabisselliella)、粉夜蛾(trichoplusiahi)、缨突野螟(udearubigalis)、柑橘夜盗虫(xylomygescuriails)、以及苹果巢蛾(yponomeutapadella)。害虫还包括选自双翅目、膜翅目、鳞翅目、羽虱目、同翅目、半翅目、直翅目、缨尾目、革翅目、等翅目、虱目、蚤目、毛翅目、鞘翅目的昆虫。主要作物的昆虫害虫包括但不限于：玉米：玉米螟虫(ostrinianubilalis)，欧洲玉米螟；小地老虎(agrotisipsilon)，黑色夜蛾；玉米穗虫(helicoverpazeae)，玉米穗虫；草地贪夜蛾(spodopterafrugiperda)，草地贪夜蛾；西南玉米螟(diatraeagrandiosella)，西南玉米螟；南美玉米苗斑螟(elasmopalpuslignosellus)，南美玉米苗斑螟；甘蔗螟虫(diatraeasaccharalis)，甘蔗螟虫；西方玉米根虫，例如，西方玉米根虫(diabroticavirgiferavirgifera)；北方玉米根虫，例如，北方玉米根虫(diabroticalongicornisbarberi)；南方玉米根虫，例如，南部玉米根虫(diabroticaundecimpunctatahowardi)；金针虫属(melanotusspp.)，铁线虫(wireworms)；北方蒙面金龟子(cyclocephalaborealis)，北方蒙面金龟子(白蛴螬)；南方蒙面金龟子(cyclocephalaimmaculata)，南方蒙面金龟子(白蛴螬)；日本金甲虫(popilliajaponica)，日本甲虫；玉米跳甲(chaetocnemapulicaria)，玉米跳甲；玉米象虫(sphenophorusmaidis)，玉米象虫；玉米叶蚜(rhopalosiphummaidis)，玉米叶蚜；玉米根蚜(anuraphismaidiradicis)，玉米根蚜；高粱长蝽(blissusleucopterusleucopterus)，高粱长蝽；红腿蝗(melanoplusfemurrubrum)，红腿蝗；血黑蝗(melanoplussanguinipes)，血黑蝗(migratorygrasshopper)；玉米种蝇(hylemyaplatura)，玉米种蝇；玉米斑潜蝇(agromyzaparvicornis)，玉米斑点斑潜蝇；玉米黄蓟马(anaphothripsobscrurus)，玉米黄蓟马；窃叶蚁(solenopsismilesta)，窃叶蚁；二斑叶螨(tetranychusurticae)，二斑叶螨；高粱：高粱螟(chilopartellus)，高粱螟；草地贪夜蛾(spodopterafrugiperda)，秋夜蛾(fallarmyworm)；玉米穗虫(helicoverpazeae)，棉铃虫(cornearworm)；南美玉米苗斑螟(elasmopalpuslignosellus)，小玉米杆螟(lesercornstalkborer)；粒伏地老虎(feltiasubterranea)，粒伏地老虎；长毛镂龟(phyllophagacrinite)，白蛴螬(whitegrub)；金针虫属(eleodes)、叩头虫属(conoderus)和aeolus属，铁线虫(wireworms)；麦芽汁虫(oulemamelanopus)，麦芽汁虫；玉米跳甲(chaetocnemapulicaria)，玉米跳甲；玉米象虫(sphenophorusmaidis)，玉米象虫；玉米叶蚜(rhopalosiphummaidis)，玉米叶蚜；蔗黄伪毛蚜(siphaflava)，黄甘蔗蚜(yellowsugarcaneaphid)；长蝽(chinchbug)，例如，麦长蝽(blissusleucopterusleucopterus)；高粱瘿蚊(contariniasorghicola)，高粱瘿蚊；朱砂叶螨(tetranychuscinnabarinus)，朱砂叶螨(carminespidermite)；二斑叶螨(tetranychusurticae)，二斑叶螨；小麦：小麦一点粘虫(pseudaletiaunipunctata)，粘虫；草地贪夜蛾(spodopterafrugiperda)，秋夜蛾(fallarmyworm)；南美玉米苗斑螟(elasmopalpuslignosellus)，南美玉米苗斑螟；灰地老虎(agrotisorthogonia)，灰地老虎；南美玉米苗斑螟(elasmopalpuslignosellus)，南美玉米苗斑螟；麦芽汁虫(oulemamelanopus)，麦芽汁虫；苜蓿象甲(hyperapunctata)，苜蓿象甲；南方玉米根虫，例如，南方玉米根虫(diabroticaundecimpunctatahowardi)；俄罗斯小麦蚜虫(russianwheataphid)；麦二叉蚜(schizaphisgraminum)，麦二叉蚜；麦长管蚜(macrosiphumavenae)，麦长管蚜(englishgrainaphid)；红腿蝗(melanoplusfemurrubrum)，红腿蝗；殊种蝗(melanoplusdifferentialis)，殊种蝗；血黑蝗(melanoplussanguinipes)，血黑蝗；黑森瘿蚊(mayetioladestructor)，黑森瘿蚊；红吸浆虫(sitodiplosismosellana)，红吸浆虫；美州麦杆蝇(meromyzaamericana)，美州麦杆蝇；小麦球蝇(hylemyacoarctata)，小麦球蝇；烟蓟马(frankliniellafusca)，烟蓟马；麦秆蜂(cephuscinctus)，麦秆蜂；郁金香痿螨(aceriatulipae)，郁金香痿螨；向日葵：向日葵茎象鼻虫(cylindrocupturusadspersus)，向日葵茎象鼻虫；红色向日葵种子象鼻虫(smicronyxfulus)，红色向日葵种子象鼻虫；灰色向日葵种子象鼻虫(smicronyxsordidus)，灰色向日葵种子象鼻虫；向日葵芽蛾(suleimahelianthana)，向日葵芽蛾；向日葵螟(homoeosomaelectellum),向日葵螟；向日葵叶甲(zygogrammaexclamationis)，向日葵叶甲：胡萝卜金龟(bothyrusgibbosus)，胡萝卜金龟；向日葵籽瘿蚊(neolasiopteramurtfeldtiana)，向日葵籽瘿蚊；棉花：烟草夜蛾(heliothisvirescens)，烟草夜蛾；玉米穗虫(helicoverpazeae)，棉铃虫(cottonbollworm)；甜菜夜蛾(spodopteraexigua)，甜菜夜蛾；棉红铃虫(pectinophoragossypiella)，棉红铃虫；棉铃象甲(bollweevil)，例如，苜蓿叶象甲(anthonomusgrandis)；棉蚜(aphisgossypii)，棉蚜；棉盲蝽(pseudatomoscelisseriatus)，棉盲蝽；带状翅白粉虱(trialeurodesabutilonea)，带状翅白粉虱；美国牧草盲蝽(lyguslineolaris)，美国牧草盲蝽(tarnishedplantbug)；红腿蝗(melanoplusfemurrubrum)，红腿蝗；殊种蝗(melanoplusdifferentialis)，殊种蝗；葱蓟马(thripstabaci)，葱蓟马；烟蓟马(franklinkiellafusca)，烟蓟马；朱砂叶螨(tetranychuscinnabarinus)，朱砂叶螨；二斑叶螨(tetranychusurticae)，二斑叶螨；水稻：甘蔗螟虫(diatraeasaccharalis)，甘蔗螟虫；草地贪夜蛾(spodopterafrugiperda)，秋夜蛾(fallarmyworm)；玉米穗虫(helicoverpazeae)，玉米穗虫(cornearworm)；葡萄肖叶甲(colaspisbrunnea)，葡萄肖叶甲；稻水象(lissorhoptrusoryzophilus)，稻水象；米象(sitophilusoryzae)，米象；二条斑黑尾叶蝉(nephotettixnigropictus)，二条斑黑尾叶蝉；长蝽，例如，麦长蝽(blissusleucopterusleucopterus)；拟绿蝽(acrosternumhilare)，拟绿蝽；大豆：大豆夜蛾(pseudoplusiaincludes)，大豆夜蛾；藜豆夜蛾(anticarsiagemmatalis)，藜豆夜蛾；苜蓿绿夜蛾(plathypenascabra)，苜蓿绿夜蛾；欧洲玉米螟(ostrinianubilalis)，欧洲玉米螟；小地老虎(agrotisipsilon)，小地老虎；甜菜夜蛾(spodopteraexigua)，甜菜夜蛾；烟草夜蛾(heliothisvirescens)，烟草夜蛾；玉米穗虫(helicoverpazeae)，棉铃虫；墨西哥豆象(epilachnavarivestis)，墨西哥豆象；桃蚜(myzuspersicae)，桃蚜；马铃薯小绿叶蝉(empoascafabae)，马铃薯小绿叶蝉；拟绿蝽(acrosternumhilare)，拟绿蝽；红腿蝗(melanoplusfemurrubrum)，红腿蝗；殊种蝗(melanoplusdifferentialis)，殊种蝗；玉米种蝇(hylemyaplatura)，玉米种蝇；大豆蓟马(sericothripsvariabilis)，大豆蓟马(soybeanthrips)；葱蓟马(thripstabaci)，葱蓟马；土格斯坦红叶螨(tetranychusturkestani)，土格斯坦红叶螨；二斑叶螨(tetranychusurticae)，二斑叶螨；大麦：玉米螟虫(ostrinianubilalis)，欧洲玉米螟；小地老虎(agrotisipsilon)，小地老虎；麦二叉蚜(schizaphisgraminum),麦二叉蚜；长蝽，例如，麦长蝽(blissusleucopterusleucopterus)；拟绿蝽(acrosternumhilare)，拟绿蝽；褐臭蝽(euschistusservus)，褐臭蝽；玉米种蝇(jylemyaplatura)，玉米种蝇；黑森瘿蚊(mayetioladestructor)，黑森瘿蚊；麦岩螨(petrobialatens)，麦岩螨；油菜籽油菜：甘蓝蚜(vrevicorynebrassicae)，甘蓝蚜(cabbageaphid)；十字花科跳甲(phyllotretacruciferae)，十字花科跳甲(cruciferfleabeetle)；黄曲条跳甲(phyllotretastriolata)，黄曲条跳甲(stripedfleabeetle)；芫菁淡足跳甲(phyllotretanemorum)，芫菁淡足跳甲(stripedturnipfleabeetle)；油菜花露尾甲(meligethesaeneus)，油菜花露尾甲；和露尾甲meligethesrufimanus、meligethesnigrescens、meligethescanadianus和meligethesviridescens；马铃薯：leptinotarsadecemlineata，科罗拉多马铃薯甲虫(coloradopotatobeetle)。本文提供的方法和组合物可有效对抗半翅目，例如豆荚草盲蝽(lygushesperus)、美国牧草盲蝽(lyguslineolaris)、牧草盲蝽(lyguspratensis)、长毛草盲蝽(lygusrugulipennispopp)、绿盲蝽(lyguspabulinus)、马铃薯盲蝽(calocorisnorvegicus)、orthopscompestris、苹盲椿(plesiocorisrugicollis)、cyrtopeltismodestus、cyrtopeltisnotatus、白斑盲蝽(spanagonicusalbofasciatus)、阜荚树蝽(diaphnocorischlorinonis)、洋葱蝽(labopidicolaallii)、棉盲蝽(pseudatomoscelisseriatus)、苜蓿褐盲蝽(adelphocorisrapidus)、四线盲蝽(poecilocapsuslineatus)、麦长蝽(blissusleucopterus)、小长蝽(nysiusericae)、谷长蝽若虫(nysiusraphanus)、褐臭椿(euschistusservus)、稻绿蝽(nezaraviridula)、扁盾蝽属(eurygaster)、缘蝽科(coreidae)、红蝽科(pyrrhocoridae)、谷蛾科(tinidae)、负子蝽科(blostomatidae)、猎蝽科(reduviidae)和臭蝽科(cimicidae)。目的害虫还包括咖啡豆象鼻虫(araecerusfasciculatus)，咖啡豆象鼻虫(coffeebeanweevil)；敏豆象鼻虫(acanthoscelidesobtectus)，豆象(beanweevil)；蚕豆象鼻虫(bruchusrufmanus)，蚕豆象鼻虫(broadbeanweevil)；豌豆象鼻虫(bruchuspisorum)，豌豆象鼻虫(peaweevil)；巴西豆象(zabrotessubfasciatus)，墨西哥豆象鼻虫(mexicanbeanweevil)；黄瓜条叶甲(diabroticabalteata)，黄瓜条叶甲(bandedcucumberbeetle)；菜豆萤叶甲(cerotomatrifurcata)，豆叶甲虫(beanleafbeetle)；玉米根叶甲(diabroticavirgifera)，墨西哥玉米根虫；马铃薯跳甲(epitrixcucumeris)，马铃薯跳甲(potatofleabeetle)；甘薯跳甲(chaetocnemaconfinis)，甘薯跳甲(sweetpotatofleabeetle)；苜蓿叶象(hyperapostica)，苜蓿叶象(alfalfaweevil)；苹果象(anthonomusquadrigibbus)，苹果象(applecurculio)；豆茎象(sternechuspaludatus)，豆茎象(beanstalkweevil)；埃及苜蓿叶象(hyperabrunnipennis)，埃及苜蓿叶象(egyptianalfalfaweevil)；谷象(sitophilusgranaries)，谷象(granaryweevil)；葡萄象(craponiusinaequalis)，葡萄象(grapecurculio)；玉米象(sitophiluszeamais)，玉米象(maizeweevil)；李象(conotrachelusnenuphar)，李象(plumcurculio)；西印度群岛甘薯象(euscepespostfaciatus)，西印度群岛甘薯象(westindiansweetpotatoweevil)；紫绒鳃角金龟(maladeracastanea)，亚洲花园甲虫；欧洲切跟鳃金龟(rhizotrogusmajalis)，欧洲金龟子(europeanchafer)；蔷薇鳃角金龟(macrodactylussubspinosus)，玫瑰金龟子(rosechafer)；杂拟谷盗(triboliumconfusum)，杂拟谷盗(confusedflourbeetle)；黑粉虫(tenebrioobscurus)，黑粉虫(darkmealworm)；赤谷拟盗(triboliumcastaneum)，红粉虫(redflourbeetle)；黄粉甲(tenebriomolitor)，黄粉虫(yellowmealworm)。线虫包括寄生线虫，例如根结线虫、包囊线虫和根腐线虫，包括包囊线虫属(heteroderaspp.)、根结线虫属(meloidogynespp.)和球异皮线虫属(globoderaspp.)，特别是包囊线虫的成员，包括但不限于大豆孢囊线虫(heteroderaglycines，soybeancystnematode)；甜菜包囊线虫(heteroderaschachtii，beetcystnematode)；禾谷包囊线虫(heteroderaavenae，cerealcystnematode)；和马铃薯金线虫(globoderarostochiensis)和根腐线虫(globoderapailida，potatocystnematodes)。包括短体线虫属(pratylenchusspp.)。可以在害虫的早期发育阶段，例如还是幼虫或其它未成熟形式时，测试本发明组合物的杀虫活性。可以在约20℃至约30℃和约30％至约70％相对湿度的全黑暗中饲养昆虫。可按照czaplaandlang(1990)j.econ.entomol.83(6):2480-2485描述的方法进行生物测定。另见本文的实验部分。iii.表达盒可以以表达盒形式提供编码本文提供的杀虫蛋白的多核苷酸，以在目的生物中表达。该表达盒包括与编码本文提供的杀虫多肽的多核苷酸可操作地连接的5'和3'调节序列，以允许所述多核苷酸的表达。表达盒可以另外包含至少一个待共转化入生物的另外的基因或遗传元件。当包括额外的基因或元件时，这些组分可操作地连接。备选地，可以在多个表达盒上提供另外的(多个)基因或元件。提供这种表达盒，其具有多个限制性位点和/或重组位点，用于插入多核苷酸置于调节区的转录调节之下。此外，表达盒可以另外含有选择标记基因。表达盒在转录的5'-3'方向依次包括在目的生物(即植物或细菌)中有功能的转录和翻译起始区(即启动子)、本发明的杀虫多核苷酸以及转录和翻译终止区(即终止区)。本发明的启动子能够指导或驱动编码序列在宿主细胞中的表达。调节区(即启动子、转录调节区和翻译终止区)可以对宿主细胞或彼此之间是内源的或异源的。如本文所用，对于序列是“异源的”是指源于外来物种的序列，或者如果来自相同物种，则是通过有意的人为干预在组合物和/或基因组基因座上对其天然形式进行了本质上的修饰。如本文所用，嵌合基因包含编码序列，其可操作地连接至与所述编码序列异源的转录起始区。可从根癌土壤杆菌(a.tumefaciens)的ti-质粒可获得方便的终止区，例如章鱼碱合酶和胭脂碱合酶终止区。另见guerineau等人(1991)mol.gen.genet.262:141-144；proudfoot(1991)cell64:671-674；sanfacon等人(1991)genesdev.5:141-149；mogen等人(1990)plantcell2:1261-1272；munroe等人(1990)gene91:151-158；ballas等人(1989)nucleicacidsres.17:7891-7903；以及joshi等人(1987)nucleicacidsres.15:9627-9639。另外的调节信号包括但不限于转录起始起始位点、操纵子、激活子、增强子、其它调控元件、核糖体结合位点、起始密码子、终止信号等等。参见，例如，美国专利号5,039,523和4,853,331；epo0480762a2；sambrook等人(1992)molecularcloning：alaboratorymanual，ed.，maniatis等人(coldspringharborlaboratorypress，coldspringharbor，ny)(以下称为“sambrook11”)；davis等人，eds.(1980)advancedbacterialgenetics(coldspringharborlaboratorypress)，coldspringharbor，ny，及其中引用的参考文献。在制备表达盒时，可以操作多种dna片段，从而以适当的方向和适当的方式在适当的阅读框中提供dna序列。为此，可以使用转接子或连接子连接dna片段，或者可能涉及其它操作以提供方便的限制性位点，去除多余的dna，去除限制性位点等。为此，可能涉及体外诱变、引物修复、限制性酶切、退火、重新置换，例如转变和转换。许多启动子可用于本发明的实践中。可以基于期望的结果来选择启动子。核酸可以与组成型启动子、诱导型启动子、组织优选的启动子或其它启动子组合以在目的生物中表达。参见例如在wo99/43838和美国专利号8,575,425；7790846；8147856；8,586832；7772369；7534939；6,072,050；5,659,026；5,608,149；5,608,144；5,604,121；5,569,597；5,466,785；5,399,680；5,268,463；5,608,142和6,177,611中的启动子；这些都通过引用并入本文。为了用于在植物中表达，组成型启动子还包括camv35s启动子(odell等人(1985)nature313：810-812)；水稻肌动蛋白启动子(mcelroy等人(1990)plantcell2：163-171)；泛素启动子(christensen等人(1989)plantmol.biol.12：619-632和christensen等人(1992)plantmol.biol.18：675-689)；pemu启动子(last等人(1991)theor.appl.genet.81:581-588)；mas启动子(velten等人(1984)emboj.3：2723-2730)。诱导型启动子包括驱动发病机制相关蛋白(pr蛋白)表达的那些，其在受病原体感染后被诱导。参见，例如，redolfi等人(1983)neth.j.plantpathol.89:245-254；uknes等人(1992)plantcell4:645-656；及vanloon(1985)plantmol.virol.4:111-116；和wo99/43819，其各自通过引用并入本文。也可以使用在病原体感染位点或其附近表达的启动子(marineau等人(1987)plantmol.biol.9：335-342；matton等人(1989)molecularplant-microbeinteractions2：325-331；somsisch等人(1986)proc.natl.acad.sci.usa83:2427-2430；somsisch等人(1988)mol.gen.genet.2:93-98；和yang(1996)proc.natl.acad.sci.usa93:14972-14977；chen等人(1996)plantj.10:955-966；zhang等人(1994)proc.natl.acad.sci.usa91:2507-2511；warner等人(1993)plantj.3:191-201；siebertz等人(1989)plantcell1:961-968；cordero等人(1992)physiol.mol.plantpath.41:189-200；美国专利号5,750,386(线虫诱导型)；及其中引用的参考文献)。伤口诱导型启动子可用于本发明的构建体。这种伤口诱导型启动子包括pinii启动子(ryan(1990)ann.revphytopath.28：425-449；duan等人(1996)naturebiotechnology14：494-498)；wun1和wun2启动子(美国专利号5,428,148)；win1和win2启动子(stanford等人(1989)mol.gen.genet.215:200-208)；系统素启动子(mcgurl等人(1992)science225:1570-1573)；wip1启动子(rohmeier等人(1993)plantmol.biol.22：783-792；eckelkamp等(1993)febsletters323：73-76)；mpi基因启动子(corderok等人(1994)plantj.6(2)：141-150)；等等，其通过引用并入本文。用于本发明的组织优选启动子包括描述于yamamoto等人(1997)plantj.12(2)：255-265；kawamata等人(1997)plantcellphysiol.38(7)：792-803；hansen等人(1997)mol.gengenet.254(3)：337-343；russell等人(1997)transgenicres.6(2)：157-168；rinehart等人(1996)plantphysiol.112(3)：1331-1341；vancamp等人(1996)plantphysiol.112(2)：525-535；canevascini等人(1996)plantphysiol.112(2)：513-524；yamamoto等人(1994)plantcellphysiol.35(5)：773-778；lam(1994)resultsprobl.celldiffer.20：181-196；orozco等人(1993)plantmolbiol.23(6)：1129-1138；松冈等人(1993)procnatl.acad.sci.usa90(20):9586-9590；和guevara-garcia等人(1993)plantj.4(3)：495-505中的那些。叶优选的启动子包括描述于yamamoto等人(1997)plantj.12(2)：255-265；kwon等人(1994)plantphysiol.105:357-67；yamamoto等人(1994)plantcellphysiol.35(5):773-778；gotor等人(1993)plantj.3:509-18；orozco等人(1993)plantmol.biol.23(6):1129-1138；及matsuoka等人(1993)proc.natl.acad.sci.usa90(20):9586-9590中的那些。根优选的启动子是已知的，并且包括在hire等人(1992)plantmol.biol.20(2):207-218(大豆根特异性谷氨酰胺合成酶基因)；keller和baumgartner(1991)plantcell3(10):1051-1061(根特异性控制元件)；sanger等人(1990)plantmol.biol.14(3):433-443(根癌农杆菌甘露碱合酶(mas)基因)；和miao等人(1991)plantcell3(1):11-22(胞质谷氨酰胺合成酶(gs))；bogusz等人(1990)plantcell2(7):633-641；leach和aoyagi(1991)plantscience(limerick)79(1):69-76(rolc和rold)；teeri等人(1989)emboj.8(2):343-350；kuster等人(1995)plantmol.biol.29(4):759-772(vfenod-grp3基因启动子)；及capana等人(1994)plantmol.biol.25(4):681-691(rolb启动子)中的那些。另见美国专利号5,837,876；5,750,386；5,633,363；5,459,252；5,401,836；5,110,732和5,023,179。“种子优选的”启动子包括“种子特异性的”启动子(在种子发育过程中有活性的启动子，如种子储存蛋白的启动子)以及“种子发芽”启动子(在种子发芽过程中有活性的启动子)。参见thompson等人(1989)bioessays10：108。种子优选的启动子包括但不限于cim1(细胞分裂素诱导的信号)；cz19b1(玉米19kda玉米蛋白)；mi1ps(肌醇-1-磷酸合成酶)(参见wo00/11177和us专利号6,225,529)。γ-玉米蛋白是一种胚乳特异性启动子。球蛋白1(glb-1)是代表性的胚胎特异性启动子。对于双子叶植物，种子特异性启动子包括但不限于豆β-菜豆素、油菜籽蛋白、β-伴大豆球蛋白、大豆凝集素、十字花科素等。对于单子叶植物，种子特异性启动子包括但不限于玉米15kda玉米蛋白、22kda玉米蛋白、27kda玉米蛋白、γ-玉米蛋白、waxy、shrunken1、shrunken2、球蛋白1等，另见wo00/12733，其中公开了来自end1和end2基因的种子优选启动子。为了在细菌宿主中表达，在细菌中起作用的启动子是本领域公知的。这样的启动子包括任何已知的晶体蛋白基因启动子，包括本发明的任何杀虫蛋白的启动子和对苏云金芽孢杆菌sigma因子特异的启动子。备选地，可以对经诱变的或重组的晶体蛋白编码基因启动子进行重组工程化并用于促进本文公开的新基因区段的表达。表达盒还可以包含用于选择转化的细胞的选择标记物基因。可选择的标记物基因用于选择转化的细胞或组织。标记物基因包括编码抗生素抗性的基因，如编码新霉素磷酸转移酶ii(neo)和潮霉素磷酸转移酶(hpt)的基因，以及赋予针对除草化合物(如草铵膦、溴苯腈、咪唑啉酮和2,4-二氯苯氧基乙酸(2,4-d))抗性的基因。其它的选择性标记物是已知的，并且可以使用其中的任何标记物。参见例如2014年12月19日提交的美国临时申请62/094,697，其中公开了可用作可选择标记物的草铵膦抗性序列，其全部内容通过引用并入本文。iv.方法，宿主细胞及植物细胞如所指出的，包含编码杀虫蛋白或者其活性变体或片段的核苷酸序列的dna构建体可用于转化目的植物或其它目的生物。转化方法包括将核苷酸构建体引入植物中。“引入”旨在将核苷酸构建体引入植物或其它宿主细胞以使构建体能进入植物或宿主细胞的细胞内部。本发明的方法不需要某一特定方法将核苷酸构建体引入植物或宿主细胞，只需要核苷酸构建体能到达植物或宿主生物中至少一个细胞内部。将核苷酸构建体引入植物和其它宿主细胞的方法是本领域已知的，包括但不限于稳定转化方法、瞬时转化方法及病毒介导的方法。这些方法产生转化的生物，例如植物，包括整株植物，以及植物器官(例如叶、茎、根等)、种子、植物细胞、繁殖体、胚胎和其后代。植物细胞可以是分化或未分化的(例如愈伤组织、悬浮的培养细胞、原生质体、叶细胞、根细胞、韧皮部细胞、花粉)。“转基因植物”或“转化的植物”或“稳定转化的”植物或细胞或组织是指已经并入或整合了编码本发明的至少一种杀虫多肽的多核苷酸的植物。人们认识到，其它外源的或内源的核酸序列或dna片段也可以并入植物细胞中。农杆菌和基因枪介导的转化仍然是所采用的两个主要方法。然而，转化还可以通过以下方法进行：感染、转染、显微注射、电穿孔、微喷射、基因枪或粒子轰击、电穿孔、二氧化硅/碳纤维、超声介导、peg介导、磷酸钙共沉淀、聚阳离子dmso技术、deae葡聚糖程序、农杆菌和病毒介导(花椰菜花叶病毒、双联体病毒、rna植物病毒)、脂质体介导，等等。转化方案以及将多肽或多核苷酸序列引入植物中的方案可以根据植物或植物细胞的类型而变化，即根据用于转化的是单子叶植物或双子叶植物而变化。转化方法是本领域已知的，包括在美国专利号8,575,425；7692068；8802934和7,541,517中的那些；其各自通过引用并入本文。另见rakoczy-trojanowska，m.(2002)cellmolbiollett.7:849-858；jones等人(2005)plantmethods1:5；rivera等人(2012)physicsoflifereviews9:308-345；bartlett等人(2008)plantmethods4:1-12；bates,g.w.(1999)methodsinmolecularbiology111:359-366；binns和thomashow(1988)annualreviewsinmicrobiology42:575-606；christou,p.(1992)theplantjournal2:275-281；christou,p.(1995)euphytica85:13-27；tzfira等人(2004)trendsingenetics20:375-383；yao等人(2006)journalofexperimentalbotany57:3737-3746；zupan和zambryski(1995)plantphysiology107:1041-1047及jones等人(2005)plantmethods1:5。转化可导致核酸稳定或瞬时并入细胞。“稳定转化”旨在表示引入宿主细胞的核苷酸构建体整合到宿主细胞的基因组中，并且能够被其后代遗传。“瞬时转化”旨在表示多核苷酸被引入宿主细胞且不整合到宿主细胞的基因组中。转化叶绿体的方法是本领域已知的。参见，例如，svab等人(1990)proc.natl.acad.sci.usa87:8526-8530；svab和maliga(1993)proc.natl.acad.sci.usa90:913-917；svab和maliga(1993)emboj.12:601-606。该方法依赖于通过粒子枪递送含有选择标记物的dna，并通过同源重组将dna靶向质体基因组。此外，通过组织优选的表达核编码、质体定向的rna聚合酶，可以反式激活沉默的质体携带的转基因来完成质体转化。mcbride等人(1994)proc.natl.acad.sci.usa91:7301-7305中已经报道有这样的系统。经转化的细胞可以由常规方式生长进入植物中。参见，例如，mccormick等人(1986)plantcellreports5：81-84。然后可以使这些植物生长，并用相同的转化株或不同的株进行授粉，鉴定所得的具有所需表型特征的组成型表达的杂交体。可以使其两代或更多代生长，以确保所期望的表型特征的表达得到稳定地保持和遗传，然后收获种子以确保达成了所需表型特征的表达。以这种方式，本发明提供了具有稳定并入其基因组的本发明的核苷酸构建体(例如本发明的表达盒)的转化的种子(也称为“转基因种子”)。在具体实施方案中，本文提供的序列可以靶向宿主细胞或植物细胞的基因组内的特定位点。这些方法包括但不限于针对植物基因组目的序列设计的大范围核酸酶(d'halluin等人，2013plantbiotechnolj)；crispr-cas9、talens及其他的基因组精确编辑技术(feng等人，cellresearch23：1229-1232，2013，podevin,等人trendsbiotechnology,在线发表，2013,wei等人,jgengenomics,2013,zhang等人(2013)wo2013/026740)；cre-lox位点特异性重组(dale等人(1995)plantj7：649-659；lyznik等人(2007)transgenicplantj1：1-9；flp-frt重组(li等人(2009)plantphysiol151：1087-1095)；bxb1介导整合(yau等人，plantj(2011)701：147-166)；锌指介导整合(wright等人(2005)plantj44：693-705)；cai等人(2009)plantmolbiol69：699-709)；以及同源重组(lieberman-lazarovich和levy(2011)methodsmolbiol701：51-65)；puchta(2002)plantmolbiol48：173-182)。本文提供的序列可用于转化任何植物物种，包括但不限于单子叶植物和双子叶植物。目的植物的实例包括但不限于玉米(玉蜀黍)、高粱、小麦、向日葵、番茄、十字花科植物、辣椒、马铃薯、棉花、水稻、大豆、甜菜、甘蔗、烟草、大麦、和油菜籽油菜、芸苔属、苜蓿、黑麦、小米、红花、花生、甘薯、木薯、咖啡、椰子、菠萝、柑橘树、可可豆、茶、香蕉、鳄梨、无花果、番石榴、芒果、橄榄、番木瓜、腰果、澳洲坚果、扁桃仁、燕麦、蔬菜、观赏植物和针叶树。蔬菜包括但不限于西红柿、莴苣、青豆、利马豆、豌豆和黄瓜属的成员，如黄瓜、哈密瓜和甜瓜。观赏植物包括但不限于杜鹃花、绣球花、木槿、玫瑰、郁金香、水仙、矮牵牛、康乃馨、一品红和菊花。优选地，本发明的植物是作物植物(例如玉米、高粱、小麦、向日葵、番茄、十字花科植物、辣椒、马铃薯、棉花、水稻、大豆、甜菜、甘蔗、烟草、大麦、油籽油菜等)。如本文所用，术语植物包括植物细胞、植物原生质体、可以再生出植物的植物细胞组织培养物、植物愈伤组织、植物团块和在植物中完整的植物细胞，或植物的部分(如胚胎、花粉、胚珠、种子、叶、花、枝、果实、核、穗、轴、壳、茎、根、根尖、花药)等。谷物是指商业种植者为种植或繁殖该物种以外的目的生产的成熟种子。再生植物的后代、变体和突变体也包括在本发明的范围内，前提是这些部分包含引入的多核苷酸。还提供了保留本文公开的序列的经加工的植物产物或副产物，包括例如豆粕。在另一个实施方案中，编码杀虫蛋白的基因可用于转化昆虫的病原生物。这些生物包括杆状病毒、真菌、原生动物、细菌和线虫。可以选择已知占据一种或多种目的作物的“植物圈”(叶面、叶际、根际和/或根面)的微生物宿主。选择这些微生物的目的是为了能够在特定环境中成功地与野生型微生物竞争，提供表达杀虫蛋白的基因的稳定维持和表达，并且理想地提供更好的保护杀虫剂不受环境的降解和灭活。这些微生物包括古细菌、细菌、藻类和真菌。特别感兴趣的是微生物如细菌，例如芽孢杆菌属、假单胞菌属、欧文氏菌属、沙雷氏菌属、克雷伯菌属、黄单胞菌属、链霉菌属、根瘤菌属、红假单胞菌属、甲基硫菌属、农杆菌属、醋杆菌属、乳杆菌属、节杆菌属、固氮菌属、明串珠菌属和产碱菌属。真菌包括酵母，例如酿酒酵母属、隐球菌属、克鲁维酵母属、丝孢霉属、红酵母属和短梗霉属。特别感兴趣的是植物圈细菌物种，诸如丁香假单胞菌(pseudomonassyringae)、铜绿假单胞菌(pseudomonasaeruginosa)、荧光假单胞菌(pseudomonasfluorescens)、粘质沙雷氏菌(serratiamarcescens)、木醋杆菌(acetobacterxylinum)、农杆菌属、红假单胞菌(rhodopseudomonasspheroides)、野油菜黄单胞菌(xanthomonascampestris)、苜蓿根瘤菌(rhizobiummelioti)、真养产碱杆菌(alcaligenesentrophus)、木质棒形杆菌(clavibacterxyli)和维涅兰德固氮菌(azotobactervinlandir)，以及植物圈酵母物种，诸如深红酵母(rhodotorularubra)、粘红酵母(r.glutinis)、海滨红酵母(r.marina)、橙黄红酵母(r.aurantiaca)、浅白隐球酵母(cryptococcusalbidus)、流散隐球酵母(c.diffluens)、罗伦隐球酵母(c.laurentii)、罗氏酵母(saccharomycesrosei)、球酵母(s.pretoriensis)、酿酒酵母(s.cerevisiae)、粉红掷孢酵母(sporobolomycesrosues)、气味掷孢酵母(s.odorus)、kluyveromycesveronae、出芽毛孢子短梗霉(aureobasidiumpollulans)、苏云金芽孢杆菌、大肠埃希氏菌(escherichiacoli)、枯草芽孢杆菌(bacillussubtilis)，等等。用于示例说明的原核生物(包括革兰氏阴性和革兰氏阳性)包括肠杆菌科，如埃希氏菌属、欧文氏菌属、志贺氏菌属、沙门氏菌属和变形杆菌属；芽孢杆菌科；根瘤菌科，如根瘤菌属；螺旋藻科，如光杆菌属、发酵单胞菌属、沙雷氏菌属、气单胞菌属、弧菌属、脱硫弧菌属、螺旋藻属；乳酸杆菌科；假单胞菌科，如假单胞菌属和醋杆菌属；固氮菌科和硝杆菌科。真菌包括植物菌纲和子囊菌纲，例如酵母，如酿酒酵母属和裂殖酵母属；及担子菌酵母，例如红酵母属、短梗霉属、掷孢酵母属等。编码杀虫蛋白的基因可以通过电转化、peg诱导的转化、热休克、转导、接合等方式引入。具体地，可以将编码杀虫蛋白的基因克隆到穿梭载体中。示例性穿梭载体为pht3101(lerecius等人(1989)femsmicrobiol.lett.66：211-218)。例如可以通过电穿孔将包含特定杀虫蛋白基因的编码序列的穿梭载体pht3101转化至根定殖的芽孢杆菌中(lerecius等人(1989)femsmicrobiol.lett.60：211-218)。可以将表达系统设计为通过向杀虫蛋白氨基末端融合适当的信号肽使杀虫蛋白被分泌到革兰氏阴性细菌的细胞质外。由大肠杆菌识别的信号肽包括ompa蛋白(ghrayeb等人(1984)emboj，3：2437-2442)。杀虫蛋白及其活性变体可以在细菌宿主中发酵，所得细菌被加工为微生物喷雾形式并使用，这与苏云金芽孢杆菌菌株用作杀虫喷雾剂的方式相同。在杀虫蛋白从芽孢杆菌分泌出来的情况下，使用本领域已知的方法除去或突变分泌信号。这种突变和/或删除防止杀虫蛋白在发酵过程中分泌到生长培养基中。将杀虫蛋白保留在细胞内，然后处理细胞以产生封装的杀虫蛋白质。备选地，通过将异源基因引入细胞宿主来产生杀虫蛋白。异源基因的表达直接或间接地导致了杀虫剂的细胞内生产和维持。然后在一定条件下处理细胞，所述条件使得当将细胞应用于(多种)靶标害虫时细胞中产生的毒素的活性可得到延长。所得产品保留有毒素的毒性。然后可以根据常规技术配制这些天然封装的杀虫蛋白，以应用于承载有目标害虫的环境，例如土壤、水和植物的叶子。参见，例如美国专利号6,468,523和美国公开号20050138685以及其中引用的参考文献。在本发明中，可将经转化的微生物(包括整个生物、细胞、孢子、杀虫蛋白、杀虫组分、影响害虫的组分、突变体、活着或死的细胞及细胞组分(包括破碎的细胞和细胞组分))或分离的杀虫蛋白与可接受载体一同配制到杀虫或农业组合物中，也即，例如悬浮液、溶液、乳液、撒粉、可分散的颗粒、可湿润的粉剂、和可乳化的浓缩物、气溶胶、浸渍颗粒、佐剂、可包被的糊剂，以及例如在多聚体物质的情况下还可以为包囊剂。农业组合物可以包含本文公开的多肽、重组发生多肽或者其变体或片段。本文公开的农业组合物可以应用于植物或栽培区域的环境，或应用于植物、植物部分、植物细胞或种子。可以通过添加以下物质获得上述公开的组合物：表面活性剂、惰性载体、防腐剂、保湿剂、进食刺激剂、引诱剂、包封剂、粘合剂、乳化剂、染料、uv保护剂、缓冲剂、流动剂或肥料、微量营养素供体或其他影响植物生长的制剂。一种或多种农业化学品包括但不限于除草剂、杀虫剂、杀真菌剂、杀细菌剂、杀线虫剂、杀软体动物剂、杀螨剂、植物生长调节剂、落叶剂和肥料，其可与本领域制剂常规使用的载体、表面活性剂或佐剂或其他组分组合，以促进产品处理及应用于特定目标害虫。合适的载体和佐剂可以是固体或液体，并且对应于通常在制剂技术中使用的物质，例如天然的或再生的矿物质、溶剂、分散剂、润湿剂、增粘剂、粘合剂或肥料。本发明的活性成分通常以组合物的形式应用，并且可以应用于要处理的作物区域、植物或种子。例如，本发明的组合物可以应用于制备用于储存或已储存在谷仓或料仓等中的谷物。本发明的组合物可以与其它化合物同时或相继地应用。本发明的活性成分或含有由本发明的细菌菌株产生的至少一种杀虫蛋白的本发明的农药组合物的应用方法包括但不限于叶面应用、种子包衣和土壤应用。应用的数量和应用的比率取决于相应害虫的侵染强度。合适的表面活性剂包括但不限于阴离子化合物，例如金属的羧酸盐；长链脂肪酸的羧酸盐；n-酰基肌氨酸盐；磷酸与脂肪醇乙氧基化物的单酯或二酯或这些酯的盐；脂肪醇硫酸盐如十二烷基硫酸钠、十八烷基硫酸钠或十六烷基硫酸钠；乙氧基化脂肪醇硫酸盐；乙氧基化烷基酚硫酸盐；木质素磺酸盐；石油磺酸盐；烷基芳基磺酸盐如烷基苯磺酸盐或低级烷基萘磺酸盐，例如丁基-萘磺酸盐；磺化萘-甲醛缩合物的盐；磺化苯酚-甲醛缩合物的盐；更复杂的磺酸盐例如酰胺磺酸盐，例如，油酸和n-甲基牛磺酸的磺化缩合产物；或二烷基磺基琥珀酸盐，例如琥珀酸二辛酯的磺酸钠。非离子试剂包括脂肪酸酯、脂肪醇、脂肪酸酰胺或者脂肪-烷基-或链烯基取代的酚与环氧乙烷的缩合产物；多元醇醚的脂肪酯，例如山梨糖醇脂肪酸酯；这些酯与环氧乙烷的缩合产物，例如聚氧乙烯山梨糖醇脂肪酸酯；环氧乙烷和环氧丙烷的嵌段共聚物，炔二醇如2,4,7,9-四乙基-5-癸炔-4,7-二醇或乙氧基化炔二醇。阳离子表面活性剂的实例包括例如脂族单、二-或多胺如乙酸盐、环烷酸盐或油酸盐；或含氧胺如聚氧乙烯烷基胺的氧化胺；通过羧酸与二胺或多胺的缩合制备的酰胺连接的胺；或季铵盐。惰性材料的实例包括但不限于无机矿物如高岭土、层状硅酸盐、碳酸盐、硫酸盐、磷酸盐或植物材料，所述植物材料如软木、粉末玉米芯、花生壳、稻壳和核桃壳。本发明的组合物可以是适合直接应用的形式或作为主要组合物的浓缩物形式，其在应用前需要用适量的水或其它稀释剂稀释。杀虫剂浓度将根据具体制剂的性质，特别是根据其是浓缩物还是直接使用而变化。组合物含有1至98％的固体或液体惰性载体、0至50％或0.1至50％的表面活性剂(根据需要可以是w/w、v/v或w/v)。这些组合物将以为商业产品标注的比率施用，例如当干燥形式时为约0.01lb-5.0lb/英亩，当为液体形式时为约0.01pts-10pts/英亩。在另一个实施方案中，可以在配制之前对本文提供的组合物以及转化的微生物和杀虫蛋白进行处理，以在应用于目标害虫的环境时延长杀虫活性，只要预处理对于杀虫活性没有不利影响。所述处理可以是化学方法，物理方法或二者，只要处理对于组合物的性质没有不利影响。化学试剂的实例包括但不限于卤化剂；醛如甲醛和戊二醛；抗感染药，如氯地昔芬；醇，如异丙醇和乙醇；和组织学固定剂，如bouin’s固定剂和helly’s固定剂(参见例如，humason(1967)animaltissuetechniques(w.h.freemanandco.))。在一方面，通过将本文提供的杀虫蛋白应用于给定区域，可以在该区域杀灭害虫或减少其数量。备选地，可以预防性地将杀虫蛋白应用于环境区域，以防止易感染害虫的侵染。优选地，害虫摄入杀虫有效量的多肽或与之接触。“杀虫有效量”是指能够导致至少一种害虫死亡或者明显减少害虫生长、进食或正常生理发育的杀虫剂的量。该量将根据例如待控制的具体目标害虫、具体环境、位置、植物、作物、或待处理的农业场所、环境条件、和方法、比率、浓度、稳定性及有杀虫效果的多肽组合物的应用量等因素而变化。制剂或组合物也可以根据气候条件，环境考虑因素和/或应用频率和/或害虫侵袭的严重程度而变化。活性成分通常以组合物的形式应用，并且可以应用于待处理的作物区域、植物或种子。因此，提供了用于向植物、植物细胞、种子、植物部分或栽培区域提供有效量的包含多肽、重组发生多肽或者其活性变体或片段的农业组合物的方法。“有效量”是指蛋白质或组合物的量具有足以杀灭或控制害虫或者导致害虫生长、进食或正常生理发育显著降低的杀虫活性。所述害虫数量、害虫生长、害虫进食或害虫正常发育的降低可以包括任何统计学上显著的降低，包括例如降低约5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、85％、90％、95％或更高。例如，可以将组合物应用于制备用于储存或已储存在谷仓或料仓等中的谷物。组合物可以与其它化合物同时或相继应用。应用活性成分或包含至少一种多肽、重组发生多肽或者其变体或片段的农药组合物的方法包括但不限于叶面应用、种子包衣和土壤应用。提供了提高植物产量的方法。所述方法包括提供表达编码本文公开的杀虫多肽序列的多核苷酸的植物或植物细胞，并且在受所述多肽对其具有杀虫活性的害虫侵害(或易受其侵害)的田地中培养植物或其种子。在一些实施方案中，多肽具有针对鳞翅目、鞘翅目、双翅目、半翅目或线虫害虫的杀虫活性，并且所述田地被鳞翅目、半翅目、鞘翅目、双翅目或线虫害虫侵染。如本文所定义，植物的“产量”指植物产生的生物质的质量和/或数量。“生物质”是指任何测量的植物产品。生物质产量的增加是测量的植物产品的产量的任何改善。提高植物产量有数种商业应用。例如，增加植物叶片生物质可以增加用于人类或动物消费的叶用蔬菜的产量。此外，增加叶片生物质可用于增加植物衍生的药物或工业产品的生产。产量的增加可以包括与不表达杀虫序列的植物相比任何统计学显著的增加，包括但不限于，产量的至少1％的增加、至少3％的增加、至少5％的增加、至少10％的增加、至少20％的增加、至少30％、至少50％、至少70％、至少100％或更多的增加。在具体方法中，由于表达本文公开的杀虫蛋白的植物的害虫抗性提高，其植物产量增加。杀虫蛋白的表达导致害虫侵染或进食的能力减小。还可以用一种或多种化学组合物处理植物，包括一种或多种除草剂、杀虫剂或杀真菌剂，或者其两种或更多种的组合。非限制性实施例包括：1.具有杀虫活性的分离的多肽，包含(a)多肽，其包含选自seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的序列的氨基酸序列；或(b)多肽，其包含与选自seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的序列的氨基酸序列相比具有至少如表1所示的序列同一性百分比的氨基酸序列。2.实施方案1的多肽，其中所述多肽包含seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的氨基酸序列。3.组合物，包含实施方案1或2的多肽。4.实施方案2的多肽，进一步包含异源氨基酸序列。5.编码实施方案1的多肽的重组核酸分子，其中所述重组核酸分子不是编码所述多肽的天然存在的序列。6.实施方案5的重组核酸，其中所述核酸分子是设计用于在植物中表达的合成序列。7.实施方案6的重组核酸分子，其中所述核酸分子可操作地连接至能够指导在植物细胞中表达的启动子。8.实施方案5的重组核酸分子，其中所述核酸分子可操作地连接至能够指导在细菌中表达的启动子。9.宿主细胞，包含实施方案8的重组核酸分子。10.实施方案9的宿主细胞，其中所述宿主细胞是细菌宿主细胞。11.dna构建体，包含驱动在植物细胞中表达的启动子，所述启动子可操作地连接至重组核酸分子，所述重组核酸分子包含(a)核苷酸序列，其编码包含seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的任一氨基酸序列的多肽；或，(b)编码多肽的核苷酸序列，所述多肽包含与选自seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的序列的氨基酸序列相比具有至少如表1所示的序列同一性百分比的氨基酸序列。12.实施方案11的dna构建体，其中所述核苷酸序列是设计用于在植物中表达的合成dna序列。13.载体，包含实施方案11的dna构建体。14.宿主细胞，包含实施方案11-13中任一项的dna构建体。15.实施方案14的宿主细胞，其中宿主细胞是植物细胞。16.转基因植物，包含实施方案15的宿主细胞。17.组合物，包含实施方案10的宿主细胞。18.实施方案17的组合物，其中所述组合物选自粉末、粉尘、弹丸(pellet)、颗粒、喷雾、乳液、胶体和溶液。19.实施方案17的组合物，其中所述组合物包含占重量约1％至约99％所述的多肽。20.用于控制害虫群体的方法，包括使所述群体与杀虫有效量的实施方案3或17的组合物接触。21.用于杀灭害虫群体的方法，包括使所述群体与杀虫有效量的实施方案3或17的组合物接触。22.生产具有杀虫活性的多肽的方法，包括在表达编码该多肽的核酸分子的条件下培养实施方案9的宿主细胞。23.在其基因组中稳定并入dna构建体的植物，所述dna构建体包含编码具有杀虫活性的蛋白质的核苷酸序列的，其中所述核苷酸序列包含(a)核苷酸序列，其编码包含seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的任一氨基酸序列的多肽；或，(b)编码多肽的核苷酸序列，所述多肽包含与选自seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的序列的氨基酸序列相比具有至少如表1所示的序列同一性百分比的氨基酸序列。24.实施方案23的植物的转基因种子。25.用于保护植物免受昆虫害虫的方法，包括在植物或其细胞中表达编码杀虫多肽的核苷酸序列，其中所述核苷酸序列包含(a)核苷酸序列，其编码包含seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的任一氨基酸序列的多肽；或，(b)编码多肽的核苷酸序列，所述多肽包含与选自seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的序列的氨基酸序列相比具有至少如表1所示的序列同一性百分比的氨基酸序列。26.根据实施方案25的方法，其中所述植物产生具有针对鳞翅目或鞘翅目害虫或者半翅目害虫的杀虫性的杀虫多肽。27.用于提高植物产量的方法，包括在田地中培养在其基因组中稳定并入dna构建体的植物或其种子，所述构建体包含驱动在植物中表达的启动子，所述启动子可操作地连接至编码杀虫多肽的核苷酸序列，其中所述核苷酸序列包括(a)核苷酸序列，其编码包含seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的任一氨基酸序列的多肽；或，(b)编码多肽的核苷酸序列，所述多肽包含与选自seqidno：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158和/或159的序列的氨基酸序列相比具有至少如表1所示的序列同一性百分比的氨基酸序列。提供以下实施例进行说明但不产生限制。实施例实施例1：通过测序和dna分析发现新基因在标准实验室培养基中以液体培养物的方式培养微生物培养物。在dna制备前使培养物生长至饱和(16至24小时)。通过去污剂裂解从细菌细胞中提取dna，然后与二氧化硅基质结合并用乙醇缓冲液洗涤。用弱碱缓冲水溶液将纯化的dna从二氧化硅基质中洗脱。通过分光光度法测试用于测序的dna的纯度和浓度。根据制造商的操作流程，使用nexteraxt文库制备试剂盒制备测序文库。根据illuminahiseq2000系统用户指南的操作流程在hiseq2000上生成序列数据。使用clcbioassemblycell软件包将测序读数聚类到基因组草图中。在聚类之后，通过几种方法进行基因调用，并且询问所得基因序列以鉴定杀虫基因的新型同源物。通过blast、通过结构域组成以及通过与杀虫基因靶组的成对比对来鉴定新基因。这些序列的概述列于表1中。通过pcr从细菌dna扩增在同源性检索中鉴定的基因，并克隆到含有融合的框内纯化标签的细菌表达载体中。在大肠杆菌中表达克隆的基因并通过柱色谱进行纯化。在昆虫饮食生物测定研究中评估纯化的蛋白质以鉴定活性蛋白质。使用小麦胚芽和琼脂人造饮食并向其中加入作为表面处理的纯化的蛋白质进行昆虫饮食生物测定。将昆虫幼虫加至经处理的饮食上并监测死亡率。使用包含在添加有纯化蛋白质的膜袋中的蔗糖液体饮食进行昆虫饮食生物测定。允许昆虫若虫在饮食袋上饲养，并监测其死亡率。在生物测定中测试的昆虫包括褐色臭虫(bsb)，褐臭蝽和南方绿色臭虫(sgsb)，稻绿蝽。数据见下文表5。表5.生物测定结果基因表达水平测试1测试2测试3apg00059极低(<10ppm)+bsb+sgsb+sgsbapg00046高(>500ppm)+bsb+bsbapg00002低(50ppm)+sgsb+sgsb+sgsbbsb＝褐色臭虫,sgsb＝南部绿色臭虫实施例2.大肠杆菌中异源表达将表6和表7所示的每个开放阅读框克隆到含有麦芽糖结合蛋白(pmbp)的大肠杆菌表达载体中。将表达载体转化到bl21*ripl中。用单个菌落接种补充有羧苄青霉素的lb培养物，并使用0.5％的过夜培养物在37℃下培养过夜，接种新鲜培养物并在37℃下培养至对数期。使用250mm的iptg在16℃诱导培养物18小时。将细胞沉淀并重新悬浮于补充有蛋白酶抑制剂的10mmtrisph7.4和150mmnacl中。通过sds-page评估蛋白质表达。实施例3.对鞘翅目和鳞翅目的杀虫活性蛋白质表达：如实施例2所述，将表6中列出的每个序列在大肠杆菌中表达。在400ml的lb中接种并培养至od600为0.6。在16℃下用0.25mm的iptg诱导培养物过夜。将细胞离心下来，将细胞沉淀重悬于5ml缓冲液中。将重悬液在冰上超声处理2分钟。生物测定：从商业昆虫饲养所(benzonresearchinc.,carlisle,pa)购买了草地夜蛾(faw)、玉米穗虫(cew)、欧洲玉米螟(ecb)、西南玉米螟(swcb)和钻石背蛾(dbm)卵。将faw、cew、ecb和bcw卵孵化至在测定进行的12小时内发生羽化的程度。将swcb和dbm以新生幼虫引入测定中。在含有多物种鳞翅目饮食(southlandproductsinc.,lakevillage,ar)的24孔盘中进行测定。将超声裂解物的样品加到饮食的表面(叠加于饮食上)并允许蒸发并浸入饮食中。对于cew、faw、bcw、ecb和swcb，将125μl超声处理的裂解物加入到饮食表面中并干燥。对于dbm，将50μl超声处理的裂解物的1:2稀释液加入到饮食表面。用打孔出针孔的平板密封膜密封生物测定板。将平板在26℃及65％的相对湿度(rh)下于percival中以16:8的光照黑暗周期孵育5天。评估测定中的死亡率、生长抑制和进食抑制的水平。对于西方玉米根虫的生物测定，在昆虫测定中评估蛋白质构建体/裂解物，通过在24孔板(cellstar，24孔，greinerbioone)的孔中饮食顶部表面分配60μl体积并使其干燥。每个孔含有500μl饮食(marrone等人，1985)。使用细尖涂料刷向每个孔中引入15至20个新生幼虫，并用膜(viewseal，greinerbioone)覆盖平板。在室温下储存生物测定样品，并在第4天对死亡率和/或生长/进食抑制进行评分。图7提供了玉米根虫生物测定的测定评分指南。对于科罗拉多马铃薯甲虫(cpb)，从马铃薯叶切下软木塞孔径8号的叶盘，并将其浸入蛋白质构建体/裂解物中直至彻底湿润并置于过滤盘顶部(millipore，玻璃纤维过滤器，13mm)。将60μl的dh2o加入到每个过滤盘中并置于24孔板(cellstar，24孔，greinerbioone)的每个孔中。使叶片干燥，并使用细尖涂料刷向每个孔中引入5至7个第一龄幼虫。将板用膜(viewseal，greinerbioone)覆盖，并在每个孔的膜中刺出小孔。对该构建体进行了四次重复评估，并在第3天对死亡率和叶损伤进行评分。表6提供了多种序列对鞘翅目和鳞翅目的杀虫活性的概述。表中符号：“-”表示没有活性；“nt”表示未测试；“s”表示发育障碍；“ss”表示轻微的发育障碍；“lf”表示进食量低。表6.针对鞘翅目和鳞翅目的杀虫活性概述。实施例4.针对半翅目的杀虫活性蛋白质表达：如实施例2所述，将表7中列出的每个序列在大肠杆菌中表达。在400ml的lb中接种并培养至od600为0.6。在16℃用0.25mm的iptg诱导培养物过夜。将细胞离心下来，将细胞沉淀重悬于5ml缓冲液中。将重悬液在冰上超声处理2分钟。从商业昆虫饲养所(benzonresearchinc.，carlisle，pa.)获得二龄sgsb。在生物测定中使用超声处理的裂解物样品与20％蔗糖的比为50％v/v。使用拉伸的封口膜作为进食膜使sgsb接触到饮食/样品混合物。将板在25℃:21℃，16:8的光照:黑暗循环及65％的rh中孵育5天。对每个样本的死亡率进行评分。结果列于表7中。虚线表示未检测到死亡率。蛋白质(apg00034)对南方绿臭虫具有25％的死亡率(4例中1例臭虫死亡)。阴性对照(只表达结合结构域的空载体和缓冲液)均未显示死亡率(4中0个臭虫死亡)。表7.针对半翅目的杀虫活性概述agb参比号seqidno对sgsb的测试apg000341525％死亡率apg000109---apg0007693---apg0003917----apg000087---apg0005225---apg0006532---apg0012471---apg0013094---实施例5.大豆转化将包含可操作地连接至在植物中有活性的启动子的seqidno：1-159的每个或者其变体或片段的dna构建体克隆到转化载体中，并引入农杆菌中，如美国临时申请号62/094,782(提交于2015年12月19日)中描述的，其全部内容通过引用并入本文。在接种前四天，将几个接种环的农杆菌划线至补充有适当抗生素**(壮观霉素、氯霉素和卡那霉素)的新鲜yep*培养基的新鲜平板上。使细菌在28℃的黑暗条件下生长两天。两天后，将数个几个接种环的细菌转移到125ml锥形瓶中，其中含有3ml的yep液体培养基及抗生素。将烧瓶放置在旋转振荡器上，在28℃以250rpm振荡过夜。接种前一天，将2-3ml的过夜培养物转移到500ml的锥形瓶中，其中含有125ml的yep及抗生素。将烧瓶放置在旋转振荡器上，在28℃以250rpm振荡过夜。接种之前，检查细菌培养物在od620处的od值。od值为0.8-1.0表示培养物处于对数期。将培养物在oakridge管中以4000rpm离心10分钟。弃去上清液，将沉淀物重悬于一定体积的大豆感染培养基(si)中以达到所需的od。定时混合培养物维持至需要接种时。在接种前两三天，用氯气对大豆种子表面进行灭菌。在通风橱中，将装有种子的培养皿放在未盖盖子钟形瓶中。将1.75ml的12nhcl缓慢加入钟形瓶内的含100ml漂白剂的250ml锥形烧瓶中。立即将盖子放置在钟形瓶顶部。对种子进行14-16小时(过夜)的灭菌。将顶部从钟形瓶上移除，并更换培养皿的盖子。然后将表面灭菌的培养皿在层流中开放约30分钟，以驱散任何剩余的氯气。用无菌di水或大豆感染培养基(si)对种子进行1-2天的浸渗。在100×25mm的培养皿中用液体覆盖20至30粒种子，并在24℃黑暗中孵育。浸渗后，弃去不发芽的种子。在无菌滤纸平板上，用si培养基润湿的无菌滤纸处理子叶外植体，使用美国专利号7,473,822的方法，其通过引用并入本文。通常，在每次处理中接种16-20个子叶。弃去用于容纳外植体的si培养基，并用25ml农杆菌培养物(od620＝0.8-20)代替。在所有的外植体都被浸没后，进行30分钟的接种，其中对培养皿进行定期旋转。30分钟后，移除农杆菌培养物。通过将一片无菌纸覆盖在大豆共培养培养基(scc)上制备共培养平板。不经过印迹，将接种的子叶侧面朝下在滤纸上培养。在每个平板上可以培养大约20个外植体。板用封口膜密封，并在24℃和约120μmolem-2s-1(在percival培养箱中)培养4-5天。共培养后，将子叶在25ml的大豆洗涤培养基(含有200mg/l的升头孢噻肟和泰米尔汀)中洗涤3次。将子叶印迹于无菌滤纸上，然后转移到大豆出芽诱导培养基(ssi)中。将外植体的节端部稍微压入培养基，其远端保持在表面之上，倾斜约45度。每块板上培养不超过10个外植体。将板用微孔带包裹，并在percival培养箱中于24℃和约120μmolem-2s-1培养。14天后，将外植体转移到新鲜的ssi培养基中。丢弃从芽顶点和子叶节点的出芽。在相同的条件下，连续14天诱导出芽。在诱导出芽4周后，将子叶与节点端部分离，并在出芽诱导区(芽垫)下面进行平行切割。将平行切割的区域放置在大豆出芽延伸培养基(sse)上，将外植体在percival培养箱中于24℃，约120μmolem-2s-1培养。只要芽继续伸长，就每两周重复一次该步骤，可长达8周。当芽达到长度2-3cm时，将它们转移到含大豆生根培养基(sr)的plantcon容器中，并在相同的条件下孵育2周，或直到根达到长度3-4cm左右。之后，将植物转移到土壤中。注意，所有关于大豆转化的培养基都可以在paz等人(2010)的著作中找到：agrobacterium-mediatedtransformationofsoybeanandrecoveryoftransgenicsoybeanplants；planttransformationfacilityofiowastateuniversity，其全部内容通过引用并入本文。(参见，agron-www.agron.iastate.edu/ptf/protocol/soybean.pdf。)实施例6.玉米转化玉米穗最好在授粉后8-12天收集。从穗中分离胚胎，并优选尺寸为0.8-1.5mm的胚胎用于转化。在适当的孵育培养基，例如dn62a5s培养基(3.98g/ln6盐；1ml/l(1000×储液)n6维生素)；800mg/ll-天冬酰胺；100mg/l肌醇；1.4g/ll-脯氨酸；100mg/l酪蛋白氨基酸；50g/l蔗糖；1ml/l(1mg/ml储液)2,4-d上以盾片侧向上铺胚胎。然而，除dn62a5s之外的介质和盐也是合适的并且在本领域已知。将胚胎在25℃于黑暗中孵育过夜。然而，将胚胎孵育过夜并不是必需的。将得到的外植体转移到网格方格(每片30-40个)，转移到渗透培养基上放置约30-45分钟，然后转移到光照平板(参见例如pct公开号wo/0138514和美国专利号5,240,842)。使用基本上如pct公开号wo/0138514中所述的条件使用气溶束加速器，使设计用于在植物细胞中表达本发明的grg蛋白质的dna构建体加速进入植物组织中。光照后，将胚胎在渗透培养基上孵育约30分钟，并在25℃在黑暗中置于孵育培养基上过夜。为了避免过度光照损伤的外植体，将它们在转移至回收培养基之前孵育至少24小时。然后将胚胎分散在恢复期培养基上，在25℃在黑暗中约5天，然后转移到选择培养基。根据所使用的具体的择的性质和特征，外植体可在选择培养基中孵育长达八周。在选择期后，将所得的愈伤组织转移到胚胎成熟培养基，直到观察到成熟体细胞胚的形成。然后将所得的成熟体细胞胚放置在低光照下，并且通过本领域已知的方法开始再生过程。使所得的出芽在生根培养基上生根，并将所得的植株转移到苗圃作为转基因植物繁殖。实施例7.对线虫的杀虫活性大豆孢囊线虫(heteroderaglycine)的体外测定将大豆孢囊线虫分配到96孔测定板中，其总体积为100ul，每孔加入100j2。将seqidno：1-159的任一目的蛋白质分配到孔中并保持在室温下用于评估。最后，分析含有scnj2的96孔板的活性。数据报告为与对照相比的抑制％。将命中定义为大于或等于70％抑制。大豆孢囊线虫的植物测定如本文其它地方所述生产表达seqidno：1-159的一项或多项的大豆植物。对3周的大豆插枝每株接种5000只scn卵。感染持续70天，然后收获用于计数在植株上发育的scn孢囊。数据报告为与对照相比的抑制％。将命中定义为大于或等于90％抑制。根结线虫(meloidogyneincognita)的体外测定将根结线虫分配到96孔测定板中，其总体积为100ul，每孔加入100j2。将包含seqidno：1-159的任一目的蛋白质分配到孔中并保持在室温下用于评估。最后分析含有rknj2的96孔板的活性。数据报告为与对照相比的抑制％。将命中定义为大于或等于70％抑制。根结线虫的植物测定如本文其它地方所述生产表达seqidno：1-159的一项或多项的大豆植物。对3周的大豆插枝每株接种5000只rkn卵。感染持续70天，然后收获用于计数在植株上发育的rkn孢囊。数据报告为与对照相比的抑制％。将命中定义为大于或等于90％抑制。实施例8.杀虫活性的其他检测可以用大量方式测试seqidno：1-159的多种多肽对害虫作为杀虫剂的作用。这些方法之一是进行进食测定。在所述进食测定中，使害虫接触含有待测试的化合物的样品或对照样品。通常这是通过将待测试材料或这种材料的合适稀释物放置在害虫将要摄取的材料上，例如人工饮食上来进行的。待测试的材料可以由液体、固体或浆料组成。可以将待测试的材料放置在表面上，然后使其干燥。备选地，可以将待测试的材料与熔化的人工饮食混合，然后分配到测定小室中。测定小室可以是例如杯、盘或微量滴定板的孔。用于吸吮害虫(例如蚜虫)的测定可以包括通过隔离将测试材料与昆虫分开，理想地是可以被吸吮昆虫的吸嘴部分刺穿的部分，以允许其摄取测试材料。通常将测试材料与进食刺激剂如蔗糖混合以促进对测试化合物的摄取。其他类型的测定可以包括将测试材料显微注射到害虫的口腔或肠道中，以及培育转基因植物，随后测试害虫进食转基因植物的能力。植物测试可以涉及分离通常被食用的植物部分，例如连接至叶子上的小笼子，或分离包含昆虫的笼子中的整个植株。测定害虫的其它方法和方法是本领域已知的，并且可以在例如robertson和preislereds.(1992)pesticidebioassayswitharthropods,crc,bocaraton,fla.中找到。备选地，测定通常在arthropodmanagementtests和journalofeconomicentomology这些期刊或通过(esa)的成员讨论而得到描述。在本文实施例3和4所述的测定中表达并使用的可以是seqidno：1-159的任一项。本说明书中提到的所有出版物和专利申请指示了本发明所属领域的技术人员的水平。所有出版物和专利申请均以引用方式并入本文，所引用的程度就如同每个单独的出版物或者专利申请被具体地和独立地指出以引用方式并入本文一样。虽然为了清楚地理解起见已经通过举例说明和实例详细地描述了本发明，但显然可以在权利要求书范围内实施一些改变和修饰。序列表<110>parks,jessicaroberts,kirabulazelthayer,rebeccae<120>杀虫基因和使用方法<130>098699-0965873<150>us60/095,524<151>2014-12-22<160>167<170>patentinversion3.5<210>1<211>885<212>prt<213>未知<220><223>从环境获得的芽孢杆菌<400>1metalaasnglythrlyslyshisvalilelyslystyrleugluleu151015ilelysproiletyrtyrlysglnasnserleuleupheserprophe202530ileleuserileproglycysaspileserglnhisglyserilepro354045alametlysargasncysphethrleuleuleutyrtyrargargtyr505560valmetalaglnleuprogluleuglnalaglnserpheileprotyr65707580asnvalleualaasnprovalthrglyasnseralaglythrasptrp859095glyglutrpleuserserleulysglualatrpthrglupheglnlys100105110thrglyserasplyspheleuglnservalleuserthrglytyrasp115120125alaalaglnglyglyserpheasntyrilealavalleuglnalagly130135140valalaileleuglythrvalleuglyalaglupheproglyilethr145150155160alailealavalproalaleuthrpheleuilelystrpvaltrppro165170175lysleuthrglyglyseralaglygluseraspthrglnlysmetleu180185190aspleuileasplysgluileglnglnlysleuaspglnalaleuser195200205glnglnaspileasnasntrpargalatyrleuthrserilepheglu210215220alaalathrasnleuserglnserileileaspalaglnphethrgly225230235240thrgluargserseraspargglnproargvalprothrserserasp245250255tyrgluaspvaltyrthrlysleuasnilealaaspalavalleuser260265270proserthrasnasnileleuthrglyasnpheaspvalleualaile275280285protyrtyrthrileglyalathrvalargilethralatyrglnser290295300phevallysphealaasnglntrpileaspvalvaltyrproaspgln305310315320thrserasnthrtrpleulysalahisglnasnleuaspasnalalys325330335asplysmetilealametileglnglntyrthralaasnvalleuarg340345350valphethraspprolysasnmetproproleuglylysasplyslys355360365serileasnalatyrasnlysphevalargglymetvalilesergly370375380leuaspleuvalalavaltrpproserleutyrproaspasptyrgln385390395400valglnthraspleuglulysthrargvalilepheseraspleuile405410415glylysaspglnthrileserasnaspvalthrleuileasnmetala420425430aspglylysaspglyserglyalapheasnglnhisalaalaileasp435440445leuasnserilephetyrtyrhisglugluleusersermetglnleu450455460glyilehisaspglyserargglyasnthrthrasncystyrasptyr465470475480glyileilealaasntyrproasnhissertyrphetyrglyaspleu485490495tyrserproserglyaspalathrpheseralaproleuasnvalleu500505510asnalaglnthrglntyrserserglytyrvalaspserglupheile515520525tyrasnseraspproseralatyrglyleuglycyshisileleugly530535540glycysthrlysglyaspcystyrasnglnsersercysserglugly545550555560pheglytyrsercysasnthrserleuproserglnlysileasnala565570575phetyrprophelysmetasnleuasnargserglythrserasplys580585590leuglyleumettyrserleuvalproleuaspleuserproasnasn595600605valpheglygluleuaspseraspthrargasnvalileglylysgly610615620pheproalaglulysglythrleusertyrglyargglnprometleu625630635640valargglutrpvalasnglyalaasnalavallysleuseraspval645650655proleuthrleuglnmetthrasnmetthrserglylystyrtyrile660665670argvalargtyralaasnthrserasnalagluileserileasngln675680685glnvaltyralaglyasnglnasnvalglnasnglyglytrpserleu690695700proserthrthralaserasnvalalathrasnpheproasnglnleu705710715720tyrilethrglyileasnglyasntyrvalleugluaspilethrtrp725730735glyvalaspglyglnglylysproilethrileproserglyaspval740745750thrvalthrleuserargthrasnmetserglnserglyaspleuphe755760765ileaspargvalgluleuvalproalaalaproglnserasptyrile770775780asntyrserilethrserglnileproasncysasnasnpheaspthr785790795800ileiletrpaspglyasnglyalaglnargserthralathrvalthr805810815valserglyleuproaspasnasnglyileleuileserglyleuser820825830sertyrglytrpglnproleuglnaspvalasnglyhisglyasnasn835840845trpvalthrasnglyglyprophethrleuserasnasphisthrphe850855860thralaileserilecysglypheglnasnvalserserglylysile865870875880thrglylysleuser885<210>2<211>777<212>prt<213>人工序列<220><223>天然序列的变体<400>2metalaasnglythrlyslyshisvalilelyslystyrleugluleu151015ilelysproiletyrtyrlysglnasnserleuleupheserprophe202530ileleuserileproglycysaspileserglnhisglyserilepro354045alametlysargasncysphethrleuleuleutyrtyrargargtyr505560valmetalaglnleuprogluleuglnalaglnserpheileprotyr65707580asnvalleualaasnprovalthrglyasnseralaglythrasptrp859095glyglutrpleuserserleulysglualatrpthrglupheglnlys100105110thrglyserasplyspheleuglnservalleuserthrglytyrasp115120125alaalaglnglyglyserpheasntyrilealavalleuglnalagly130135140valalaileleuglythrvalleuglyalaglupheproglyilethr145150155160alailealavalproalaleuthrpheleuilelystrpvaltrppro165170175lysleuthrglyglyseralaglygluseraspthrglnlysmetleu180185190aspleuileasplysgluileglnglnlys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