编码具有糖转移活性的蛋白质的基因的制作方法

专利名称：编码具有糖转移活性的蛋白质的基因的制作方法
技术领域：
本发明涉及编码具有类黄酮5位糖转移活性的蛋白质的基因以及该基因的利用方法。
花卉产业正努力开发新的且品种多样的花卉。作为培育新品种的有效方法之一往往是改变花色，利用经典的育种方法，就几乎所有商业品种花卉生成范围广泛的花色都可成功。然而，因为使用这种方法每种花的基因库受到限制，所以单一的一种品种而具有范围广泛的各种着色品种还是很稀少的。
花色主要来自两种类型的色素、即类黄酮和类胡萝卜素。类黄酮的贡献是在从黄色至红色乃至绿色范围，而类胡萝卜素的贡献是在橙色或黄色的色调。对花色起主要贡献的类黄酮分子是以花青素、花翠素、3′-甲花翠素、甲基花青素、二甲花翠素以及花葵素等糖苷的花色素，不同的花色素会带来花色的显著变化。另外，花色也受到无色的类黄酮的辅助发色、金属配合物的形成、葡萄糖基化、酰化、甲基化以及液泡的pH的影响(Forkmann，植物育种(Plant Breeding),106,1,1991)。
从苯丙氨酸开始的花色苷的生物合成途径人们已经了解的很清楚(例如，植物细胞(Plant Cell),7、1071-1083,1995)、与生物合成有关的基因几乎都已经被克隆了。例如、认为与作为紫苏的花色苷的丙二酰紫苏色素(3-O-(6-O-(p-香豆酰基)-β-D-葡萄糖基)-5-O-(丙二酰-β-D-葡萄糖基)-花青素)的生物合成有关的基因中、到现在为止还没有报道的该同源基因只是类黄酮-3′-羟基化酶基因、UDP-葡萄糖花色苷(类黄酮)5-O-葡萄糖基转移酶(以下称5GT)基因、丙二酰基转移酶基因。
据推测，其中类黄酮-3′-羟基化酶与已经知道是属于细胞色素P450基因家族(植物细胞(Plant Cell),7、1071-1083,1995)的细胞色素P450基因之间表现出结构的同源性。
一般来说，类黄酮的3位羟基是通过葡萄糖修饰的，但人们认为通过以葡萄糖基化为代表的糖的修饰会使花色苷的稳定性和溶解度增大(《黄酮》The Flavonoids,Chapman &Hall,1994)。
编码催化该反应的UDP-葡萄糖花色素或类黄酮3-葡萄糖基转移酶(以下称3GT)的基因可以从玉米、大麦、金鱼草、龙胆等多种植物得到、相互间的氨基酸序列表现出显著的同源性。例如、单子叶植物玉米与双子叶植物龙胆的3GT的氨基酸序列的同源性为32％、单子叶植物玉米与大麦的3GT的氨基酸序列的同源性为73％、双子叶植物龙胆与茄子的3GT的氨基酸序列的同源性为46％。
另外、编码矮牵牛花的UDP-鼠李糖花色素3-葡萄糖苷鼠李糖基转移酶(3RT)的基因也已经克隆了。
可是、尽管很多植物的类黄酮的5位的羟基可被葡萄糖基化、但催化该反应的基因(5GT)还未得到。
另外、虽有测定将糖转移到矮牵牛花和紫罗兰的花色素5位上反应的例子(植物(Planta)160,341-347,1984、植物(Planta)168,586-591,1986)，但这些报道只是停留在用花卉的粗提取液或部分纯化的酶研究其酶学性质方面，而将该酶纯化到纯品的例子还没有。另外、一般来说、糖转移酶从生物化学角度讲是不稳定的、酶的纯化是困难的。
通过在类黄酮分子上附加糖使其色调变化的例子几乎还没有，因为对色调有很大影响的芳香族酰基可转移到花色素内的葡萄糖分子或鼠李糖分子上、那么控制糖转移反应在控制花色素的生物合成、进而控制花色上是重要的。而作为调控糖转移酶基因的表达改变花色的例子，如在转化矮牵牛花中，就是控制矮牵牛花3RT引起的反应来修饰花色的。
作为可能转化的植物，例如已知有蔷薇、菊花、石竹、大丁草花、矮牵牛花、蓝竹耳、草原龙胆、カラソュェ、郁金香、唐菖蒲等。
这里，本发明人等以获得编码具有类黄酮5位糖转移活性的蛋白质的基因作为课题、完成了本发明。
例如菊花的花色素以及蔷薇和石竹花的花色苷中的一部分结构中，5位的羟基还没有被葡萄糖基化。通过将本发明得到的5GT基因导入这些植物、可以改变花色苷的结构。
通过利用国际公开公报WO96/25500记载的酰基转移酶基因使类黄酮酰基化是有可能使花色改变、或是使类黄酮稳定、但酰基并不是直接与类黄酮结合、而是通过糖结合的、所以只是导入酰基转移酶基因的话、有时花色的变化并不充分、有时也不稳定。
然而、通过将5GT基因与酰基转移酶基因同时导入、可以使糖转移到类黄酮的5位上、进而可使其酰化、预料花色素的结构也会改变、花色也会变绿。
另外、如果利用反意义法或ュサプレシツョゥ法等抑制花色素的5位葡萄糖基化的植物的5GT基因的表达，可以抑制花色素的生物合成、其结果可以使花色改变。例如、如果在龙胆或草原龙胆中抑制5GT活性的话，可预料花色会变红。
本发明人利用基因重组技术，从紫苏、蓝竹耳、马鞭草以及矮牵牛花中分离出5GT的cDNA、确定了结构基因的碱基序列。就是说、提供编码存在于这些植物花色苷出现的组织中的5GT的DNA序列。而且由于该酶使得花色素系色素的5位上转移了糖、所以可用于花色的变化、也可增加花色素的稳定性。
为了得到编码本发明的酶的DNA，例如可以利用差别显示(Differential display)法。例如在紫苏(Perilla frutescens)中有蓄积花色苷的品种(例如紫薰)和没有蓄积花色苷的品种(青薰)、如果克隆存在于蓄积花色素的品种的而在不蓄积花色素品种中不存在的DNA的话，可以得到编码本发明的酶的DNA。
具体来说、从紫薰和青薰的叶提取RNA、按照常规方法合成cDNA、经电泳分离、可分离出存在于来自紫薰cDNA文库的而不存在于来自青薰cDNA文库的cDNA。然后以得到的cDNA作为探针、筛选来自紫薰的cDNA文库、得到编码本发明酶的基因。
在本发明中，作为上述的筛选的例子、利用差别显示法筛选来自紫苏的编码本发明酶的DNA(实施例1)、然后以得到的DNA作为探针、通过筛选来自马鞭草的cDNA、得到来自马鞭草的编码本发明酶的DNA(实施例2)、通过同样的操作可以得到来自蓝竹耳的编码本发明酶的基因(实施例3)。
然后确认了这些DNA能够表达具有本发明酶活性的蛋白质。
另外、得到了编码来自矮牵牛花的本发明酶的DNA(实施例4)。
作为本发明的DNA、例如有编码序列7～10或12中任一个序列记载的氨基酸序列的DNA。然而已知、具有通过添加、缺失数个氨基酸和/或通过其它氨基酸取代修饰的氨基酸序列的蛋白质也可维持与原来蛋白质同样的酶活性。因此、编码具有对序列号7～10或12中任一个序列记载的氨基酸序列添加、缺失一个或数个氨基酸的和/或通过其它氨基酸取代修饰的氨基酸序列、而且维持类黄酮5位糖转移活性的蛋白质的基因也属于本发明。
本发明还涉及序列1～4或6中任一个序列记载的碱基序列或编码记载的氨基酸序列的碱基序列或对那些部分、例如对编码共有区的6个氨基酸以上的氨基酸部分、例如在2至5×SSC、例如5×SSC、50℃的条件下杂化、而且编码具有类黄酮5位糖转移活性的蛋白质的基因。另外、最适的杂化温度因碱基序列或其长度的不同而不同、优选的是随着碱基序列变短、杂化温度也降低，例如编码6个氨基酸的碱基序列(18个碱基)其杂化温度优选是在50℃以下。
通过这样杂化选择的基因有来自天然的、例如来自植物的、如来自马鞭草或来自蓝竹耳的基因，也可以是来自其它植物的、例如来自矮牵牛花、蔷薇、石竹、风信子等植物的基因。另外、通过杂化选择的基因既可以是cDNA、也可以是基因组DNA。
另外本发明还涉及编码对7～10或12中任一个序列记载的氨基酸序列具有30％以上、优选是在50％以上、例如60％或70％以上的同源性、有时达90％的同源性的氨基酸序列、而且具有类黄酮5位糖转移活性的蛋白质的基因。就是说、就象实施例所给出的那样、编码本发明的酶的DNA与其它糖转移酶基因比较表现出20～30％的同源性。因此、本发明也包含编码表现出与7～10或12中记载的氨基酸序列30％以上的同源性、而且具有类黄酮5位糖转移活性的蛋白质的基因。
就象由实施例1～4的结果的比较所表明的那样、本发明酶的氨基酸序列因品种而异、种间的同源性在50％以上(参照实施例3和4)、例如60～70％(参照实施例2)、而来自同一品种的酶的氨基酸序列的同源性在90％以上(参照实施例1)。因此、编码对序列7～10或12中记载的氨基酸序列具有50％以上、例如60～70％以上、例如60％或70％以上的同源性、有时达90％的同源性的氨基酸序列、而且维持本发明的糖转移酶活性的蛋白质的基因也包括在本发明的范围。
具有天然的碱基序列的DNA、就象实施例具体记载的那样，例如可以通过cDNA文库筛选得到。
而编码具有修饰的氨基酸序列的酶的DNA是可以以具有天然的碱基序列的DNA为基础、利用常用的特定部位的诱导变异和PCR法合成。例如、将含有希望导入修饰的部位的DNA片段、是从上述得到的cDNA或基因组DNA经限制性内切酶消化得到的、作为模板、利用导入了所希望的变异的引物实施特定部位的诱导变异或PCR法、得到了导入了所希望的修饰的DNA片段、如果将该片段与编码目的酶的其它部分DNA连接就可以合成出编码具有修饰的氨基酸序列的酶的DNA了。
或者、为了得到编码具有缩短了的氨基酸序列的酶的DNA，例如可以通过所希望的限制性内切酶消化编码比目的氨基酸序列长的氨基酸序列的DNA、例如编码全长氨基酸序列的DNA，得到的DNA片段不能编码整个目的氨基酸序列时、不足部分可以通过连接合成DNA予以补足。
使用大肠杆菌以及酵母中的基因表达体系使该克隆表达、通过测定酶活、确认得到的基因是编码糖转移酶的基因，通过弄清楚于类黄酮的5位转移糖的糖转移酶基因的翻译区，就得到编码与本发明有关的糖转移酶的基因，另外、通过使该基因表达可以得到作为基因产物的于类黄酮5位转移糖的糖转移酶目的蛋白质。
或者是利用相应于序列7～10或12中任一个序列记载的氨基酸序列的抗体也可得到上述蛋白质。
因此本发明还涉及含有上述DNA的重组载体、特别是表达载体、以及通过该载体转化的宿主。作为宿主可以用原核生物或真核生物。作为原核生物可以利用细菌、例如属于Escherichia菌属的细菌、例如大肠杆菌(Escherichia coli)、属于Bacillus属的微生物、例如枯草杆菌(Bacillus subtilis)等常用的宿主。
作为真核的宿主可以使用低等真核生物、例如真核微生物、例如作为真菌的酵母或霉菌。作为酵母菌有属于Saccharomyces属的微生物、例如酿酒酵母(Saccharomyces cerevesiae)等、而作为霉菌有属于Aspergillus属的微生物、例如米曲霉(Aspergillus oryzae)、黑曲霉(Aspergillus niger)、以及属于Penicillium属的微生物等。也可以使用动物细胞或植物细胞、作为动物细胞可以使用小鼠、大鼠、猴、人等的细胞系。而也可以使用昆虫细胞、例如蚕细胞、或蚕的成虫本身作为宿主。
本发明的表达载体依赖于应导入该载体的宿主的种类，含有表达调控区、例如启动子以及终止子、复制起点等。作为细菌用的表达载体的启动子使用常用的启动子、例如trc启动子、tac启动子、lac启动子等，作为酵母用的启动子、例如可使用甘油醛-3-磷酸脱氢酶启动子、PHO5启动子等、作为霉菌用启动子、例如可使用淀粉酶、trp C等。而作为动物细胞宿主用启动子可以使用病毒性启动子、例如SV40早期启动子、SV40晚期启动子等。
表达载体的制备可以使用限制性内切酶、连接酶等按照常规方法进行。另外、表达载体对宿主的转化也是按照常规方法进行。
上述蛋白质的制造方法中、通过对上述表达载体转化的宿主进行培育、栽培或饲养、按照常规方法、例如过滤、离心分离、细胞破碎、凝胶过滤层析、离子交换层析等从培养物中回收目的蛋白质、进行纯化。
另外在本说明书中虽然叙述了来自紫苏、马鞭草、蓝竹耳以及矮牵牛花的类黄酮5位糖转移的糖转移酶(本发明中有时也简单称之“糖转移酶”)。可以通过直接使用或经部分改造的该酶的纯化方法纯化其它植物的糖转移酶、通过确定与该酶系有关的氨基酸序列、克隆编码该酶的基因。另外、通过使用来自与本发明有关的紫苏的糖转移酶的cDNA作为探针、可以从紫苏中得到其它的糖转移酶的cDNA、以及从其它植物得到别的糖转移酶的cDNA。因此、如果使用糖转移酶基因的一部分或全部，可以得到其它的糖转移酶基因。
另外、就象本说明书所说明的那样，纯化来自紫苏、马鞭草、蓝竹耳以及矮牵牛花的糖转移酶、通过按照常规方法得到对应于该酶的抗体，可以克隆制造与该抗体反应的蛋白质的cDNA或染色体DNA。因此、本发明并不只限制于来自紫苏、马鞭草、蓝竹耳以及矮牵牛花的糖转移酶基因，也涉及更广泛的糖转移酶。
另外本发明也涉及通过导入糖转移酶基因，颜色受到调节的植物或其后代或它们的组织，其形态即使是切花也可以。
另外、本发明中、含有花色素的类黄酮的糖转移反应中作为糖的供体有UDP-葡萄糖。
以下根据实施例详细说明本发明。实验过程只要没有特别叙述的都是按照《分子克隆》(Molecular Cloning)(冷泉港、1989)《新生物化学实验手册》第3卷(化学同人1996)、国际公开公报WO96/25500记载的方法进行的。
实施例1、在红紫苏中特异表达的基因的克隆(1)差别显示紫苏(Perilla futescens)中，有叶子中蓄积花色苷的品种(例如、紫薰(サカタのタネ)和叶子中不蓄积花色素的品种(例如、青薰(サカタのタネ))，据报道，主要的花色素的结构是丙二酰紫苏色苷(3-O-(p-香豆基)-β-D-葡萄糖基)-5-O-(6-O-丙二酰-β-D-葡萄糖基)-花青素(农业生物化学(Agri.Biol.Chem.)53:197-198,1989)。
差别显示是《科学》(Scienc)257,967-971(1992)报道的方法，被用于获得组织特异表达的基因等研究中。
按照热酚法(《植物分子生物学手册》(Plant Molecular BiologyManual.)Kluwer Academic Publishers1994pp.D5/1-13)从两种紫苏的叶子中提取总RNA。使用mRNA分离试剂盒(Clonetech公司)从得到的总RNA中纯化聚A+RNA。使用附加了锚的oligo dT引物(GenHunter公司的H-T11G、H-T11A、H-T11C)于33μl反应液中对0.9μg的oligoA+RNA进行逆转录，得到单链cDNA。以该cDNA作为模板、以同样附加了锚的oligo dT引物和合成引物(GenHunter公司的H-AP1至8)作为引物进行PCR。
PCR反应液的体积为20μl，含有2μl的cDNA溶液，0.2μM的H-T11G、H-T11A、H-T11C中的任一种引物、0.2μM的H-AP1至8中的任一种引物、0.12μM dNTP、5或10μCi[32P]dCTP、10mMTris-HCl(pH9.0)、50mM KCl0.01％Triton X-100、1.25mM MgCl2、1u的Taq聚合酶。反应条件如下。首先于72℃下保持20秒后、进行40个循环反应，每一循环反应都是94℃30秒、40℃2分钟、72℃30秒，然后于72℃保持5分钟。
利用确定DNA碱基序列时用的聚丙烯酰胺凝胶分离上述扩增得到的DNA。凝胶干燥后、对X胶片曝光。在得到的大约2600条带中、比较两个品种的紫苏、只在紫薰中看到的带有36条。将这些带从干燥的凝胶中切下来、溶解于100μl的水中。溶出的DNA用乙醇沉淀，溶解于20μl的水中。其中一半量的DNA作为模板、分别进行上述的PCR反应，可以扩增有关33条带的DNA片段。用这些DNA片段进行文库的筛选和Northern解析。
(2)Northern解析用以上33种DNA探针按照以下方法进行Northern解析。来自紫薰和青薰的oligo A+RNA经含有1.2％琼脂糖的甲醛凝胶分离后、转移到尼龙膜上。该膜在5×SSPE、5×Denhalt液、0.5％SDS、20μg/ml的变性鲑DNA存在下，于65℃下与32P标记的上述DNA探针杂交过夜。将杂交的膜用1×SSPE、0.1％SDS于65℃下洗净，得到放射自显影的胶片。结果、只有5种探针特异地出现在紫薰中。预计这些克隆是与花色素的生物合成有关的基因。
(3)cDNA文库的筛选利用从紫薰的叶子得到的聚A+RNA，使用完全快速克隆系统(CompleteRapid Cloning System)λgt10(Amersham)，制备以入gt10为载体的cDNA文库。该cDNA文库首先使用先前讲到的5种DNA片段进行筛选，得到各个相应的cDNA。其中取名为3R5的克隆由于是用来自H-T11A和H-AP3的引物的DNA片段得到的产物，所以表现出在已报道的玉米的类黄酮-3-O-糖转移酶的氨基酸水平上大约26％的同源性。
另外通过使用了同样探针的文库的筛选可以得到称之3R4以及3R6的克隆，这些克隆都表现出与3R5有极高的同源性。推断为3R4以及3R6的总碱基序列的氨基酸序列分别表示在序列表中的序列1和序列2中。而推断的3R4和3R6编码的蛋白质的氨基酸序列表现出92％的同源性。
取名为8R6的克隆由于是用来自H-T11G和H-AP8的引物的DNA片段得到的产物，所以没有表现出与到现在为止报道的DNA碱基序列的有意义的同源性。该序列表示在序列5中。虽然，8R6极有可能是与花色苷生物合成有关的基因，但其结构由于与至今报道的基因没有同源性，所以推测该基因是与花色苷生物合成有关的新的基因。
如果考虑到紫苏的花色苷(上述的丙二酰紫苏色素)的结构的话，预料该基因是丙二酰基转移酶的基因。为了证明这一点，只要使该基因在酵母或大肠杆菌中表达，使表达的产物与作为底物的花色苷和丙二酰CoA进行反应就可证明。这样的实验可以利用诸如国际公开公报WO96/25500记载的方法进行。丙二酰基转移酶基因在人为地改变花色素的结构上也是有用的。
(4)3R4的cDNA在酵母中的表达利用T4DNA聚合酶(宝酒造公司制造)补平p3R4的BstXⅠ的酶切部位，再于连接体内的BamHⅠ酶切部位酶切得到大约1.5kb的DNA片段，该DNA片段与pYE22m的EcoRⅠ酶切末端补平，再经BamHⅠ消化得到的大约8kb的DNA片段连接得到的质粒作为pY3R4。
而带有pYE22m的大肠杆菌JM109菌株命名为Escherichia coliSBM335，以FERM BP-5435保藏于工业技术院生命工程工业技术研究所。在pY3R4中，编码糖转移酶的cDNA连接于作为酵母的启动子组成之一的甘油醛-3磷酸脱氢酶的启动子的下游，通过同一个启动子控制转录。利用伊藤等人的方法(Ito等，细菌学杂志，(J.Bacteriol.),153,163-166,1983)用pY3R4转化酵母Saccharomycescerevesiae G1315(Ashikari等应用微生物学与生物技术(Appl.Microbiol.Biotechnol.)30,515-520,1989)。转化的酵母通过色氨酸合成能力恢复进行选择。得到的转化菌株于10ml的含有1％酪蛋白水解物(Difco公司)的Burkholder培养基(Burkholder,Amer.J.Bot.30,206-210)在30℃条件下振荡培育24小时。
同时，作为对照实验，也同样培养能自然恢复色氨酸合成能力的酵母。收集这些酵母菌，将它们悬浮于悬浊缓冲液(100mM磷酸缓冲液(pH8.5)、0.1％(v/v)2-巯基乙醇、10μM APMSF、100μM UDP-葡萄糖)，通过加入玻璃珠(Glass Beads425-600microns Acid-Wash、Sigma公司)激烈振荡磨碎菌体。经15000rpm20分钟离心，将离心上清作为粗酶液，用于以下酶活测定。
(5)酶活测定使含有20μl粗酶液的50μl反应液(100mM磷酸缓冲液(pH8.5)、670μM花青素-3-葡萄糖苷、1mM UDP-葡萄糖)于30℃下反应10分钟后、加入含有0.1％TFA的50％乙腈溶液50μl，终止反应。将经15000rpm5分钟离心得到的上清通过Samprep LCR4(T)-LC(Millipore公司)除去不溶物。得到的样品利用高效液相色谱分析。分析是用反相柱(AsahipakODP-50,4.6mmφ*250mm昭和电工株式会社制造)，流动相为A溶液0.5％TFA/H2O、B溶液为0.5％TFA50％CH3CN、流速为0.6ml/min。在进行B20％→B100％(20min)梯度洗脱后，再维持5min B100％的洗脱。
用于分析的反应液为20μl。检测是通过A520nm,AUFS0.5(岛津SPD-10A)和光敏二极管阵列检测器(岛津SPD-M16A)的600-250的吸收进行的。如果使pY3R4表达的酵母的粗酶液反应，就将它加到底物花青素-3-葡萄糖苷中(展开时间17分钟)，于14.5分钟展开时生成新出现的物质。由于该物质在对照实验的酵母粗酶液反应中没有看到，所以认为该物质是通过来自pY3R4的蛋白质活性产生的物质。与花青素-3,5-二葡萄糖苷的混合色谱分析的结果表明，该反应生成物的展开时间与花青素-3,5-二葡萄糖苷的展开时间一致，两者的吸收光谱也一致。由以上事实可知，紫苏的3R4的cDNA编码5GT。
实施例2．马鞭草(Verbena hybrida)的5GT基因的克隆(1)eDNA文库的制备从马鞭草品种花球紫(三得利)收集花瓣、于液氮中用乳钵磨碎。通过利用胍硫氰酸盐/氯化铯的方法从该磨碎物中提取RNA，再使用Oligotex(宝酒造制造)，用制造者推荐的方法得到聚A+RNA。使用了胍硫氰酸盐/氯化铯的方法是依据R.McGookin,Robert J.Slater等人、分子生物学方法(Methods in Molecular Biology)vol2,(Humana PressInc.1984)详细给出的方法。
以得到的聚A+RNA为模板，利用Stratagene公司的2AP-cDNA合成试剂盒合成双链cDNA，然后利用Uni-ZAP XR克隆试剂盒(Stratagene公司)用制造者推荐的方法制备cDNA文库。
(2)5GT cDNA的克隆以紫苏的p3R4的cDNA作为探针，按以下操作筛选上述得到的λ嗜菌体文库。将膜于42℃放置于杂交缓冲液(5×SSC,30％甲酰胺、50mM磷酸钠缓冲液(pH7.0)、1％SDS、2％Blocking reagent(Boehringer公司)、0.1％月桂酰肌氨酸、80g/ml鲑鱼精子DNA)中1小时。然后将DIG标记的紫苏的5GT基因、p3R4的DNA片段加入杂交溶液中，再温育16小时。
用洗净液(5×SSC50℃、1％SDS)将滤膜洗净、然后通过依据碱性磷酸酶标记的DIG特异的抗体的免疫测定法(Boehringer公司)在5-溴-4-氯-3-吲哚磷酸和硝基蓝四唑盐的发色反应中检测出探针杂交的克隆。检测方法按使用说明书进行。
检测结果得到7个阳性克隆。用Stratagene公司推荐的方法将这些cDNA回收到质粒pBluescript SK中。通过琼脂糖凝胶电泳研究cDNA的长度时，确认了插入最长2.0kb。
(3)碱基序列的确定从得到的克隆中提取质粒、使用测序仪ABI373A(Perkin Elmer公司)、利用该公司推荐的依据荧光试剂的双脱氧测序方法确定cDNA的3′和5′末端附近的碱基序列。结果发现7个克隆中的5个克隆之间具有同样的碱基序列，认为是cDNA长度不同的克隆。其中还确定了pSHGT8的整个碱基序列。碱基序列的确定是使用了Kilo-Sequence用deletion试剂盒(宝酒造制)得到一系列的缺失克隆、或利用对pSHGT8内部序列特异的寡核苷酸引物、按上述那样进行。
(4)碱基序列和氨基酸序列的比较插入pSHGT8的cDNA是2062bp，其中存在由1386bp(含终止码)构成的开放读框(ORF)。序列3给出了该序列。该ORF的氨基酸序列表现出与紫苏p3R4编码的5GT的氨基酸序列有68％、与p3R6编码的有64％的同源性。而与单子叶植物以及双子叶植物的3GT有22～25％、与矮牵牛花的3RT有21％的同源性。
(5)在酵母中的表达和酶活的测定将BamHⅠ/XhoⅠ消化pSHGT8后得到的大约2.0kb的DNA片段与BamHⅠ/SalⅠ消化pYE22m得到的大约8kb的DNA片段连接得到的质粒作为pYHGT8。与实施例1一样、在酵母菌体内表达pYHGT8、测定有关pYHGT8编码的蛋白质的酶活。结果、如果使导入pYHGT8的酵母粗酶液反应的话，可生成与花青素-3,5-二葡萄糖苷的展开时间、吸收光谱都一致的产物。由这一事实可知，马鞭草的pSHGT8的cDNA编码5GT。
实施例3．蓝竹耳的5GT基因的克隆(1)cDNA文库的制备从蓝竹耳品种Summer Wave Blue(三得利(株))收集花瓣、于液氮中用乳钵磨碎。通过利用胍硫氰酸盐/氯化铯的方法从该磨碎物中提取RNA，再使用oligotex(宝酒制造)，用制造者推荐的方法得到聚A+RNA。使用的胍硫氰酸盐/氯化铯方法是依据R.McGookin,Robert J.Slater等人于分子生物学方法(Methods in Molecular Biology)vol2,(HumanaPress Inc.1984)中详细给出的方法。
以得到的聚A+RNA为模板，利用Stratagene公司的ZAP-cDNA合成试剂盒合成双链cDNA，然后利用Uni-ZAP XR克隆试剂盒(Stratagene公司)用制造者推荐的方法制备cDNA文库。
(2)5GT cDNA的克隆以紫苏的p3R4的cDNA作为探针，与实施例2一样筛选上述得到的λ嗜菌体文库。结果得到8个阳性克隆。将cDNA回收到质粒pBluescript SK中，通过琼脂糖凝胶电泳研究该cDNA的长度，确认插入了最长1.6kb。
(3)碱基序列的确定从得到的克隆中提取质粒、与实施例2一样确定两末端附近的碱基序列。结果发现其中的6个克隆之间具有同样的碱基序列，认为是cDNA长度不同的克隆。其中还确定了该6个克隆中pSTGT5的整个碱基序列。
(4)碱基序列和氨基酸序列的比较插入pSTGT5的cDNA是1671bp，其中存在由1437bp(含终止码)构成的开放读框(ORF)。序列4给出了该序列。该ORF的氨基酸序列表现出与紫苏p3R4编码的5GT的氨基酸序列有58％、与p3R6编码的有57％的同源性。而与马鞭草pSHGT8编码的有57％的同源性。而与单子叶植物以及双子叶植物的3GT有19～23％、与矮牵牛花的3RT有20％的同源性。
(5)5GT基因在酵母中的表达将SamⅠ/KpnⅠ消化pSTGT5后得到的大约1.6kb的DNA片段与pYE22m的EcoRⅠ酶切部位补平后被KpnⅠ消化得到的大约8kb的DNA片段连接得到的质粒作为pYTGT5。与实施例1一样、在酵母菌体内表达pYTGT5、测定有关pSTGT5编码的蛋白质的酶活。结果、如果使导入pYTGT5的酵母粗酶液反应的话，可得到与花青素-3,5-二葡萄糖苷的展开时间、吸收光谱都一致的产物。由这一事实可知，蓝竹耳的pSTGT5的cDNA编码5GT。
实施例4．矮牵牛花的5GT基因的克隆(1)cDNA文库的制备在从矮牵牛花品种Old Glory Blue的花瓣中提取RNA的基础上，按照T.Holton等人的报道(植物学报Plant Journal,1993 4:1003-1010)详细记载的那样制备cDNA文库。
(2)5GT cDNA的克隆以上述那样操作得到的紫苏、蓝竹耳、马鞭草的cDNA作为探针，与实施例2一样进行筛选。将结果得到的阳性克隆中的4个克隆回收到质粒pBluescript SK-中，通过琼脂糖凝胶电泳研究该cDNA的长度，确认插入了最长2.0kb的cDNA。
(3)碱基序列的确定确定5’末端附近的碱基序列。结果表明这些克隆中的2个克隆的pSPGT1编码表现出与到目前为止得到的紫苏、蓝竹耳、马鞭草的5GT有很高同源性的氨基酸序列。于是确定了pSPGT1的整个碱基序列。
(4)碱基序列和氨基酸序列的比较插入pSPGT1的cDNA是2105bp，其中存在由1407bp(含终止码)构成的ORF。序列6给出了该序列。该ORF的氨基酸序列表现出与紫苏p3R4编码的5GT的氨基酸序列有57％、与p3R6编码的有54％、与马鞭草pSHGT8编码的有55％、与蓝竹耳的pTGT5编码的有51％的同源性。而与单子叶植物以及双子叶植物的3GT有20～29％、与矮牵牛花的3RT有20％的同源性。由这一事实可以认为从矮牵牛花得到的pSPGT1的cDNA编码5GT。如上所述，进行了编码来自紫苏、马鞭草、蓝竹耳以及矮牵牛花的向类黄酮5位上转移糖的酶的cDNA的克隆并确定了其碱基序列。通过酵母中活性的表达阐明了分离到的cDNA是编码5GT的基因。通过将该cDNA与适当的植物表达载体连接，导入植物中，使其有时具有5GT的活性、有时使其活性增加、有时又使其活性降低，用来调节植物的花色成为可能。而通过利用该酶的活性，在植物体中或在试管中改变花色素的结构可以合成更稳定的花色素。
序列序列1序列长度1507序列类型核酸链数双链拓扑类型直链形起源生物名称紫苏(Perilla frutescens)组织种类叶直接起源文库名cDNA library克隆名p3R4序列GAAAATTTCC ACAAAA ATG GTC CGC CGC CGC GTG CTG CTA GCA ACG TTT 49Met Val Arg Arg Arg Val Leu Leu Ala Thr Phe1 5 10CCT GCG CAA GGC CAC ATA AAT CCC GCC CTC CAA TTC GCC AAG AGA CTC97Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln Phe Ala Lys Arg Leu15 20 25CTA AAA GCC GGC ACT GAC GTC ACA TTT TTC ACG AGC GTT TAT GCA TGG 145Leu Lys Ala Gly Thr Asp Val Thr Phe Phe Thr Ser Val Tyr Ala Trp30 35 40CGC CGC ATG GCC AAC ACA GCC TCC GCC GCT GCC GGA AAC CCA CCG GGC 193Arg Arg Met Ala Asn Thr Ala Ser Ala Ala Ala Gly Asn Pro Pro Gly45 50 55CTC GAC TTC GTG GCG TTC TCC GAC GGC TAC GAC GAC GGG CTG AAG CCC 241Leu Asp Phe Val Ala Phe Ser Asp Gly Tyr Asp Asp Gly Leu Lys Pro60 65 70 75TGC GGC GAC GGG AAG CGC TAC ATG TCC GAG ATG AAA GCC CGC GGC TCC 289Cys Gly Asp Gly Lys Arg Tyr Met Ser Glu Met Lys Ala Arg Gly Ser80 85 90GAG GCC TTA AGA AAC CTC CTT CTC AAC AAC CAC GAC GTC ACG TTC GTC 337Glu Ala Leu Arg Asn Leu Leu Leu Asn Asn His Asp Val Thr Phe Val95 100 105GTC TAC TCC CAC CTC TTT GCA TGG GCG GCG GAG GTG GCG CGT GAG TCC 385Val Tyr Ser His Leu Phe Ala Trp Ala Ala Glu Val Ala Arg Glu Ser110 115 120CAG GTC CCG AGC GCC CTT CTC TGG GTC GAG CCC GCC ACC GTG CTG TGC 433Gln Val Pro Ser Ala Leu Leu Trp Val Glu Pro Ala Thr Val Leu Cys125 130 135ATA TAT TAC TTC TAC TTC AAC GGC TAC GCA GAC GAG ATC GAC GCC GGT 481Ile Tyr Tyr Phe Tyr Phe Asn Gly Tyr Ala Asp Glu Ile Asp Ala Gly140 145 150 155TCC GAC GAA ATT CAG CTC CCT CGG CT7 CCA CCC CTG GAG CAG CGC AGT 529Ser Asp Glu Ile Gln Leu Pro Arg Leu Pro Pro Leu Glu Gln Arg Ser160 165 170CTT CCG ACC TTT CTG CTG CCG GAG ACA CCG GAG AGA TTC CGG TTG ATG 577Leu Pro Thr Phe Leu Leu Pro Glu Thr Pro Glu Arg Phe Arg Leu Met175 180 185ATG AAG GAG AAG CTG GAA ACT TTA GAC GGT GAA GAG AAG GCG AAA GTG 625Met Lys Glu Lys Leu Glu Thr Leu Asp Gly Glu Glu Lys Ala Lys Val190 195 200TTG GTG AAC ACG TTT GAT GCG TTG GAG CCC GAT GCA CTC ACG GCT ATT 673Leu Val Asn Thr Phe Asp Ala Leu Glu Pro Asp Ala Leu Thr Ala Ile205 210 215GAT AGG TAT GAG TTG ATC GGG ATC GGG CCG TTG ATT CCC TCC GCC TTC 721Asp Arg Tyr Glu Leu Ile Gly Ile Gly Pro Leu Ile Pro Ser Ala Phe220 225 230 235TTG GAC GGC GGA GAT CCC TCC GAA ACG TCT TAC GGC GGC GAT CTT TTC 769Leu Asp Gly Gly Asp Pro Ser Glu Thr Ser Tyr Gly Gly Asp Leu Phe240 245 250GAA AAA TCG GAG GAG AAT AAC TGC GTG GAG TGG TTG GAC ACG AAG CCG 817Glu Lys Ser Glu Glu Asn Asn Cys Val Glu Trp Leu Asp Thr Lys Pro255 260 265AAA TCT TCG GTG GTG TAT GTG TCG TTT GGG AGC GTT TTG AGG TTT CCA 865Lys Ser Ser Val Val Tyr Val Ser Phe Gly Ser Val Leu Arg Phe Pro270 275 280AAG GCA CAA ATG GAA GAG ATT GGG AAA GGG CTA TTA GCC TGC GGA AGG 913Lys Ala Gln Met Glu Glu Ile Gly Lys Gly Leu Leu Ala Cys Gly Arg285 290 295CCG TTT TTA TGG ATG ATA CGA GAA CAG AAG AAT GAC GAC GGC GAA GAA 961Pro Phe Leu Trp Met Ile Arg Glu Gln Lys Asn Asp Asp Gly Glu Glu300 305 310 315GAA GAA GAA GAG TTG AGT TGC ATT GGG GAA TTG AAA AAA ATG GGG AAA 1009Glu Glu Glu Glu Leu Ser Cys Ile Gly Glu Leu Lys Lys Met Gly Lys320 325 330ATA GTT TCG TGG TGC TCG CAG TTG GAG GTT CTG GCG CAC CCT GCG TTG 1057Ile Val Ser Trp Cys Ser Gln Leu Glu Val Leu Ala His Pro Ala Leu335 340 345GGA TGT TTC GTG ACG CAT TGT GGG TGG AAC TCG GCT GTG GAG AGC TTG 1105Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser Ala Val Glu Ser Leu350 355 360AGT TGC GGG GTT CCG GTG GTG GCG GTG CCG CAG TGG TTT GAT CAG ACG 1153Ser Cys Gly Val Pro Val Val Ala Val Pro Gln Trp Phe Asp Gln Thr365 370 375ACG AAT GCG AAG CTG ATT GAG GAT GCG TGG GGG ACA GGG GTG AGA GTG 1201Thr Asn Ala Lys Leu Ile Glu Asp Ala Trp Gly Thr Gly Val Arg Val380 385 390 395AGA ATG AAT GAA GGG GGT GGG GTT GAT GGA TCT GAG ATA GAG AGG TGT 1249Arg Met Asn Glu Gly Gly Gly Val Asp Gly Ser Glu Ile Glu Arg Cys400 405 410GTG GAG ATG GTG ATG GAT GGG GGT GAG AAG AGC AAA CTA GTG AGA GAA 1297Val Glu Met Val Met Asp Gly Gly Glu Lys Ser Lys Leu Val Arg Glu415 420 425AAT GCC ATA AAA TGG AAG ACT TTG GCC AGA GAA GCC ATG GGA GAG GAT 1345Asn Ala Ile Lys Trp Lys Thr Leu Ala Arg Glu Ala Met Gly Glu Asp430 435 440GGA TCT TCA CTC AAG AAT CTC AAC GCC TTT CTT CAT CAA GTT GCA CGT 1393Gly Ser Ser Leu Lys Asn Leu Asn Ala Phe Leu His Gln Val Ala Arg445 450 455GCT TAATACACAA AATGGCTTTC CACTTTTAAT CTACTCAAAC ACCGGTTCAA 1446Ala460ATAAATATCC CCTTCCACTT CTTTCTATTT CACTATCACA TTTATAATTT TAGTAACAAA 1506A 1507序列2序列长度1470序列类型核酸链数双链拓扑类型直链形起源生物名称紫苏(Perilla frutescens)组织种类叶直接起源文库名cDNA library克隆名p3R6序列ACCAAACCAA AACAAAATTT CCACAAAA ATG GTC CGC CGC CGC GTG CTG CTA 48Met Val Arg Arg Arg Val Leu Leu1 5GCA ACG TTT CCG GCG CAA GGC CAC ATA AAT CCC GCC CTC CAA TTC GCC96Ala Thr Phe Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln Phe Ala10 15 20AAG AGA CTC CTA AAA GCC GGC ACT GAC GTC ACG TTT TTC ACG AGC GTT 144Lys Arg Leu Leu Lys Ala Gly Thr Asp Val Thr Phe Phe Thr Ser Val25 30 35 40TAT GCA TGG CGC CGC ATG GCC AAC ACA GCC TCC GCC GCT GCC GGA AAC 192Tyr Ala Trp Arg Arg Met Ala Asn Thr Ala Ser Ala Ala Ala Gly Asn45 50 55CCA CCG GGC CTC GAC TTC GTG GCG TTC TCC GAC GGC TAC GAC GAC GGG 240Pro Pro Gly Leu Asp Phe Val Ala Phe Ser Asp Gly Tyr Asp Asp Gly60 65 70CTG AAG CCC GGC GGC GAC GGG AAG CGC TAC ATG TCC GAG ATG AAA GCC 288Leu Lys Pro Gly Gly Asp Gly Lys Arg Tyr Met Ser Glu Met Lys Ala75 80 85CGC GGC TCC GAG GCC TTA AGA AAC CTC CTT CTC AAC AAC GAC GAC GTC 336Arg Gly Ser Glu Ala Leu Arg Asn Leu Leu Leu Asn Asn Asp Asp Val90 95 100ACT TTC GTC GTC TAC TCC CAC CTC TTT GCA TGG GCG GCG GAG GTG GCG 384Thr Phe Val Val Tyr Ser His Leu Phe Ala Trp Ala Ala Glu Val Ala105 ll0 115 120CGT TTG TCC CAC GTC CCG ACC GCC CTT CTC TGG GTC GAG CCC GCC ACC 432Arg Leu Ser His Val Pro Thr Ala Leu Leu Trp Val Glu Pro Ala Thr125 130 135GTG CTG TGC ATA TAC CAC TTC TAC TTC AAC GGC TAC GCA GAC GAG ATC 480Val Leu Cys Ile Tyr His Phe Tyr Phe Asn Gly Tyr Ala Asp Glu Ile140 145 150GAC GCC GGT TCC AAT GAA ATT CAG CTC CCT CGG CTT CCA TCC CTG GAG 528Asp Ala Gly Ser Ash Glu Ile Gln Leu Pro Arg Leu Pro Ser Leu Glu155 160 165CAG CGC AGT CTT CCG ACG TTT CTG CTG CCT GCG ACG CCG GAG AGA TTC 576Gln Arg Ser Leu Pro Thr Phe Leu Leu Pro Ala Thr Pro Glu Arg Phe170 175 180CGG TTG ATG ATG AAG GAG AAG CTG GAA ACT TTA GAC GGT GAA GAG AAG 624Arg Leu Met Met Lys Glu Lys Leu Glu Thr Leu Asp Gly Glu Glu Lys185 190 195 200GCG AAA GTA TTG GTG AAC ACG TTT GAT GCG TTG GAG CCC GAT GCA CTC 672Ala Lys Val Leu Val Asn Thr Phe Asp Ala Leu Glu Pro Asp Ala Leu205 210 215ACG GCT ATT GAT AGG TAT GAG TTG ATC GGG ATC GGG CCG TTG ATT CCC 720Thr Ala Ile Asp Arg Tyr Glu Leu Ile Gly Ile Gly Pro Leu Ile Pro220 225 230TCC GCC TTC TTG GAC GGC GAA GAT CCC TCC GAA ACG TCT TAC GGC GGC 768Ser Ala Phe Leu Asp Gly Glu Asp Pro Ser Glu Thr Ser Tyr Gly Gly235 240 245GAT CTT TTC GAA AAA TCG GAG GAG AAT AAC TGC GTG GAG TGG TTG AAC 816Asp Leu Phe Glu Lys Ser Glu Glu Asn Asn Cys Val Glu Trp Leu Asn250 255 260TCG AAG CCG AAA TCT TCG GTG GTG TAT GTG TCG TTT GGG AGC GTT TTG 864Ser Lys Pro Lys Ser Ser Val Val Tyr Val Ser Phe Gly Ser Val Leu265 270 275 280AGG TTT CCA AAG GCA CAA ATG GAA GAG ATT GGG AAA GGG CTA TTA GCC 912Arg Phe Pro Lys Ala Gln Met Glu Glu Ile Gly Lys Gly Leu Leu Ala285 290 295TGC GGA AGG CCC TTT TTA TGG ATG ATA CGA GAA CAG AAG AAT GAC GAC 960Cys Gly Arg Pro Phe Leu Trp Met Ile Arg Glu Gln Lys Asn Asp Asp300 305 310GGC GAA GAA GAA GAA GAA GAA GAA GAG TTG AGT TGC ATT GGG GAA TTG 1008Gly Glu Glu Glu Glu Glu Glu Glu Glu Leu Ser Cys Ile Gly Glu Leu315 320 325AAA AAA ATG GGG AAA ATA GTG TCG TGG TGC TCG GAG TTG GAG GTT CTG 1056Lys Lys Met Gly Lys Ile Val Ser Trp Cys Ser Gln Leu Glu Val Leu330 335 340GCG CAC CCT GCG TTG GGA TGT TTC GTG ACG CAT TGT GGG TGG AAC TCG 1104Ala His Pro Ala Leu Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser345 350 355 360GCT GTG GAG AGC TTG AGT TGC GGG ATT CCG GTG GTG GCG GTG CCG CAG 1152Ala Val Glu Ser Leu Ser Cys Gly Ile Pro Val Val Ala Val Pro Gln365 370 375TGG TTT GAT CAG ACG ACG AAT GCG AAG CTG ATT GAG GAT GCG TGG GGG 1200Trp Phe Asp Gln Thr Thr Asn Als Lys Leu Ile Glu Asp Ala Trp Gly380 385 390ACA GGG GTG AGA GTG AGA ATG AAT GAA GGG GGT GGG GTT GAT GGA TGT 1248Thr Gly Val Arg Val Arg Met Asn Glu Gly Gly Gly Val Asp Gly Cys395 400 405GAG ATA GAA AGG TGT GTG GAG ATG GTG ATG GAT GGG GGT GAC AAG ACC 1296Glu Ile Glu Arg Cys Val Glu Met Val Met Asp Gly Gly Asp Lys Thr410 415 420AAA CTA GTG AGA GAA AAT GCC ATC AAA TGG AAG ACT TTG GCC AGA CAA 1344Lys Leu Val Arg Glu Asn Ala Ile Lys Trp Lys Thr Leu Ala Arg Gln425 430 435 440GCC ATG GGA TAGGATGGAT CTTCACTCAA CAATCTCAAC GCCTTTCTTC 1393Ala Met Gly443GTCAAGTTGC ACACTTTTAA TCTGCTCAAA CAGCGGTTCA AATAAATATC CCCTTCCACT 1453TAAAAAAAAA AAAAAAA1470序列3序列长度2062序列类型核酸链数双链拓扑类型直链形起源生物名称马鞭草(Verbena hybrida)
组织种类花瓣直接起源文库名cDNA library克隆名pSHGT8序列ATTTTACCAA AAAAATAAAA AAAAA ATG AGC AGA GCT CAC GTC CTC TTG GCC 52Met Ser Arg Ala His Val Leu Leu Ala1 5ACA TTC CCA GCA CAG GGA CAC ATA AAT CCC GCC CTT CAA TTC GCC AAG100Thr Phe Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln Phe Ala Lys10 15 20 25CGT CTC GCA AAT GCC GAC ATT CAA GTC ACA TTC TTC ACC AGC GTC TAC148Arg Leu Ala Asn Ala Asp Ile Gln Val Thr Phe Phe Thr Ser Val Tyr30 35 40GCA TGG CGC CGC ATG TCC AGA ACC GCC GCT GGC TCA AAC GGG CTC ATC196Ala Trp Arg Arg Met Ser Arg Thr Ala Ala Gly Ser Asn Gly Leu Ile45 50 55AAT TTT GTG TCG TTT TCC GAC GGG TAT GAC GAC GGG TTA CAG CCC GGA244Asn Phe Val Ser Phe Ser Asp Gly Tyr Asp Asp Gly Leu Gln Pro Gly60 65 70GAC GAT GGG AAG AAC TAC ATG TCG GAG ATG AAA AGC AGA GGT ATA AAA292Asp Asp Gly Lys Asn Tyr Met Ser Glu Met Lys Ser Arg Gly Ile Lys75 80 85GCC TTG AGC GAT ACT CTT GCA GCC AAT AAT GTC GAT CAA AAA AGC AGC340Ala Leu Ser Asp Thr Leu Ala Ala Asn Asn Val Asp Gln Lys Ser Ser90 95 100 105AAA ATC ACG TTC GTG GTG TAC TCC CAC CTC TTT GCA TGG GCG GCC AAG 388Lys Ile Thr Phe Val Val Tyr Ser His Leu Phe Ala Trp Ala Ala Lys110 115 120GTG GCG CGT GAG TTC CAT CTC CGG AGC GCG CTA CTC TGG ATT GAG CCA 436Val Ala Arg Glu Phe His Leu Arg Ser Ala Leu Leu Trp Ile Glu Pro125 130 135GCT ACG GTG TTG GAT ATA TTT TAC TTT TAT TTC AAC GGC TAT AGC GAC 484Ala Thr Val Leu Asp Ile Phe Tyr Phe Tyr Phe Asn Gly Tyr Ser Asp140 145 150GAA ATC GAT GCG GGT TCG GAT GCT ATT CAC TTG CCC GGA GGA CTC CCA 532Glu Ile Asp Ala Gly Ser Asp Ala Ile His Leu Pro Gly Gly Leu Pro155 160 165GTG CTG GCC CAG CGT GAT TTA CCG TCT TTC CTT CTT CCT TCC ACG CAT 580Val Leu Ala Gln Arg Asp Leu Pro Ser Phe Leu Leu Pro Ser Thr His170 175 180 185GAG AGA TTC CGT TCA CTG ATG AAG GAG AAA TTG GAA ACT TTA GAA GGT 628Glu Arg Phe Arg Ser Leu Met Lys Glu Lys Leu Glu Thr Leu Glu Gly190 195 200GAA GAA AAA CCT AAG GTC TTG GTG AAC AGC TTT GAT GCG TTG GAG CCT 676Glu Glu Lys Pro Lys Val Leu Val Asn Ser Phe Asp Ala Leu Glu Pro205 210 215GAT GCG CTC AAG GCC ATT GAT AAG TAC GAG ATG ATT GCA ATC GGG CCG 724Asp Ala Leu Lys Ala Ile Asp Lys Tyr Glu Met Ile Ala Ile Gly Pro220 225 230TTG ATT CCT TCC GCA TTC TTG GAC GGT AAA GAT CCT TCG GAC AGG TCT 772Leu Ile Pro Ser Ala Phe Leu Asp Gly Lys Asp Pro Ser Asp Arg Ser235 240 245TTC GGC GGA GAT TTG TTC GAG AAA GGG TCG AAT GAC GAC GAT TGC CTC 820Phe Gly Gly Asp Leu Phe Glu Lys Gly Ser Asn Asp Asp Asp Cys Leu250 255 260 265GAA TGG TTG AGC ACG AAT CCT CGA TCT TCG GTG GTT TAC GTT TCG TTC 868Glu Trp Leu Ser Thr Asn Pro Arg Ser Ser Val Val Tyr Val Ser Phe270 275 280GGA AGC TTC GTT AAT ACG ACG AAG TCG CAA ATG GAA GAG ATA GCA AGA 916Gly Ser Phe Val Asn Thr Thr Lys Ser Gln Met Glu Glu Ile Ala Arg285 290 295GGG CTG TTA GAT TGT GGG AGG CCG TTT TTG TGG GTG GTA AGA GTA AAC 964Gly Leu Leu Asp Cys Gly Arg Pro Phe Leu Trp Val Val Arg Val Asn300 305 310GAA GGA GAA GAG GTA TTG ATA AGT TGC ATG GAG GAG TTG AAA CGA GTG 1012Glu Gly Glu Glu Val Leu Ile Ser Cys Met Glu Glu Leu Lys Arg Val315 320 325GGG AAA ATT GTA TCT TGG TGT TCT CAA TTG GAA GTC CTG ACG CAT CCC 1060Gly Lys Ile Val Ser Trp Cys Ser Gln Leu Glu Val Leu Thr His Pro330 335 340 345TCG TTG GGA TGT TTC GTG ACA CAC TGC GGG TGG AAT TCG ACT CTA GAG 1108Ser Leu Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser Thr Leu Glu350 355 360AGT ATA TCT TTC GGG GTT CCG ATG GTG GCT TTT CCG CAG TGG TTC GAT 1156Ser Ile Ser Phe Gly Val Pro Met Val Ala Phe Pro Gln Trp Phe Asp365 370 375CAA GGG ACG AAT GCG AAG CTG ATG GAG GAT GTG TGG AGG ACG GGT GTG 1204Gln Gly Thr Asn Ala Lys Leu Met Glu Asp Val Trp Arg Thr Gly Val380 385 390AGA GTG AGA GCT AAT GAG GAG GGT AGC GTC GTT GAT GGT GAT GAA ATT 1252Arg Vsl Arg Ala Asn Glu Glu Gly Ser Val Val Asp Gly Asp Glu Ile395 400 405AGG AGA TGT ATT GAG GAG GTT ATG GAT GGG GGA GAA AAG AGT AGG AAA 1300Arg Arg Cys Ile Glu Glu Val Met Asp Gly Gly Glu Lys Ser Arg Lys410 415 420 425CTT AGA GAG AGT GCT GGC AAG TGG AAG GAT TTG GCA AGA AAA GCT ATG 1348Leu Arg Glu Ser Ala Gly Lys Trp Lys Asp Leu Als Arg Lys Ala Met430 435 440GAG GAA GAT GGA TCT TCA GTT AAC AAC CTC AAG GTC TTT CTT GAT GAG 1396Glu Glu Asp Gly Ser Ser Val Asn Asn Leu Lys Val Phe Leu Asp Glu445 450 455GTT GTA GGT ATC TAAAGACGTA AATGAGGTCC CCATAGGCAA AATTGCAAAT 1448Val Vsl Gly Ile460 461TTCATCTCGT AAGTTGAATA CTTTTTGGCT TTAATTTTGT TCGAGTTTGT TTTTCAAAAT 1508TTATCTTGTA ATTTTACATT GAGTGTAAAT TTAGTCTGAT TTTAACTGGA AAAATATAAA 1568ATTCATTGTT GAGACTCTTC ATCAAAATCA TCTGATTTCC TTTATTGTCT TGGTCAAAAT 1628TCTCATATCA ATTGGAAAAA ATAAATTTCA AAATCGTCCA ATTTTGAACC AAGAAAGAAG 1688TATAATTTGA CCAAAATAAT AAAAGGATTC AAGTGATCTT GATGAAGTGT CTGAGCGACG 1748AGTTCTATAT TTTTCCACCG AATTTCTAAC GAGTTTTTGA ATTTTTTTTA GCCAAAATCG 1808GACTAACTTT GTACAAAATG AAAAGTTATA TGATGAAATT TTAAAAAACA AACTCAGACA 1868ATAATAAAGC CCGAAAGTAG TAAAATTACC TGACGAAATT TGCAATTTCG CCTCCTATTT 1928TAATTTTTTT GGTGTGTTTA ATAAATCGGT TATTTTACTT TTAATTAAAA TAAAAGTGAG 1988ATGCATGATA GCTTGGTGAG TATATATGAG TTGATGGTAA TGTACGATAT TTTCTAAAAA 2048AAAAAAAAAA AAAA 2062序列4序列长度1671序列类型核酸链数双链拓扑类型直链形起源生物名称蓝竹耳组织种类花瓣直接起源文库名cDNA library克隆名pSTGT5序列AACACATAAA AAAAAAATAA AAGAAGAAAT AATTAAAAAA AAAA ATG GTT AAG53Met Val Asn1AAA CGC CAT ATT CTA CTA GCA ACA TTC CCA GCA CAA GGC CAC ATA AAC101Lys Arg His Ile Leu Leu Ala Thr Phe Pro Ala Gln Gly His Ile Asn5 10 15CCT TCT CTC GAG TTC GCC AAA AGG CTC CTC AAC ACC GGA TAC GTC GAC149Pro Ser Leu Glu Phe Ala Lys Arg Leu Leu Asn Thr Gly Tyr Val Asp20 25 30 35CAA GTC ACA TTC TTC ACG AGT GTA TAC GCA TTG AGA CGC ATG CGC TTC197Gln Val Thr Phe Phe Thr Ser Val Tyr Ala Leu Arg Arg Met Arg Phe40 45 50GAA ACC GAT CCG AGC AGC AGA ATC GAT TTC GTG GCA TKT YCA GAT TCT245Glu Thr Asp Pro Ser Sar Ara Ile Asp Phe Val Ala X X Asp Ser55 60 65TAC GAT GAT GGC TTA AAG AAA GGC GAC GAT GGC AAA AAC TAC ATG TCG 293Tyr Asp Asp Gly Leu Lys Lys Gly Asp Asp Gly Lys Asn Tyr Met Ser70 75 80GAG ATG AGA AAG CGC GGA ACG AAG GCC TTA AAG GAC ACT CTTATT AAG341Glu Met Arg Lys Arg Gly Thr Lys Ala Leu Lys Asp Thr Leu Ile Lys85 90 95CTC AAC GAT GCT GCG ATG GGA AGT GAA TGT TAC AAT CGC GTG AGC TTT 389Leu Asn Asp Ala Ala Met Gly Ser Glu Cys Tyr Asn Arg Val Ser Phe100105 110 115GTG GTG TAC TCT CAT CTA TTT TCG TGG GCA GCT GAA GTG GCG CGT GAA 437Val Val Tyr Ser His Leu Phe Ser Trp Ala Ala Glu Val Ala Arg Glu120 125 130GTC GAC GTG CCG AGT GCC CTT CTT TGG ATT GAA CCG GCT ACG GTT TTC 485Val Asp Val Pro Ser Ala Leu Leu Trp Ile Glu Pro Ala Thr Val Phe135 140 145GAT GTG TAC TAT TTT TAC TTC AAT GGG TAT GCC GAT GAT ATC GAT GCG 533Asp Val Tyr Tyr Phe Tyr Phe Asn Gly Tyr Ala Asp Asp lle Asp Ala150 155 160GGC TCA GAT CAA ATC CAA CTG CCC AAT CTT CCG CAG CTC TCC AAG CAA 581Gly Ser Asp Gln Ile Gln Leu Pro Asn Leu Pro Gln Leu Ser Lys Gln165 170 175GAT CTC CCC TCT TTC CTA CTC CCT TCG AGC CCC GCG AGA TTC CGA ACC 629Asp Leu Pro Ser Phe Leu Leu Pro Ser Ser Pro Ala Arg Phe Arg Thr180 185 190 195CTA ATG AAA GAA AAG TTC GAC ACG CTC GAC AAA GAA CCG AAA GCG AAG 677Leu Met Lys Glu Lys Phe Asp Thr Leu Asp Lys Glu Pro Lys Ala Lys200 205 210GTC TTG ATA AAC ACG TTC GAC GCA TTA GAA ACC GAA CAA CTC AAA GCC 725Val Leu Ile Asn Thr Phe Asp Ala Leu Glu Thr Glu Gln Leu Lys Ala215 220 225ATC GAC AGG TAT GAA CTA ATA TCC ATC GGC CCA TTA ATC CCA TCA TCG 773Ile Asp Arg Tyr Glu Leu Ile Ser Ile Gly Pro Leu Ile Pro Ser Ser230 235 240ATA TTC TCA GAT GGC AAC GAC CCC TCA TCA AGC AAC AAA TCC TAC GGT 821Ile Phe Ser Asp Gly Asn Asp Pro Ser Ser Ser Asn Lys Ser Tyr Gly245 250 255GGA GAC CTC TTC AGA AAA GCC GAT GAA ACT TAC ATG GAC TGG CTA AAC 869Gly Asp Leu Phe Arg Lys Ala Asp Glu Thr Tyr Met Asp Trp Leu Asn260265 270 275TCA AAA CCC GAA TCA TCG GTC GTT TAC GTT TCG TTC GGG AGC CTC CTG 917Ser Lys Pro Glu Ser Ser Val Val Tyr Val Ser Phe Gly Ser Leu Leu280 285 290AGG CTC CCG AAA CCC CAA ATG GAA GAA ATA GCA ATA GGG CTT TCA GAC 965Arg Leu Pro Lys Pro Gln Met Glu Glu Ile Ala Ile Gly Leu Ser Asp295 300 305ACC AAA TCG CCA GTT CTC TGG GTG ATA AGA AGA AAC GAA GAG GGC GAC 1013Thr Lys Ser Pro Val Leu Trp Val Ile Arg Arg Asn Glu Glu Gly Asp310 315 320GAA CAA GAG CAA GCA GAA GAA GAA GAG AAG CTG CTG AGC TTC TTT GAT 1061Glu Gln Glu Gln Ala Glu Glu Glu Glu Lys Leu Leu Ser Phe Phe Asp325 330 335CGT CAC GGA ACT GAA CGA CTC GGG AAA ATC GTG ACA TGG TGC TCA CAA 1109Arg His Gly Thr Glu Arg Leu Gly Lys Ile Val Thr Trp Cys Ser Gln340 345 350 355TTG GAT GTT CTG ACG CAT AAG TCG GTG GGA TGC TTC GTG ACG CAT TGC 1157Leu ASp Val Leu Thr His Lys Ser Val Gly Cys Phe Val Thr His Cys360 365 370GGT TGG AAT TCT GCT ATC GAG AGC CTG GCT TGT GGT GTG CCC GTG GTG 1205Gly Trp Asn Ser Ala Ile Glu Ser Leu Ala Cys Gly Val Pro Val Val375 380 385TGC TTT CCT CAA TGG TTC GAT CAA GGG ACT AAT GCG AAG ATG ATC GAA 1253Cys Phe Pro Gln Trp Phe Asp Gln Gly Thr Asn Ala Lys Met Ile Glu390 395 400GAT GTG TGG AGG AGT GGT GTG AGA GTC AGA GTG AAT GAG GAA GGC GGC 1301Asp Val Trp Arg Ser Gly Val Arg Val Arg Val Asn Glu Glu Gly Gly405 410 415GTT GTT GAT AGG CGT GAG ATT AAG AGG TGC GTC TCG GAG GTT ATA AAG 1349Val Val Asp Arg Arg Glu Ile Lys Arg Cys Val Ser Glu Val Ile Lys420 425 430 435AGT CGA GAG TTG AGA GAA AGC GCA ATG ATG TGG AAG GGT TTG GCT AAA 1397Ser Arg Glu Leu Arg Glu Ser Ala Met Met Trp Lys Gly Leu Ala Lys440 445 450GAA GCT ATG GAT GAA GAA CGT GGA TCA TCA ATG AAC AAT CTG AAG AAT 1445Glu Ala Met Asp Glu Glu Arg Gly Ser Ser Met Asn Asn Leu Lys Asn455 460 465TTT ATT ACT AGG ATT ATT AAT GAA AAT GCC TCA TAAGTTGTAC1488Phe Ile Thr Arg Ile Ile Asn Glu Asn Ala Ser470 475 478TATATATGTT ATTATTGTTG TTATGGACGT CGAATTAAGT ATTAGTTAAA TGATATGTAT 1548TTAGAGGAAG GCCAAAACGG GCTACACCCG GCAGGCCACG GGTTGGAAAA GCCCGCCATG 1608ATTTAAAATA TATATTTTAA AATAAATATT TTCTACTATT AAACTAAAAA AAAAAAAAAA 1668AAA 1671序列5序列长度1437序列类型核酸链数双链拓扑类型直链形起源生物名称紫苏(Perilla frutescens)组织种类叶直接起源文库名cDNA library克隆名p8R6序列TTCAAAACTC ATAACGTGAT TGAGCTAATG TGCACATCTT CCTCTTCAAA GTCTACAGTG 60TCATCCTACC AGCATCATCA TGATCAATCT CTTTATAATG AGGAGAATGG AGTAACAAGG 120AGTGGGTTTT GTTACTCAGC TTCAACCTAC GTACGTACTA CTACTGACTC AACTCTCAAG 180AGAATGAATA TAATATATAA TGGGCGATAG ATCTTTGTAG ATATGTAGGT GTAGCCTGCA 240GGTGGTTAAT TAATTTCCGG TGTGGGAAAATAAATAAATA AATAAATATA GCG ATG AGC 299Met Ser1AGC AGC AGC AGC AGA AGG TGG AGA GAG AAT GAG GGG ATG CGA AGG ACA 347Ser Ser Ser Ser Arg Arg Trp Arg Glu Asn Glu Gly Met Arg Arg Thr5 10 15TTG CTG GGG TTG GGT TTG GGG CAG TTG GTT TCT TTC GAT TTG GCT ATC 395Leu Leu Gly Leu Gly Leu Gly Gln Leu Val Ser Phe Asp Leu Ala Ile20 25 30ATG ACC TTT TCT GCT TCT TTG GTT TCA ACC ACA GTG GAT GCA CCA CTT 443Met Thr Phe Ser Ala Ser Leu Val Ser Thr Thr Val Asp Ala Pro Leu35 40 45 50ACT ATG TCG TTC ACT ACA TAC ACT GTT GTG GCC CTG CTC TAT GGA ACC 491Thr Met Ser Phe Thr Thr Tyr Thr Val Val Ala Leu Leu Tyr Gly Thr55 60 65ATC TTG CTT TAC CGC CGC CAC AAA TTC TTG GTT CCA TGG TAC TGG TAT 539Ile Leu Leu Tyr Arg Arg His Lys Phe Leu Val Pro Trp Tyr Trp Tyr70 75 80GCT CTC CTG GGG TTC GTG GAC GTC CAC GGC AAT TAT CTT GTT AAT AAA 587Ala Leu Leu Gly Phe Val Asp Val His Gly Asn Tyr Leu Val Asn Lys85 90 95GCA TTC GAG TTG ACA TCG ATT ACG AGT GTG AGC ATA CTG GAT TGT TGG 635Ala Phe Glu Leu Thr Ser Ile Thr Ser Val Ser Ile Leu Asp Cys Trp100 105 110ACA ATC GTG TGG TCC ATC ATC TTT ACA TGG ATG TTC CTA GGC ACA AAA 683Thr Ile Val Trp Ser Ile Ile Phe Thr Trp Met Phe Leu Gly Thr Lys115 120 125 130TAC TCT GTA TAC CAG TTT GTC GGT GCT GCT ATT TGT GTA GGA GGC CTC 731Tyr Ser Val Tyr Gln Phe Val Gly Ala Ala Ile Cys Val Gly Gly Leu135 140 145CTC CTC GTG CTT CTT TCC GAC TCA GGG GTC ACT GCT GCT GGT TCG AAT 779Leu Leu Val Leu Leu Ser Asp Ser Gly Val Thr Ala Ala Gly Ser Asn150 155 160CCT CTT TTG GGT GAT TTT CTT GTC ATA ACA GGC TCT ATT TTG TTC ACA 827Pro Leu Leu Gly Asp Phe Leu Val Ile Thr Gly Ser Ile Leu Phe Thr165 170 175CTC AGC ACT GTT GGT CAG GAA TAC TGC GTG AAG AGG AAA GAT CGT ATT 875Leu Ser Thr Val Gly Gln Glu Tyr Cys Val Lys Arg Lys Asp Arg Ile180 185 190GAA GTA GTA GCA ATG ATC GGT GTA TTT GGT ATG CTC ATC AGT GCA ACC 923Glu Val Val Ala Met Ile Gly Val Phe Gly Met Leu Ile Ser Ala Thr195 200 205 210GAG ATT ACT GTG CTG GAG AGG AAT GCC CTC TCA TCA ATG CAG TGG TCT 971Glu Ile Thr Val Leu Glu Arg Asn Ala Leu Ser Ser Met Gln Trp Ser215 220 225ACT GGA CTT TTG GCA GCC TAT GTT GTT TAT GCA CTG TCC AGC TTC CTC 1019Thr Gly Leu Leu Ala Ala Tyr Val Val Tyr Ala Leu Ser Ser Phe Leu230 235 240TTC TGC ACA CTC ACC CCT TTT CTT CTC AAG ATG AGT GGC GCT GCA TTT 1067Phe Cys Thr Leu Thr Pro Phe Leu Leu Lys Met Ser Gly Ala Ala Phe245 250 255TTC AAT CTT TCC ATG CTT ACA TCT GAT ATG TGG GCT GTT GCA ATT AGG 1115Phe Asn Leu Ser Met Leu Thr Ser Asp Met Trp Ala Val Ala Ile Arg260 265 270ACA TTC ATA TAC AAC CAG GAG GTT GAT TGG TTA TAC TAT TTG GCC TTT 1163Thr Phe Ile Tyr Asn Gln Glu Val Asp Trp Leu Tyr Tyr Leu Ala Phe275 280 285 290TGT CTC GTT GTT GTT GGA ATA TTC ATA TAT ACA AAA ACA GAG AAG GAT 1211Cys Leu Val Val Val Gly Ile Phe Ile Tyr Thr Lys Thr Glu Lys Asp295 300 305CCT AAC AAT ACG AGA GCC CTT GAG AAT GGA AAC TTG GAT CAT GAA TAT 1259Pro Asn Asn Thr Arg Ala Leu Glu Asn Gly Asn Leu Asp His Glu Tyr310 315 320AGT CTC CTT GAG GAT CAA GAT GAC ACA CCA AGA AAA CCA TAGCTAGCTT1308Ser Leu Leu Glu Asp Gln Asp Asp Thr Pro Arg Lys Pro325 330335TGCCCACAAT CTTTTCATCA ACAGTTTTAA ATAATTCGTG AGGGGGAGAG AGATCGAGAT 1368ACTAATTAAT GGACGTCTAT TATATAGTTG GAGGTTTTTG TTTTATTTAT TTATTTGAGT 1428AAAAAAAAA 1437序列6序列长度2105序列类型核酸链数双链拓扑类型直链形起源生物名称矮牵牛花组织种类花瓣直接起源文库名cDNA library克隆名pSPGTl序列AGTGAGCGCA ACGCAATTAA TGTGAGTTAG CTCACTCATT AGGCACCCCA GGCTTTACAC 60TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATTGTGAGCG GATAACAATT TCACACAGGA 120AACAGCTATG ACCATGATTA CGCCAAGCTC GAAATTAACC CTCACTAAAG GGAACAAAAG 180CTGGAGCTCC ACGCGGTGGC GGCCGCTCTA GAACTAGTGG ATCCCCCGGG CTGCAGGAAT 240TCCGTTGCTG TCGCCACAAT TTACAAACCA AGAAATTAAG CATCCCTTTC CCCCCCTTAA 300AAAACATACA AGTTTTTAAT TTTTCACTAA GCAAGAAAAT ATG GTG CAG CCT CAT GTC 358Met Val Gln Pro His Val1 5ATC TTA ACA ACA TTT CCA GCA CAA GGC CAT ATT AAT CCA GCA CTT CAA 406Ile Leu Thr Thr Phe Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln10 15 20TTT GCC AAG AAT CTT GTC AAG ATG GGC ATA GAA GTG ACA TTT TCT ACA 454Phe Ala Lys Asn Leu Val Lys Met Gly Ile Glu Val Thr Phe Ser Thr25 30 35AGC ATT TAT GCC CAA AGC CGT ATG GAT GAA AAA TCC ATT CTT AAT GCA 502Ser Ile Tyr Ala Gln Ser Arg Met Asp Glu Lys Ser Ile Leu Asn Ala40 45 50CCA AAA GGA TTG AAT TTC ATT CCA TTT TCC GAT GGC TTT GAT GAA GGT 550Pro Lys Gly Leu Asn Phe Ile Pro Phe Ser Asp Gly Phe Asp Glu Gly55 60 65 70TTT GAT CAT TCA AAA GAC CCT GTA TTT TAC ATG TCA CAA CTT CGT AAA 598Phe Asp His Ser Lys Asp Pro Val Phe Tyr Met Ser Gln Leu Arg Lys75 80 85TGT GGA AGT GAA ACT GTC AAA AAA ATA ATT CTC ACT TGC TCT GAA AAT 646Cys Gly Ser Glu Thr Val Lys Lys Ile Ile Leu Thr Cys Ser Glu Asn90 95 100GGA CAG CCT ATA ACT TGC CTA CTT TAC TCC ATT TTC CTT CCT TGG GCA 694Gly Gln Pro Ile Thr Cys Leu Leu Tyr Ser Ile Phe Leu Pro Trp Ala105 110 115GCA GAG GTA GCA CGT GAA GTT CAC ATC CCT TCT GCT CTT CTT TGG AGT 742Ala Glu Val Ala Arg Glu Val His Ile Pro Ser Ala Leu Leu Trp Ser120 125 130CAA CCA GCA ACA ATA TTG GAC ATA TAT TAC TTC AAC TTT CAT GGA TAT 790Gln Pro Ala Thr Ile Leu Asp Ile Tyr Tyr Phe Asn Phe His Gly Tyr135 140 145 150GAA AAA GCT ATG GCT AAT GAA TCC AAT GAT CCA AAT TGG TCC ATT CAA 838Glu Lys Ala Met Ala Asn Glu Ser Asn Asp Pro Asn Trp Ser Ile Gln155 160 165CTT CCC GGG CTT CCA CTA CTG GAA ACT CGA GAT CTT CCT TCA TTT TTA 886Leu Pro Gly Leu Pro Leu Leu Glu Thr Arg Asp Leu Pro Ser Phe Leu170 175 180CTT CCT TAT GGT GCA AAA GGG AGT CTT CGA GTT GCA CTT CCA CCA TTC 934Leu Pro Tyr Gly Ala Lys Gly Ser Leu Arg Val Ala Leu Pro Pro Phe185 190 195AAA GAA TTG ATA GAC ACA TTA GAT GCT GAA ACC ACT CCT AAG ATT CTT 982Lys Glu Leu Ile Asp Thr Leu Asp Ala Glu Thr Thr Pro Lys Ile Leu200 205 210GTG AAT ACA TTT GAT GAA TTA GAG CCT GAG GCA CTC AAT GCA ATT GAA 1030Val Asn Thr Phe Asp Glu Leu Glu Pro Glu Ala Leu Asn Ala Ile Glu215 220 225 230GGT TAT AAG TTT TAT GGA ATT GGA CCG TTG ATT CCT TCT GGT TTC TTG 1078Gly Tyr Lys Phe Tyr Gly Ile Gly Pro Leu Ile Pro Ser Ala Phe Leu235 240 245GGT GGA AAT GAC CCT TTA GAT GCT TCA TTT GGT GGT GAT CTT TTT CAA 1126Gly Gly Asn Asp Pro Leu Asp Ala Ser Phe Gly Gly Asp Leu Phe Gln250 255 260AAT TCA AAT GAC TAT ATG GAA TGG TTA AAC TCA AAG CCA AAT TCA TCA 1174Asn Ser Asn Asp Tyr Met Glu Trp Leu Asn Ser Lys Pro Asn Ser Ser265 270 275GTT GTT TAT ATA TCT TTT GGG AGT CTA ATG AAT CCA TCT ATT AGC CAA 1222Val Val Tyr Ile Ser Phe Gly Ser Leu Met Asn Pro Ser Ile Ser Gln280 285 290ATG GAG GAG ATA TCA AAA GGG TTG ATA GAC ATA GGA AGG CCG TTT TTA 1270Met Glu Glu Ile Ser Lys Gly Leu Ile Asp Ile Gly Arg Pro Phe Leu295 300 305 310TGG GTG ATA AAA GAA AAT GAA AAA GGC AAA GAA GAA GAG AAT AAA AAG 1318Trp Val Ile Lys Glu Asn Glu Lys Gly Lys Glu Glu Glu Asn Lys Lys315 320 325CTT GGT TGT ATT GAA GAA TTG GAA AAA ATA GGA AAA ATA GTT CCA TGG 1366Leu Gly Cys Ile Glu Glu Leu Glu Lys Ile Gly Lys Ile Val Pro Trp330 335 340TGT TCA CAA CTT GAA GTT CTA AAA CAT CCA TCT TTA GGA TGT TTT GTT 1414Cys Ser Gln Leu Glu Val Leu Lys His Pro Ser Leu Gly Cys Phe Val345 350 355TCT CAT TGT GGA TGG AAT TCA GCC TTA GAG AGT TTA GCT TGT GGA GTG 1462Ser His Cys Gly Trp Asn Ser Ala Leu Glu Ser Leu Ala Cys Gly Val360 365 370CCA GTT GTG GCA TTT CCT CAA TGG ACA GAT CAA ATG ACA AAT GCC AAA 1510Pro Val Val Ala Phe Pro Gln Trp Thr Asp Gln Met Thr Asn Ala Lys375 380 385 390CAA GTT GAA GAT GTG TGG AAA AGT GGA GTA AGA GTG AGA ATA AAT GAA 1558Gln Val Glu Asp Val Trp Lys Ser Gly Val Arg Val Arg Ile Asn Glu395 400 405GAT GGT GTT GTT GAA AGT GAG GAA ATC AAA AGG TGT ATT GAA TTG GTA 1606Asp Gly Val Val Glu Ser Glu Glu Ile Lys Arg Cys Ile Glu Leu Val410 415 420ATG GAT GGA GGA GAG AAA GGG GAA GAA TTG AGA AAG AAT GCT AAG AAA 1654Met Asp Gly Gly Glu Lys Gly Glu Glu Leu Arg Lys Asn Ala Lys Lys425 430 435TGG AAA GAA TTG GCT AGA GAA GCT GTG AAG GAA GGT GGA TCT TCA CAC1702Trp Lys Glu Leu Ala Arg Glu Ala Val Lys Glu Gly Gly Ser Ser His440 445 450AAG AAT TTA AAG GCT TTT ATT GAT GAT GTT GCC AAA GGG TTT TAATATTTAC 1754Lys Asn Leu Lys Ala Phe Ile Asp Asp Val Ala Lys Gly Phe455 460 465 468AGGCTTTTGC CGTGATATTA CTTCCCCTAG TTGGCGATTC ACTCTTTGTG GACTTGCTTG 1814ACAAAAAACT GAGGGAATGT GCTAAGACAC GCTAATGCTT TAAGAAGTCA TTTCCAAGGC 1874TTGAAGCCTG CTTTTAAAAC TTATTAGCCA GTAATCTATA GGGTTCTCTT CTATTTTTCT 1934CTGTCTCTCT TTTTAGCCTT TTTCTTTCCA AGGTTTAAGA ATAGCGTGAA CATAGCTTAG 1994TACGTAGTCT TGGTATCTCT ATCTTACCAA GTGCAAGATT ATGCTTATGC TGTCCTCCTA 2054AATTTCTTAA TAAAATGCAA GATGAAAAAG TACAAAAAAA AAAAAAAAAA A2105
序列表<110>Suntory Limited<120>编码具有糖转移活性的蛋白质的基因<130>STY-F846-PCT<150>JP PH9-200571<151>1997-07-25<160>11<210>1<211>1507<212>DNA<213>Perilla frutescens<400>1gaaaatttcc acaaaa atg gtc cgc cgc cgc gtg ctg cta gca acg ttt 49Met Val Arg Arg Arg Val Leu Leu Ala Thr Phe1 5 10cct gcg caa ggc cac ata aat ccc gcc ctc caa ttc gcc aag aga ctc97Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln Phe Ala Lys Arg Leu15 20 25cta aaa gcc ggc act gac gtc aca ttt ttc acg agc gtt tat gca tgg145Leu Lys Ala Gly Thr Asp Val Thr Phe Phe Thr Ser Val Tyr Ala Trp30 35 40cgc cgc atg gcc aac aca gcc tcc gcc gct gcc gga aac cca ccg ggc193Arg Arg Met Ala Asn Thr Ala Ser Ala Ala Ala Gly Asn Pro Pro Gly45 50 55ctc gac ttc gtg gcg ttc tcc gac ggc tac gac gac ggg ctg aag CCC 24lLeu Asp Phe Val Ala Phe Ser Asp Gly Tyr Asp Asp Gly Leu Lys Pro60 65 70 75tgc ggc gac ggg aag cgc tac atg tcc gag atg aaa gcc cgc ggc tcc 289Cys Gly Asp Gly Lys Arg Tyr Met Ser Glu Met Lys Ala Arg Gly Ser80 85 90gag gcc tta aga aac ctc ctt ctc aac aac cac gac gtc acg ttc gtc 337Glu Ala Leu Arg Asn Leu Leu Leu Asn Asn His Asp Val Thr Phe Val95 100 105gtc tac tcc cac ctc ttt gca tgg gcg gcg gag gtg gcg cgt gag tcc 385Val Tyr Ser His Leu Phe Ala Trp Ala Ala Glu Val Ala Arg Glu Ser110 115 120cag gtc ccg agc gcc ctt ctc tgg gtc gag ccc gcc acc gtg ctg tgc 433Gln Val Pro Ser Ala Leu Leu Trp Val Glu Pro Ala Thr Val Leu Cys125 130 135ata tat tac ttc tac ttc aac ggc tac gca gac gag atc gac gcc ggt 481Ile Tyr Tyr Phe Tyr Phe Asn Gly Tyr Ala Asp Glu Ile Asp Ala Gly140 145 150 155tcc gac gaa att cag ctc cct cgg ctt cca ccc ctg gag cag cgc agt 529Ser Asp Glu Ile Gln Leu Pro Arg Leu Pro Pro Leu Glu Gln Arg Ser160 165 170ctt ccg acc ttt ctg ctg ccg gag aca ccg gag agattc cgg ttg atg577Leu Pro Thr Phe Leu Leu Pro Glu Thr Pro Glu Arg Phe Arg Leu Met175 180 185atg aag gag aag ctg gaa act tta gac ggt gaa gag aag gcg aaa gtg 625Met Lys Glu Lys Leu Glu Thr Leu Asp Gly Glu Glu Lys Ala Lys Val190 195 200ttg gtg aac acg ttt gat gcg ttg gag ccc gat gca ctc acg gct att 673Leu Val Asn Thr Phe Asp Ala Leu Glu Pro Asp Ala Leu Thr Ala Ile205 210 215gat agg tat gag ttg arc ggg atc ggg ccg ttg att ccc tcc gcc ttc 721Asp Arg Tyr Glu Leu Ile Gly Ile Gly Pro Leu Ile Pro Ser Ala Phe220 225 230 235ttg gac ggc gga gat ccc tcc gaa acg tct tac ggc ggc gat ctt ttc 769Leu Asp Gly Gly Asp Pro Set Glu Thr Set Tyr Gly Gly Asp Leu Phe240 245 250gaa aaa tcg gag gag aat aac tgc gtg gag tgg ttg gac acg aag ccg 817Glu Lys Ser Glu Glu Asn Asn Cys Val Glu Trp Leu Asp Thr Lys Pro255 260 265aaa tct tcg gtg gtg tat gtg tcg ttt ggg agc gtt ttg agg ttt cca 865Lys Ser Ser Val Val Tyr Val Ser Phe Gly Ser Val Leu Arg Phe Pro270 275 280aag gca caa atg gaa gag att ggg aaa ggg cta tta gcc tgc gga agg 913Lys Ala Gln Met Glu Glu Ile Gly Lys Gly Leu Leu Ala Cys Gly Arg285 290 295ccg ttt tta tgg atg ata cga gaa cag aag aat gac gac ggc gaa gaa 961Pro Phe Leu Trp Met Ile Arg Glu Gln Lys Asn Asp Asp Gly Glu Glu300 305 310 315gaa gaa gaa gag ttg agt tgc att ggg gaa ttg aaa aaa atg ggg aaa 1009Glu Glu Glu Glu Leu Ser Cys Ile Gly Glu Leu Lys Lys Met Gly Lys320 325 330ata gtt tcg tgg tgc tcg cag ttg gag gtt ctg gcg cac cct gcg ttg 1057Ile Val Ser Trp Cys Ser Gln Leu Glu Val Leu Ala His Pro Ala Leu335 340 345gga tgt ttc gtg acg cat tgt ggg tgg aac tcg gct gtg gag agc ttg 1105Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser Ala Val Glu Ser Leu350 355 360agt tgc ggg gtt ccg gtg gtg gcg gtg ccg cag tgg ttt gat cag acg 1153Ser Cys Gly Val Pro Val Val Ala Val Pro Gln Trp Phe Asp Gln Thr365 370 375acg aat gcg aag ctg att gag gat gcg tgg ggg aca ggg gtg aga gtg 1201Thr Asn Ala Lys Leu Ile Glu Asp Ala Trp Gly Thr Gly Val Arg Val380 385 390 395aga atg aat gaa ggg ggt ggg gtt gat gga tct gag ata gag agg tgt 1249Arg Met Asn Glu Gly Gly Gly Val Asp Gly Ser Glu Ile Glu Arg Cys400 405 410gtg gag atg gtg atg gat ggg ggt gag aag agc aaa cta gtg aga gaa 1297Val Glu Met Val Met Asp Gly Gly Glu Lys Ser Lys Leu Val Arg Glu415 420 425aat gcc ata aaa tgg aag act ttg gcc aga gaa gcc atg gga gag gat 1345Asn Ala Ile Lys Trp Lys Thr Leu Ala Arg Glu Ala Met Gly Glu Asp430 435 440gga tct tca ctc aag aat ctc aac gcc ttt ctt cat caa gtt gca cgt 1393Gly Ser Ser Leu Lys Asn Leu Asn Ala Phe Leu His Gln Val Ala Arg445 450 455gct taatacacaa aatggctttc cacttttaat ctactcaaac accggttcaa1446Ala460ataaatatcc ccttccactt ctttctattt cactatcaca tttataattt tagtaacaaa 1506a 1507<210>2<211>1470<212>DNA<213>Perilla frutescens<400>2accaaaccaa aacaaaattt ccacaaaa atg gtc cgc cgc cgc gtg ctg cta 48Met Val Arg Arg Arg Val Leu Leu1 5gca acg ttt ccg gcg caa ggc cac ata aat ccc gcc ctc caa ttc gcc96Ala Thr Phe Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln Phe Ala10 15 20aag aga ctc cta aaa gcc ggc act gac gtc acg ttt ttc acg agc gtt 144Lys Arg Leu Leu Lys Ala Gly Thr Asp Val Thr Phe Phe Thr Ser Val25 30 35 40tat gca tgg cgc cgc atg gcc aac aca gcc tcc gcc gct gcc gga aac 192Tyr Ala Trp Arg Arg Met Ala Asn Thr Ala Ser Ala Ala Ala Gly Asn45 50 55cca ccg ggc ctc gac ttc gtg gcg ttc tcc gac ggc tac gac gac ggg 240Pro Pro Gly Leu Asp Phe Val Ala Phe Ser Asp Gly Tyr Asp Asp Gly60 65 70ctg aag ccc ggc ggc gac ggg aag cgc tac atg tcc gag atg aaa gcc 288Leu Lys Pro Gly Gly Asp Gly Lys Arg Tyr Met Ser Glu Met Lys Ala75 80 85cgc ggc tcc gag gcc tta aga aac ctc ctt ctc aac aac gac gac gtc 336Arg Gly Ser Glu Ala Leu Arg Asn Leu Leu Leu Asn Asn Asp Asp Val90 95 100act ttc gtc gtc tac tcc cac ctc ttt gca tgg gcg gcg gag gtg gcg 384Thr Phe Val Val Tyr Ser His Leu Phe Ala Trp Ala Ala Glu Val Ala105 110 115 120cgt ttg tcc cac gtc ccg acc gcc ctt ctc tgg gtc gag ccc gcc acc 432Arg Leu Ser His Val Pro Thr Ala Leu Leu Trp Val Glu Pro Ala Thr125 130 135gtg ctg tgc ata tac cac ttc tac ttc aac ggc tac gca gac gag atc 480Val Leu Cys Ile Tyr His Phe Tyr Phe Asn Gly Tyr Ala Asp Glu Ile140 145 150gac gcc ggt tcc aat gaa att cag ctc cct cgg ctt cca tcc ctg gag 528Asp Ala Gly Ser Asn Glu Ile Gln Leu Pro Arg Leu Pro Ser Leu Glu155 160 165cag cgc agt ctt ccg acg ttt ctg ctg cct gcg acg ccg gag aga ttc 576Gln Arg Ser Leu Pro Thr Phe Leu Leu Pro Ala Thr Pro Glu Arg Phe170 175 180cgg ttg atg atg aag gag aag ctg gaa act tta gac ggt gaa gag aag 624Arg Leu Met Met Lys Glu Lys Leu Glu Thr Leu Asp Gly Glu Glu Lys185 190 195 200gcg aaa gta ttg gtg aac acg ttt gat gcg ttg gag ccc gat gca ctc 672Ala Lys Val Leu Val Asn Thr Phe Asp Ala Leu Glu Pro Asp Ala Leu205 210 215acg gct att gat agg tat gag ttg atc ggg atc ggg ccg ttg att ccc 720Thr Ala Ile Asp Arg Tyr Glu Leu Ile Gly Ile Gly Pro Leu Ile Pro220 225 230tcc gcc ttc ttg gac ggc gaa gat ccc tcc gaa acg tct tac ggc ggc 768Ser Ala Phe Leu Asp Gly Glu Asp Pro Ser Glu Thr Ser Tyr Gly Gly235 240 245gat ctt ttc gaa aaa tcg gag gag aat aac tgc gtg gag tgg ttg aac 816Asp Leu Phe Glu Lys Ser Glu Glu Asn Asn Cys Val Glu Trp Leu Asn250 255 260tcg aag ccg aaa tct tcg gtg gtg tat gtg tcg ttt ggg agc gtt ttg 864Ser Lys pro Lys Ser Ser Val Val Tyr Val Ser Phe Gly Ser Val Leu265 270 275 280agg ttt cca aag gca caa atg gaa gag att ggg aaa ggg cra tta gcc 912Arg Phe Pro Lys Ala Gln Met Glu Glu Ile Gly Lys Gly Leu Leu Ala285 290 295tgc gga agg ccc ttt tta tgg atg ata cga gaa cag aag aat gac gac 960Cys Gly Arg Pro Phe Leu Trp Met Ile Arg Glu Gln Lys Asn Asp Asp300 305 310ggc gaa gaa gaa gaa gaa gaa gaa gag ttg agt tgc att ggg gaa ttg 1008Gly Glu Glu Glu Glu Glu Glu Glu Glu Leu Ser Cys Ile Gly Glu Leu135 320 325aaa aaa atg ggg aaa ata gtg tcg tgg tgc tcg cag ttg gag gtt ctg 1056Lys Lys Met Gly Lys Ile Val Ser Trp Cys Ser Gln Leu Glu Val Leu330 335 340gcg cac cct gcg ttg gga tgt ttc gtg acg cat tgt ggg tgg aac tcg 1104Ala His Pro Ala Leu Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser345 350 355 360gct gtg gag agc ttg agt tgc ggg att ccg gtg gtg gcg gtg ccg cag 1152Ala Val Glu Ser Leu Ser Cys Gly Ile Pro Val Val Ala Val Pro Gln365 370 375tgg ttt gat cag acg acg aat gcg aag ctg att gag gat gcg tgg ggg 1200Trp Phe Asp Gln Thr Thr Asn Ala Lys Leu Ile Glu Asp Ala Trp Gly380 385 390aca ggg gtg aga gtg aga atg aat gaa ggg ggt ggg gtt gat gga tgt 1248Thr Gly Val Arg Val Arg Met Asn Glu Gly Gly Gly Val Asp Gly Cys395 400 405gag ata gaa agg tgt gtg gag atg gtg atg gat ggg ggt gac aag acc 1296Glu Ile Glu Arg Cys Val Glu Met Val Met Asp Gly Gly Asp Lys Thr410 415 420aaa cta gtg aga gaa aat gcc atc aaa tgg aag act ttg gcc aga caa 1344Lys Leu Val Arg Glu Asn Ala Ile Lys Trp Lys Thr Leu Ala Arg Gln425 430 435 440gcc atg gga taggatggat cttcactcaa caatctcaac gcctttcttc 1393Ala Met Gly443gtcaagttgc acacttttaa tctgctcaaa cagcggttca aataaatatc cccttccact 1453taaaaaaaaa aaaaaaa1470<210>3<211>2062<212>DNA<213>Verbena hybrida<400>3attttaccaa aaaaataaaa aaaaa atg agc aga gct cac gtc ctc ttg gcc 52Met Ser Arg Ala His Val Leu Leu Ala1 5aca ttc cca gca cag gga cac ata aat ccc gcc ctt caa ttc gcc aag 100Thr Phe Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln Phe Ala Lys10 15 20 25cgt crc gca aat gcc gac att caa gtc aca ttc ttc acc agc gtc tac 148Arg Leu Ala Asn Ala Asp Ile Gln Val Thr Phe Phe Thr Ser Val Tyr30 35 40gca tgg cgc cgc atg tcc aga acc gcc gct ggc tca aac ggg ctc atc 196Ala Trp Arg Arg Met Ser Arg Thr Ala Ala Gly Ser Asn Gly Leu Ile45 50 55aat ttt gtg tcg ttt tcc gac ggg tat gac gac ggg tta cag ccc gga 244Asn Phe Val Ser Phe Ser Asp Gly Tyr Asp Asp Gly Leu Gln Pro Gly60 65 70gac gat ggg aag aac tac atg tcg gag atg aaa agc aga ggt ata aaa 292Asp Asp Gly Lys Asn Tyr Met Ser Glu Met Lys Ser Arg Gly Ile Lys75 80 85gcc ttg agc gat act ctt gca gcc aat aat gtc gat caa aaa agc agc 340Ala Leu Ser Asp Thr Leu Ala Ala Asn Asn Val Asp Gln Lys Ser Ser90 95 100 105aaa atc acg ttc gtg gtg tac tcc cac ctc ttt gca tgg gcg gcc aag 388Lys Ile Thr Phe Val Val Tyr Ser His Leu Phe Ala Trp Ala Ala Lys110 115 120gtg gcg cgt gag ttc cat ctc cgg agc gcg cta ctc tgg att gag cca 436Val Ala Arg Glu Phe His Leu Arg Ser Ala Leu Leu Trp Ile Glu Pro125 130 135gct acg gtg ttg gat ata ttt tac ttt tat ttc aac ggc tat agc gac 484Ala Thr Val Leu Asp Ile Phe Tyr Phe Tyr Phe Asn Gly Tyr Ser Asp140 145 150gaa atc gat gcg ggt tcg gat gct att cac ttg ccc gga gga ctc cca 532Glu Ile Asp Ala Gly Ser Asp Ala Ile His Leu Pro Gly Gly Leu Pro155 160 165gtg ctg gcc cag cgt gat tta ccg tct ttc ctt ctt cct tcc acg cat 580Val Leu Ala Gln Arg Asp Leu Pro Ser Phe Leu Leu Pro Ser Thr His170 175 180 185gag aga ttc cgt tca ctg atg aag gag aaa ttg gaa act tta gaa ggt 628Glu Arg Phe Arg Ser Leu Met Lys Glu Lys Leu Glu Thr Leu Glu Gly190 195 200gaa gaa aaa cct aag gtc ttg gtg aac agc ttt gat gcg ttg gag cct 676Glu Glu Lys Pro Lys Val Leu Val Asn Ser Phe Asp Ala Leu Glu Pro205 210 215gat gcg ctc aag gcc att gat aag tac gag atg att gca atc ggg ccg 724Asp Ala Leu Lys Ala Ile Asp Lys Tyr Glu Met Ile Ala Ile Gly Pro220 225 230ttg att cct tcc gca ttc ttg gac ggt aaa gat cct tcg gac agg tct 772Leu Ile Pro Ser Ala Phe Leu Asp Gly Lys Asp Pro Ser Asp Arg Ser235 240 245ttc ggc gga gat ttg ttc gag aaa ggg tcg aat gac gac gat tgc ctc 820Phe Gly Gly Asp Leu Phe Glu Lys Gly Ser Asn Asp Asp Asp Cys Leu250 255 260 265gaa tgg ttg agc acg aat cct cga tct tcg gtg gtt tac gtt tcg ttc 868Glu Trp Leu Ser Thr Asn Pro Arg Ser Ser Val Val Tyr Val Ser Phe270 275 280gga agc ttc gtt aat acg acg aag tcg caa atg gaa gag ata gca aga 916Gly Ser Phe Val Asn Thr Thr Lys Ser Gln Met Glu Glu Ile Ala Arg285 290 295ggg ctg tta gat tgt ggg agg ccg ttt ttg tgg gtg gta aga gta aac 964Gly Leu Leu Asp Cys Gly Arg Pro Phe Leu Trp Val Val Arg Val Asn300 305 310gaa gga gaa gag gta ttg ata agt tgc atg gag gag ttg aaa cga gtg 1012Glu Gly Glu Glu Val Leu Ile Ser Cys Met Glu Glu Leu Lys Arg Val315 320 325ggg aaa att gta tct tgg tgt tct caa ttg gaa gtc ctg acg cat ccc 1060Gly Lys Ile Val Ser Trp Cys Ser Gln Leu Glu Val Leu Thr His Pro330 335 340 345tcg ttg gga tgt ttc gtg aca cac tgc ggg tgg aat tcg act cta gag 1108Ser Leu Gly Cys Phe Val Thr His Cys Gly Trp Asn Ser Thr Leu Glu350 355 360agt ata tct ttc ggg gtt ccg atg gtg gct ttt ccg cag tgg ttc gat 1156Ser Ile Ser Phe Gly Val Pro Met Val Ala Phe Pro Gln Trp Phe Asp365 370 375caa ggg acg aat gcg aag ctg atg gag gat gtg tgg agg acg ggt gtg 1204Gln Gly Thr Asn Ala Lys Leu Met Glu Asp Val Trp Arg Thr Gly Val380 385 390aga gtg aga gct aat gag gag ggt agc gtc gtt gat ggt gat gaa att 1252Arg Val Arg Ala Asn Glu Glu Gly Ser Val Val Asp Gly Asp Glu Ile395 400 405agg aga tgt att gag gag gtt atg gat ggg gga gaa aag agt agg aaa 1300Arg Arg Cys Ile Glu Glu Val Met Asp Gly Gly Glu Lys Ser Arg Lys410 415 420 425ctt aga gag agt gct ggc aag tgg aag gat ttg gca aga aaa gct atg 1348Leu Arg Glu Ser Ala Gly Lys Trp Lys Asp Leu Ala Arg Lys Ala Met430 435 440gag gaa gat gga tct tca gtt aac aac ctc aag gtc ttt ctt gat gag 1396Glu Glu Asp Gly Ser Ser Val Asn Asn Leu Lys Val Phe Leu Asp Glu445 450 455gtt gta ggt atc taaagacgta aatgaggtcc ccataggcaa aattgcaaat1448Val Val Gly Ile460 461ttcatctcgt aagttgaata ctttttggct ttaattttgt tcgagtttgt ttttcaaaat 1508ttatcttgta attttacatt gagtgtaaat ttagtctgat tttaactgga aaaatataaa 1568attcattgtt gagactcttc atcaaaatca tctgatttcc tttattgtct tggtcaaaat 1628tctcatatca attggaaaaa ataaatttca aaatcgtcca attttgaacc aagaaagaag 1688tataatttga ccaaaataat aaaaggattc aagtgatctt gatgaagtgt ctgagcgacg 1748agttctatat ttttccaccg aatttctaac gagtttttga atttttttta gccaaaatcg 1808gactaacttt gtacaaaatg aaaagttata tgatgaaatt ttaaaaaaca aactcagaca 1868ataataaagc ccgaaagtag taaaattacc tgacgaaatt tgcaatttcg cctcctattt 1928taattttttt ggtgtgttta ataaatcggt tattttactt ttaattaaaa taaaagtgag 1988atgcatgata gcttggtgag tatatatgag ttgatggtaa tgtacgatat tttctaaaaa 2048aaaaaaaaaa aaaa2062<210>4<211>1671<212>DNA<213>Torenia hybrira<220><221><222><223>Xaa(64)is Cys or Phe,Xaa(65)is Ser or Pro<400>4aacacataaa aaaaaaataa aagaagaaat aattaaaaaa aaaa atg gtt aac 53Met Val Asn1aaa cgc cat att cta cta gca aca ttc cca gca caa ggc cac ata aac 101Lys Arg His Ile Leu Leu Ala Thr Phe Pro Ala Gln Gly His Ile Asn5 10 15cct tct ctc gag ttc gcc aaa agg ctc ctc aac acc gga tac gtc gac 149Pro Ser Leu Glu Phe Ala Lys Arg Leu Leu Asn Thr Gly Tyr Val Asp20 25 30 35caa gtc aca ttc ttc acg agt gta tac gca ttg aga cgc atg cgc ttc 197Gln Val Thr Phe Phe Thr Set Val Tyr Ala Leu Arg Arg Met Arg Phe40 45 50gaa acc gat ccg agc agc aga atc gat ttc gtg gca tkt yca gat tct 245Glu Thr Asp Pro Ser Ser Arg Ile Asp Phe Val Ala Xaa Xaa Asp Ser55 60 65tac gat gat ggc tta aag aaa ggc gac gat ggc aaa aac tac atg tcg 293Tyr Asp Asp Gly Leu Lys Lys Gly Asp Asp Gly Lys Asn Tyr Met Ser70 75 80gag atg aga aag cgc gga acg aag gcc tta aag gac act ctt att aag 341Glu Met Arg Lys Arg Gly Thr Lys Ala Leu Lys Asp Thr Leu Ile Lys85 90 95ctc aac gat gct gcg atg gga agt gaa tgt tac aat cgc gtg agc ttt 389Leu Asn Asp Ala Ala Met Gly Ser Glu Cys Tyr Asn Arg Val Ser Phe100 105 110 115gtg gtg tac tct cat cta ttt tcg tgg gca gct gaa gtg gcg cgt gaa 437Val Val Tyr Ser His Leu Phe Ser Trp Ala Ala Glu Val Ata Arg Glu120 125 130gtc gac gtg ccg agt gcc ctt ctt tgg att gaa ccg gct acg gtt ttc 485Val Asp Val Pro Ser Ala Leu Leu Trp Ile Glu Pro Ala Thr Val Phe135 140 145gat gtg tac tat ttt tac ttc aat ggg tat gcc gat gat atc gat gcg 533Asp Val Tyr Tyr Phe Tyr Phe Asn Gly Tyr Ala Asp Asp Ile Asp Ala150 155 160ggc tca gat caa atc caa ctg ccc aat ctt ccg cag ctc tcc aag caa 581Gly Ser Asp Gln Ile Gln Leu Pro Asn Leu Pro Gln Leu Ser Lys Gln165 170 175gat ctc ccc tct ttc cta ctc cct tcg agc ccc gcg aga ttc cga acc 629Asp Leu Pro Ser Phe Leu Leu Pro Ser Ser Pro Ala Arg Phe Arg Thr180 185 190 195cta atg aaa gaa aag ttc gac acg ctc gac aaa gaa ccg aaa gcg aag 677Leu Met Lys Glu Lys Phe Asp Thr Leu Asp Lys Glu Pro Lys Ala Lys200 205 210gtc ttg ata aac acg ttc gac gca tta gaa acc gaa caa ctc aaa gcc 725Val Leu Ile Asn Thr Phe Asp Ala Leu Glu Thr Glu Gln Leu Lys Ala215 220 225atc gac agg tat gaa cta ata tcc atc ggc cca tta atc cca tca tcg 773Ile Asp Arg Tyr Glu Leu Ile Ser Ile Gly Pro Leu Ile Pro Ser Ser230 235 240ata ttc tca gat ggc aac gac ccc tca tca agc aac aaa tcc tac ggt 821Ile Phe Sar Asp Gly Asn Asp Pro Ser Ser Ser Asn Lys Ser Tyr Gly245 250 255gga gac ctc ttc aga aaa gcc gat gaa act tac atg gac tgg cta aac 869Gly Asp Leu Phe Arg Lys Ala Asp Glu Thr Tyr Met Asp Trp Leu Asn260 265 270 275tca aaa ccc gaa tca tcg gtc gtt tac gtt tcg ttc ggg agc ctc ctg 917Ser Lys Pro Glu Ser Ser Val Val Tyr Val Ser Phe Gly Ser Leu Leu280 285 290agg ctc ccg aaa ccc caa atg gaa gaa ata gca ata ggg ctt tca gac 965Arg Leu Pro Lys Pro Gln Met Glu Glu Ile Ala Ile Gly Leu Ser Asp295 300 305acc aaa tcg cca gtt ctc tgg gtg ata aga aga aac gaa gag ggc gac 1013Thr Lys Ser Pro Val Leu Trp Val Ile Arg Arg Asn Glu Glu Gly Asp310 315 320gaa caa gag caa gca gaa gaa gaa gag aag ctg ctg agc ttc ttt gat 1061Glu Gln Glu Gln Ala Glu Glu Glu Glu Lys Leu Leu Ser Phe Phe Asp325 330 335cgt cac gga act gaa cga ctc ggg aaa atc gtg aca tgg tgc tca caa 1109Arg His Gly Thr Glu Arg Leu Gly Lys Ile Val Thr Trp Cys Ser Gln340 345 350 355ttg gat gtt ctg acg cat aag tcg gtg gga tgc ttc gtg acg cat tgc 1157Leu Asp Val Leu Thr His Lys Ser Val Gly Cys Phe Val Thr His Cys360 365 370ggt tgg aat tct gct atc gag agc ctg gct tgt ggt gtg ccc gtg gtg 1205Gly Trp Asn Ser Ala Ile Glu Ser Leu Ala Cys Gly Val Pro Val Val375 380 385tgc ttt cct caa tgg ttc gat caa ggg act aat gcg aag atg atc gaa 1253Cys Phe Pro Gln Trp Phe Asp Gln Gly Thr Asn Ala Lys Met Ile Glu390 395 400gat gtg tgg agg agt ggt gtg aga gtc aga gtg aat gag gaa ggc ggc 1301Asp Val Trp Arg Ser Gly Val Arg Val Arg Val Asn Glu Glu Gly Gly405 410 415gtt gtt gat agg cgt gag att aag agg tgc gtc tcg gag gtt ata aag 1349Val Val Asp Arg Arg Glu Ile Lys Arg Cys Val Ser Glu Val Ile Lys420 425 430 435agt cga gag ttg aga gaa agc gca atg atg tgg aag ggt ttg gct aaa1397Ser Arg Glu Leu Arg Glu Ser Ala Met Met Trp Lys Gly Leu Ala Lys440 445 450gaa gct atg gat gaa gaa cgt gga tca tca atg aac aat ctg aag aat1445Glu Ala Met Asp Glu Glu Arg Gly Ser Ser Met Asn Asn Leu Lys Asn455 460 465ttt att act agg att att aat gaa aat gcc tca taagttgtac 1488Phe Ile Thr Arg Ile Ile Asn Glu Asn Ala Ser470 475 478tatatatgtt attattgttg ttatggacgt cgaattaagt attagttaaa tgatatgtat 1548ttagaggaag gccaaaacgg gctacacccg gcaggccacg ggttggaaaa gcccgccatg 1608atttaaaata tatattttaa aataaatatt ttctactatt aaactaaaaa aaaaaaaaaa 1668aaa1671<210>5<211>1437<212>DNA<213>Perilla frutescens<400>5ttcaaaactc ataacgtgat tgagctaatg tgcacatctt cctcttcaaa gtctacagtg 60tcatcctacc agcatcatca tgatcaatct ctttataatg aggagaatgg agtaacaagg 120agtgggtttt gttactcagc ttcaacctac gtacgtacta ctactgactc aactctcaag 180agaatgaata taatatataa tgggcgatag atctttgtag atatgtaggt gtagcctgca 240ggtggttaat taatttccgg tgtgggaaaa taaataaata aataaatata gcg atg agc 299Met Ser1agc agc agc agc aga agg tgg aga gag aat gag ggg atg cga agg aca 347Ser Ser Ser Ser Arg Arg Trp Arg Glu Asn Glu Gly Met Arg Arg Thr5 10 15ttg ctg ggg ttg ggt ttg ggg cag ttg gtt tct ttc gat ttg gct atc 395Leu Leu Gly Leu Gly Leu Gly Gln Leu Val Ser Phe Asp Leu Ala Ile20 25 30atg acc ttt tct gct tct ttg gtt tca acc aca gtg gat gca cca ctt 443Met Thr Phe Ser Ala Ser Leu Val Ser Thr Thr Val Asp Ala Pro Leu35 40 45 50act atg tcg ttc act aca tac act gtt gtg gcc ctg ctc tat gga acc 491Thr Met Ser Phe Thr Thr Tyr Thr Val Val Ala Leu Leu Tyr Gly Thr55 60 65atc ttg ctt tac cgc cgc cac aaa ttc ttg gtt cca tgg tac tgg tat 539Ile Leu Leu Tyr Arg Arg His Lys Phe Leu Val Pro Trp Tyr Trp Tyr70 75 80gct ctc ctg ggg ttc gtg gac gtc cac ggc aat tat ctt gtt aat aaa 587Ala Leu Leu Gly Phe Val Asp Val His Gly Asn Tyr Leu Val Asn Lys85 90 95gca ttc gag ttg aca tcg att acg agt gtg agc ata ctg gat tgt tgg 635Ala Phe Glu Leu Thr Ser Ile Thr Ser Val Ser Ile Leu Asp Cys Trp100 105 110aca atc gtg tgg tcc atc atc ttt aca tgg atg ttc cta ggc aca aaa 683Thr Ile Val Trp Ser Ile Ile Phe Thr Trp Met Phe Leu Gly Thr Lys115 120 125 130tac tct gta Lac cag ttt gtc ggt gct gct att tgt gta gga ggc ctc 731Tyr Ser Val Tyr Gln Phe Val Gly Ala Ala Ile Cys Val Gly Gly Leu135 140 145ctc ctc gtg ctt ctt tcc gac tca ggg gtc act gct gct ggt tcg aat 779Leu Leu Val Leu Leu Ser Asp Ser Gly Val Thr Ala Ala Gly Ser Asn150 155 160cct ctt ttg ggt gat ttt ctt gtc ata aca ggc tct att ttg ttc aca 827Pro Leu Leu Gly Asp Phe Leu Val Ile Thr Gly Ser Ile Leu Phe Thr165 170 175ctc agc act gtt ggt cag gaa tac tgc gtg aag agg aaa gat cgt att 875Leu Ser Thr Val Gly Gln Glu Tyr Cys Val Lys Arg Lys Asp Arg Ile180 185 190gaa ata gta gca atg atc ggt gta ttt ggt atg ctc atc agt gca acc 923Glu Val Val Ala Met Ile Gly Val Phe Gly Met Leu Ile Ser Ala Thr195 200 205 210gag att act gtg ctg gag agg aat gcc ctc tca tca atg cag tgg tct 971Glu Ile Thr Val Leu Glu Arg Asn Ala Leu Ser Ser Met Gln Trp Ser215 220 225act gga ctt ttg gca gcc tat gtt gtt tat gca ctg tcc agc ttc ctc 1019Thr Gly Leu Leu Ala Ala Tyr Val Val Tyr Ala Leu Ser Ser Phe Leu230 235 240ttc tgc aca ctc acc cct ttt ctt ctc aag atg agt ggc gct gca ttt 1067Phe Cys Thr Leu Thr Pro Phe Leu Leu Lys Met Ser Gly Ala Ala Phe245 250 255ttc aat ctt tcc atg ctt aca tct gat atg tgg gct gtt gca att agg 1115Phe Asn Leu Ser Met Leu Thr Ser Asp Met Trp Ala Val Ala Ile Arg260 265 270aca ttc ata tac aac cag gag gtt gat tgg tta Lac tat ttg gcc ttt 1163Thr Phe Ile Tyr Asn Gln Glu Val Asp Trp Leu Tyr Tyr Leu Ala Phe275 280 285 290tgt ctc gtt gtt gtt gga ata ttc ata tat aca aaa aca gag aag gat1211Cys Leu Val Val Val Gly Ile Phe Ile Tyr Thr Lys Thr Glu Lys Asp295 300 305cct aac aat acg aga gcc ctt gag aat gga aac ttg gat cat gaa tat1259Pro Asn Asn Thr Arg Ala Leu Glu Asn Gly Asn Leu Asp His Glu Tyr310 315 320agt ctc ctt gag gat caa gat gac aca cca aga aaa cca tagctagctt 1308Ser Leu Leu Glu Asp Gln Asp Asp Thr Pro Arg Lys Pro325 330 335tgcccacaat cttttcatca acagttttaa ataattcgtg agggggagag agatcgagat 1368actaattaat ggacgtctat tatatagttg gaggtttttg ttttatttat ttatttgagt 1428aaaaaaaaa 1437<210> 6<211>2105<212>DNA<213>Petunia hybrida<400>6agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac60tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga 120aacagctatg accatgatta cgccaagctc gaaattaacc ctcactaaag ggaacaaaag 180ctggagctcc acgcggtggc ggccgctcta gaactagtgg atcccccggg ctgcaggaat 240tccgttgctg tcgccacaat ttacaaacca agaaattaag catccctttc ccccccttaa 300aaaacataca agtttttaat ttttcactaa gcaagaaaat atg gtg cag cct cat gtc 358Met Val Gln Pro His Val1 5atc tta aca aca ttt cca gca caa ggc cat att aat cca gca ctt caa 406lle Leu Thr Thr Phe Pro Ala Gln Gly His Ile Asn Pro Ala Leu Gln10 15 20ttt gcc aag aat ctt gtc aag atg ggc ata gaa gtg aca ttt tct aca 454Phe Ala Lys Asn Leu Val Lys Met Gly Ile Glu Val Thr Phe Ser Thr25 30 35agc att tat gcc caa agc cgt atg gat gaa aaa tcc att ctt aat gca 502Ser Ile Tyr Ala Gln Ser Arg Met Asp Glu Lys Ser Ile Leu Asn Ala40 45 50cca aaa gga ttg aat ttc att cca ttt tcc gat ggc ttt gat gaa ggt 550Pro Lys Gly Leu Asn Phe Ile Pro Phe Ser Asp Gly Phe Asp Glu Gly55 60 65 70ttt gat cat tca aaa gac cct gta ttt tac atg tca caa ctt cgt aaa 598Phe Asp His Ser Lys Asp Pro Val Phe Tyr Met Ser Gln Leu Arg Lys75 80 85tgt gga agt gaa act gtc aaa aaa ata att ctc act tgc tct gaa aat 646Cys Gly Ser Glu Thr Val Lys Lys Ile Ile Leu Thr Cys Ser Glu Asn90 95 100gga cag cct ata act tgc cta ctt tac tcc att ttc ctt cct tgg gca 694Gly Gln Pro Ile Thr Cys Leu Leu Tyr Ser Ile Phe Leu Pro Trp Ala105 110 115gca gag gta gca cgt gaa gtt cac atc cct tct gct ctt ctt tgg agt 742Ala Glu Val Ala Arg Glu Val His Ile Pro Ser Ala Leu Leu Trp Ser120125 130caa cca gca aca ata ttg gac ata tat tacttc aac ttt cat gga tat 790Gln Pro Ala Thr Ile Leu Asp Ile Tyr Tyr Phe Asn Phe His Gly Tyr135 140 145 150gaa aaa gct atg gct aat gaa tcc aat gat cca aat tgg tcc att caa 838Glu Lys Ala Met Ala Asn Glu Ser Asn Asp Pro Asn Trp Ser Ile Gln155 160 165ctt ccc ggg ctt cca cta ctg gaa act cga gat ctt cct tca ttt tta 886Leu Pro Gly Leu Pro Leu Leu Glu Thr Arg Asp Leu Pro Ser Phe Leu170 175 180ctt cct tat ggt gca aaa ggg agt ctt cga gtt gca ctt cca cca ttc 934Leu Pro Tyr Gly Ala Lys Gly Ser Leu Arg Val Ala Leu Pro Pro Phe185 190 195aaa gaa ttg ata gac aca tta gat gct gaa acc act cct aag att ctt 982Lys Glu Leu Ile Asp Thr Leu Asp Ala Glu Thr Thr Pro Lys Ile Leu200 205 210gtg aat aca ttt gat gaa tta gag cct gag gca ctc aat gca att gaa 1030Val Asn Thr Phe Asp Glu Leu Glu Pro Glu Ala Leu Asn Ala Ile Glu215 220 225 230ggt tat aag ttt tat gga att gga ccg ttg att cct tct gct ttc ttg 1078Gly Tyr Lys Phe Tyr Gly Ile Gly Pro Leu Ile Pro Ser Ala Phe Leu235 240 245ggt gga aat gac cct tta gat gct tca ttt ggt ggt gat ctt ttt caa 1126Gly Gly Asn Asp Pro Leu Asp Ala Ser Phe Gly Gly Asp Leu Phe Gln250 255 260aat tca aat gac tat atg gaa tgg tta aac tca aag cca aat tca tca 1174Asn Ser Asn Asp Tyr Met Glu Trp Leu Asn Ser Lys Pro Asn Ser Ser265 270 275gtt gtt tat ata tct ttt ggg agt cta atg aat cca tct att agc caa 1222Val Val Tyr Ile Ser Phe Gly Ser Leu Met Asn Pro Ser Ile Ser Gln280 285 290atg gag gag ata tca aaa ggg ttg ata gac ata gga agg ccg ttt tta 1270Met Glu Glu Ile Ser Lys Gly Leu Ile Asp Ile Gly Arg Pro Phe Leu295 300 305 310tgg gtg ata aaa gaa aat gaa aaa ggc aaa gaa gaa gag aat aaa aag 1318Trp Val Ile Lys Glu Asn Glu Lys Gly Lys Glu Glu Glu Asn Lys Lys315 320 325ctt ggt tgt att gaa gaa ttg gaa aaa ata gga aaa ata gtt cca tgg 1366Leu Gly Cys Ile Glu Glu Leu Glu Lys Ile Gly Lys Ile Val Pro Trp330 335 340tgt tca caa ctt gaa gtt cta aaa cat cca tct tta gga tgt ttt gtt 1414Cys Ser Gln Leu Glu Val Leu Lys His Pro Ser Leu Gly Cys Phe Val345 350 355tct cat tgt gga tgg aat tca gcc tta gag agt tta gct tgt gga gtg 1462Ser His Cys Gly Trp Asn Ser Ala Leu Glu Ser Leu Ala Cys Gly Val360 365 370cca gtt gtg gca ttt cct caa tgg aca gat caa atg aca aat gcc aaa 1510Pro Val Val Ala Phe Pro Gln Trp Thr Asp Gln Met Thr Asn Ala Lys375 380 385 390caa gtt gaa gat gtg tgg aaa agt gga gta aga gtg aga ata aat gaa 1558Gln Val Glu Asp Val Trp Lys Ser Gly Val Arg Val Arg Ile Asn Glu395 400 405gat ggt gtt gtt gaa agt gag gaa atc aaa agg tgt att gaa ttg gta 1606Asp Gly Val Val Glu Ser Glu Glu Ile Lys Arg Cys Ile Glu Leu Val410 415 420atg gat gga gga gag aaa ggg gaa gaa ttg aga aag aat gct aag aaa 1654Met Asp Gly Gly Glu Lys Gly Glu Glu Leu Arg Lys Asn Ala Lys Lys425 430 435tgg aaa gaa ttg gct aga gaa gct gtg aag gaa ggt gga tct tca cac1702Trp Lys Glu Leu Ala Arg Glu Ala Val Lys Glu Gly Gly Ser Ser His440 445 450aag aat tta aag gct ttt att gat gat gtt gcc aaa ggg ttt taatatttac 1754Lys Asn Leu Lys Ala Phe Ile Asp Asp Val Ala Lys Gly Phe455 460 465 468aggcttttgc cgtgatatta cttcccctag ttggcgattc actctttgtg gacttgcttg 1814acaaaaaact gagggaatgt gctaagacac gctaatgctt taagaagtca tttccaaggc 1874ttgaagcctg cttttaaaac ttattagcca gtaatctata gggttctctt ctatttttct 1934ctgtctctct ttttagcctt tttctttcca aggtttaaga atagcgtgaa catagcttag 1994tacgtagtct tggtatctct atcttaccaa gtgcaagatt atgcttatgc tgtcctccta 2054aatttcttaa taaaatgcaa gatgaaaaag tacaaaaaaa aaaaaaaaaa a 2105<210>7<211>460<212>PRT<213>Perilla frutescens<400>7Met Val Arg Arg Arg Val Leu Leu Ala Thr Phe Pro Ala Gln Gly His1 5 10 15Ile Asn Pro Ala Leu Gln Phe Ala Lys Arg Leu Leu Lys Ala Gly Thr20 25 30Asp Val Thr Phe Phe Thr Ser Val Tyr Ala Trp Arg Arg Met Ala Asn35 40 45Thr Ala Ser Ala Ala Ala Gly Asn Pro Pro Gly Leu Asp Phe Val Ala50 55 60Phe Ser Asp Gly Tyr Asp Asp Gly Leu Lys Pro Cys Gly Asp Gly Lys65 70 75 80Arg Tyr Met Ser Glu Met Lys Ala Arg Gly Ser Glu Ala Leu Arg Asn85 90 95Leu Leu Leu Asn Asn His Asp Val Thr Phe Val Val Tyr Ser His Leu100 105 110Phe Ala Trp Ala Ala Glu Val Ala Arg Glu Ser Gln Val Pro Ser Ala115 120 125Leu Leu Trp Val Glu Pro Ala Thr Val Leu Cys Ile Tyr Tyr Phe Tyr130 135 140Phe Asn Gly Tyr Ala Asp Glu Ile Asp Ala Gly Ser Asp Glu Ile Gln145 150 155 160Leu Pro Arg Leu Pro Pro Leu Glu Gln Arg Ser Leu Pro Thr Phe Leu165 170 175Leu Pro Glu Thr Pro Glu Arg Phe Arg Leu Met Met Lys Glu Lys Leu180 185 190Glu Thr Leu Asp Gly Glu Glu Lys Ala Lys Val Leu Val Asn Thr Phe195 200 205Asp Ala Leu Glu Pro Asp Ala Leu Thr Ala Ile Asp Arg Tyr Glu Leu210 215 220Ile Gly Ile Gly Pro Leu Ile Pro Ser Ala Phe Leu Asp Gly Gly Asp225 230 235 240Pro Ser Glu Thr Ser Tyr Gly Gly Asp Leu Phe Glu Lys Ser Glu Glu245 250 255Asn Asn Cys Val Glu Trp Leu Asp Thr Lys Pro Lys Ser Ser Val Val260 265 270Tyr Val Ser Phe Gly Ser Val Leu Arg Phe Pro Lys Ala Gln Met Glu275 280 285Glu Ile Gly Lys Gly Leu Leu Ala Cys Gly Arg Pro Phe Leu Trp Met
290 295 300Ile Arg Glu Gln Lys Asn Asp Asp Gly Glu Glu Glu Glu Glu Glu Leu305 310 315 320Ser Cys Ile Gly Glu Leu Lys Lys Met Gly Lys Ile Val Ser Trp Cys325 330 335Ser Gln Leu Glu Val Leu Ala His Pro Ala Leu Gly Cys Phe Val Thr340 345 350His Cys Gly Trp Asn Ser Ala Val Glu Ser Leu Ser Cys Gly Val Pro355 360 365Val Val Ala Val Pro Gln Trp Phe Asp Gln Thr Thr Ash Ala Lys Leu370 375 380Ile Glu Asp Ala Trp Gly Thr Gly Val Arg Val Arg Met Asn Glu Gly385 390 395 400Gly Gly Val Asp Gly Ser Glu Ile Glu Arg Cys Val Glu Met Val Met405 410 415Asp Gly Gly Glu Lys Ser Lys Leu Val Arg Glu Asn Ala Ile Lys Trp420 425 430Lys Thr Leu Ala Arg Glu Ala Met Gly Glu Asp Gly Ser Ser Leu Lys435 440 445Asn Leu Asn Ala Phe Leu His Gln Val Ala Arg Ala450 455 460<210>8<211>443<212>PRT<213>Perilla frutescens<400>8Met Val Arg Arg Arg Val Leu Leu Ala Thr Phe Pro Ala Gln Gly His1 5 10 15Ile Asn Pro Ala Leu Gln Phe Ala Lys Arg Leu Leu Lys Ala Gly Thr20 25 30Asp Val Thr Phe Phe Thr Ser Val Tyr Ala Trp Arg Arg Met Ala Asn35 40 45Thr Ala Ser Ala Ala Ala Gly Asn Pro Pro Gly Leu Asp Phe Val Ala50 55 60Phe Ser Asp Gly Tyr Asp Asp Gly Leu Lys Pro Gly Gly Asp Gly Lys65 70 75 80Arg Tyr Met Ser Glu Met Lys Ala Arg Gly Ser Glu Ala Leu Arg Asn85 90 95Leu Leu Leu Asn Asn Asp Asp Val Thr Phe Val Val Tyr Ser His Leu100 105 110Phe Ala Trp Ala Ala Glu Val Ala Arg Leu Ser His Val Pro Thr Ala115 120 125Leu Leu Trp Val Glu Pro Ala Thr Val Leu Cys Ile Tyr His Phe Tyr130 135 140Phe Asn Gly Tyr Ala Asp Glu Ile Asp Ala Gly Ser Asn Glu Ile Gln145 150 155 160Leu Pro Arg Leu Pro Ser Leu Glu Gln Arg Ser Leu Pro Thr Phe Leu165 170 175Leu Pro Ala Thr Pro Glu Arg Phe Arg Leu Met Met Lys Glu Lys Leu180 185 190Glu Thr Leu Asp Gly Glu Glu Lys Ala Lys Val Leu Val Asn Thr Phe195 200 205Asp Ala Leu Glu Pro Asp Ala Leu Thr Ala Ile Asp Arg Tyr Glu Leu210 215 220Ile Gly Ile Gly Pro Leu Ile Pro Ser Ala Phe Leu Asp Gly Glu Asp225 230 235 240Pro Ser Glu Thr Ser Tyr Gly Gly Asp Leu Phe Glu Lys Ser Glu Glu245 250 255Asn Asn Cys Val Glu Trp Leu Asn Ser Lys Pro Lys Ser Ser Val Val260 265 270Tyr Val Ser Phe Gly Ser Val Leu Arg Phe Pro Lys Ala Gln Met Glu275 280 285Glu Ile GIy Lys Gly Leu Leu Ala Cys Gly Arg Pro Phe Leu Trp Met290 295 300Ile Arg Glu Gln Lys Asn Asp Asp Gly Glu Glu Glu Glu Glu Glu Glu305 310 315 320Glu Leu Ser Cys Ile Gly Glu Leu Lys Lys Met Gly Lys Ile Val Ser325 330 335Trp Cys Ser Gln Leu Glu Val Leu Ala His Pro Ala Leu Gly Cys Phe340 345 350Val Thr His Cys Gly Trp Asn Ser Ala Val Glu Ser Leu Ser Cys Gly355 360 365Ile Pro Val Val Ala Val Pro Gln Trp Phe Asp Gln Thr Thr Asn Ala370 375 380Lys Leu Ile Glu Asp Ala Trp Gly Thr Gly Val Arg Val Arg Met Asn385 390 395 400Glu Gly Gly Gly Val Asp Gly Cys Glu Ile Glu Arg Cys Val Glu Met405 410 415Val Met Asp Gly Gly Asp Lys Thr Lys Leu Val Arg Glu Asn Ala Ile420 425 430Lys Trp Lys Thr Leu Ala Arg Gln Ala Met Gly435 440 443<210>9<211>461<212>PRT<213>Verbena hybrida<400>9Met Ser Arg Ala His Val Leu Leu Ala Thr Phe Pro Ala Gln Gly His1 5 10 15Ile Asn Pro Ala Leu Gln Phe Ala Lys Arg Leu Ala Asn Ala Asp Ile20 25 30Gln Val Thr Phe Phe Thr Ser Val Tyr Ala Trp Arg Arg Met Ser Arg35 40 45Thr Ala Ala Gly Ser Asn Gly Leu Ile Asn Phe Val Ser Phe Ser Asp50 55 60Gly Tyr Asp Asp Gly Leu Gln Pro Gly Asp Asp Gly Lys Asn Tyr Met65 70 75 80Ser Glu Met Lys Ser Arg Gly Ile Lys Ala Leu Ser Asp Thr Leu Ala85 90 95Ala Asn Asn Val Asp Gln Lys Ser Ser Lys Ile Thr Phe Val Val Tyr100 105 110Ser His Leu Phe Ala Trp Ala Ala Lys Val Ala Arg Glu Phe His Leu115 120 125Arg Ser Ala Leu Leu Trp Ile Glu Pro Ala Thr Val Leu Asp Ile Phe130 135 140Tyr Phe Tyr Phe Asn Gly Tyr Ser Asp Glu Ile Asp Ala Gly Ser Asp145 150 155 160Ala Ile His Leu Pro Gly Gly Leu Pro Val Leu Ala Gln Arg Asp Leu165 170 175Pro Ser Phe Leu Leu Pro Ser Thr His Glu Arg Phe Arg Ser Leu Met180 185 190Lys Glu Lys Leu Glu Thr Leu Glu Gly Glu Glu Lys Pro Lys Val Leu195 200 205Val Asn Ser Phe Asp Ala Leu Glu Pro Asp Ala Leu Lys Ala Ile Asp210 215 220Lys Tyr Glu Met Ile Ala Ile Gly Pro Leu Ile Pro Ser Ala Phe Leu225 230 235 240Asp Gly Lys Asp Pro Ser Asp Arg Ser Phe Gly Gly Asp Leu Phe Glu245 250 255Lys Gly Ser Asn Asp Asp Asp Cys Leu Glu Trp Leu Ser Thr Asn Pro260 265 270Arg Ser Ser Val Val Tyr Val Ser Phe Gly Ser Phe Val Asn Thr Thr275 280 285Lys Ser Gln Met Glu Glu Ile Ala Arg Gly Leu Leu Asp Cys Gly Arg290 295 300Pro Phe Leu Trp Val Val Arg Val Asn Glu Gly Glu Glu Val Leu Ile305 310 315 320Ser Cys Met Glu Glu Leu Lys Arg Val Gly Lys Ile Val Ser Trp Cys325 330 335Ser Gln Leu Glu Val Leu Thr His Pro Ser Leu Gly Cys Phe Val Thr340 345 350His Cys Gly Trp Asn Ser Thr Leu Glu Ser Ile Ser Phe Gly Val Pro355 360 365Met Val Ala Phe Pro Gln Trp Phe Asp Gln Gly Thr Asn Ala Lys Leu370 375 380Met Glu Asp Val Trp Arg Thr Gly Val Arg Val Arg Ala Asn Glu Glu385 390 395 400Gly Ser Val Val Asp Gly Asp Glu Ile Arg Arg Cys Ile Glu Glu Val405 410 415Met Asp Gly Gly Glu Lys Ser Arg Lys Leu Arg Glu Ser Ala Gly Lys420 425 430Trp Lys Asp Leu Ala Arg Lys Ala Met Glu Glu Asp Gly Ser Ser Val435 440 445Asn Asn Leu Lys Val Phe Leu Asp Glu Val Val Gly Ile450 455 460 461<210>10<211>478<212>PRT<213>Torenia hybrida<220><221><222><223>Xaa(64)is Cys or Phe,Xaa(65)is Ser or Pro<400>10Met Val Asn Lys Arg His Ile Leu Leu Ala Thr Phe Pro Ala Gln Gly1 5 10 15His Ile Asn Pro Ser Leu Glu Phe Ala Lys Arg Leu Leu Asn Thr Gly20 25 30Tyr Val Asp Gln Val Thr Phe Phe Thr Ser Val Tyr Ala Leu Arg Arg35 40 45Met Arg Phe Glu Thr Asp Pro Ser Ser Arg Ile Asp Phe Val Ala Xaa50 55 60Xaa Asp Ser Tyr Asp Asp Gly Leu Lys Lys Gly Asp Asp Gly Lys Asn65 70 75 80Tyr Met Ser Glu Met Arg Lys Arg Gly Thr Lys Ala Leu Lys Asp Thr85 90 95Leu Ile Lys Leu Asn Asp Ala Ala Met Gly Ser Glu Cys Tyr Asn Arg100 105 110Val Ser Phe Val Val Tyr Ser His Leu Phe Ser Trp Ala Ala Glu Val115 120 125Ala Arg Glu Val Asp Val Pro Ser Ala Leu Leu Trp Ile Glu Pro Ala130 135 140Thr Val Phe Asp Val Tyr Tyr Phe Tyr Phe Asn Gly Tyr Ala Asp Asp145 150 155 160Ile Asp Ala Gly Ser Asp Gln Ile Gln Leu Pro Asn Leu Pro Gln Leu165 170 175Ser Lys Gln Asp Leu Pro Ser Phe Leu Leu Pro Ser Ser Pro Ala Arg180 185 190Phe Arg Thr Leu Met Lys Glu Lys Phe Asp Thr Leu Asp Lys Glu Pro195 200 205Lys Ala Lys Val Leu Ile Asn Thr Phe Asp Ala Leu Glu Thr Glu Gln210 215 220Leu Lys Ala Ile Asp Arg Tyr Glu Leu Ile Ser Ile Gly Pro Leu Ile225 230 235 240Pro Ser Ser Ile Phe Ser Asp Gly Asn Asp Pro Ser Ser Ser Asn Lys245 250 255Ser Tyr Gly Gly Asp Leu Phe Arg Lys Ala Asp Glu Thr Tyr Met Asp260 265 270Trp Leu Asn Ser Lys Pro Glu Ser Ser Val Val Tyr Val Ser Phe Gly275 280 285Ser Leu Leu Arg Leu Pro Lys Pro Gln Met Glu Glu Ile Ala Ile Gly290 295 300Leu Ser Asp Thr Lys Ser Pro Val Leu Trp Val Ile Arg Arg Asn Glu305 310 315 320Glu Gly Asp Glu Gln Glu Gln Ala Glu Glu Glu Glu Lys Leu Leu Ser325 330 335Phe Phe Asp Arg His Gly Thr Glu Arg Leu Gly Lys Ile Val Thr Trp340 345 350Cys Ser Gln Leu Asp Val Leu Thr His Lys Ser Val Gly Cys Phe Val355 360 365Thr His Cys Gly Trp Asn Ser Ala Ile Glu Ser Leu Ala Cys Gly Val370 375 380Pro Val Val Cys Phe Pro Gln Trp Phe Asp Gln Gly Thr Asn Ala Lys385 390 395 400Met Ile Glu Asp Val Trp Arg Ser Gly Val Arg Val Arg Val Asn Glu405 410 415Glu Gly Gly Val Val Asp Arg Arg Glu Ile Lys Arg Cys Val Ser Glu420 425 430Val Ile Lys Ser Arg Glu Leu Arg Glu Ser Ala Met Met Trp Lys Gly435 440 445Leu Ala Lys Glu Ala Met Asp Glu Glu Arg Gly Ser Ser Met Asn Asn450 455 460Leu Lys Asn Phe Ile Thr Arg Ile Ile Asn Glu Asn Ala Ser465 470 475 478<210>11<211>335<212>PRT<213>Perilla frutescens<400>11Met Ser Ser Ser Ser Ser Arg Arg Trp Arg Glu Asn Glu Gly Met Arg1 5 l0 15Arg Thr Leu Leu Gly Leu Gly Leu Gly Gln Leu Val Ser Phe Asp Leu20 25 30Ala Ile Met Thr Phe Ser Ala Ser Leu Val Ser Thr Thr Val Asp Ala35 40 45Pro Leu Thr Met Ser Phe Thr Thr Tyr Thr Val Val Ala Leu Leu Tyr50 55 60Gly Thr Ile Leu Leu Tyr Arg Arg His Lys Phe Leu Val Pro Trp Tyr65 70 75 80Trp Tyr Ala Leu Leu Gly Phe Val Asp Val His Gly Asn Tyr Leu Val85 90 95Asn Lys Ala Phe Glu Leu Thr Ser Ile Thr Ser Val Ser Ile Leu Asp100 105 110Cys Trp Thr Ile Val Trp Ser Ile Ile Phe Thr Trp Met Phe Leu Gly115 120 125Thr Lys Tyr Ser Val Tyr Gln Phe Val Gly Ala Ala Ile Cys Val Gly130 135 140Gly Leu Leu Leu Val Leu Leu Ser Asp Ser Gly Val Thr Ala Ala Gly145 150 155 160Ser Asn Pro Leu Leu Gly Asp Phe Leu Val Ile Thr Gly Ser Ile Leu165 170 175Phe Thr Leu Ser Thr Val Gly Gln Glu Tyr Cys Val Lys Arg Lys Asp180 185 190Arg Ile Glu Val Val Ala Met Ile Gly Val Phe Gly Met Leu Ile Ser195 200 205Ala Thr Glu Ile Thr Val Leu Glu Arg Asn Ala Leu Ser Ser Met Gln210 215 220Trp Ser Thr Gly Leu Leu Ala Ala Tyr Val Val Tyr Ala Leu Ser Ser225 230 235 240Phe Leu Phe Cys Thr Leu Thr Pro Phe Leu Leu Lys Met Ser Gly Ala245 250 255Ala Phe Phe Asn Leu Ser Met Leu Thr Ser Asp Met Trp Ala Val Ala260 265 270Ile Arg Thr Phe Ile Tyr Asn Gln Glu Val Asp Trp Leu Tyr Tyr Leu275 280 285Ala Phe Cys Leu Val Val Val Gly Ile Phe Ile Tyr Thr Lys Thr Glu290 295 300Lys Asp Pro Asn Asn Thr Arg Ala Leu Glu Asn Gly Asn Leu Asp His305 310 315 320Glu Tyr Ser Leu Leu Glu Asp Gln Asp Asp Thr Pro Arg Lys Pro325 330 335<210>12<211>468<212>PRT<213>Petunia hybrida<400>12Met Val Gln Pro His Val Ile Leu Thr Thr Phe Pro Ala Gln Gly His1 5 10 15Ile Asn Pro Ala Leu Gln Phe Ala Lys Asn Leu Val Lys Met Gly Ile20 25 30Glu Val Thr Phe Ser Thr Ser Ile Tyr Ala Gln Ser Arg Met Asp Glu35 40 45Lys Ser Ile Leu Asn Ala Pro Lys Gly Leu Asn Phe Ile Pro Phe Ser50 55 60Asp Gly Phe Asp Glu Gly Phe Asp His Ser Lys Asp Pro Val Phe Tyr65 70 75 80Met Ser Gln Leu Arg Lys Cys Gly Ser Glu Thr Val Lys Lys Ile Ile85 90 95Leu Thr Cys Ser Glu Asn Gly Gln Pro Ile Thr Cys Leu Leu Tyr Ser100 105 110Ile Phe Leu Pro Trp Ala Ala Glu Val Ala Arg Glu Val His Ile Pro115 120 125Ser Ala Leu Leu Trp Ser Gln Pro Ala Thr Ile Leu Asp Ile Tyr Tyr130 135 140Phe Asn Phe His Gly Tyr Glu Lys Ala Met Ala Asn Glu Ser Asn Asp145 150 155160Pro Asn Trp Ser Ile Gln Leu Pro Gly Leu Pro Leu Leu Glu Thr Arg165 170 175Asp Leu Pro Ser Phe Leu Leu Pro Tyr Gly Ala Lys Gly Ser Leu Arg180 185 190Val Ala Leu Pro Pro Phe Lys Glu Leu Ile Asp Thr Leu Asp Ala Glu195 200 205Thr Thr Pro Lys Ile Leu Val Asn Thr Phe Asp Glu Leu Glu Pro Glu210 215 220Ala Leu Asn Ala Ile Glu Gly Tyr Lys Phe Tyr Gly Ile Gly Pro Leu225 230 235 240Ile Pro Ser Ala Phe Leu Gly Gly Asn Asp Pro Leu Asp Ala Ser Phe245 250 255Gly Gly Asp Leu Phe Gln Asn Ser Asn Asp Tyr Met Glu Trp Leu Asn260 265 270Ser Lys Pro Asn Ser Ser Val Val Tyr Ile Ser Phe Gly Ser Leu Met275 280 285Asn Pro Ser Ile Ser Gln Met Glu Glu Ile Ser Lys Gly Leu Ile Asp290 295 300Ile Gly Arg Pro Phe Leu Trp Val Ile Lys Glu Asn Glu Lys Gly Lys305 310 315 320Glu Glu Glu Asn Lys Lys Leu Gly Cys Ile Glu Glu Leu Glu Lys Ile325 330 335Gly Lys lle Val Pro Trp Cys Ser Gln Leu Glu Val Leu Lys His Pro340 345 350Ser Leu Gly Cys Phe Val Ser His Cys Gly Trp Asn Ser Ala Leu Glu355 360 365Ser Leu Ala Cys Gly Val Pro Val Val Ala Phe Pro Gln Trp Thr Asp370 375 380Gln Met Thr Asn Ala Lys Gln Val Glu Asp Val Trp Lys Ser Gly Val385390 395 400Arg Val Arg Ile Asn Glu Asp Gly Val Val Glu Ser Glu Glu Ile Lys405 410 415Arg Cys Ile Glu Leu Val Met Asp Gly Gly Glu Lys Gly Glu Glu Leu420 425 430Arg Lys Asn Ala Lys Lys Trp Lys Glu Leu Ala Arg Glu Ala Val Lys435 440 445Glu Gly Gly Ser Ser His Lys Asn Leu Lys Ala Phe Ile Asp Asp Val450 455 460Ala Lys Gly Phe465 468
权利要求
1．编码具有类黄酮5位糖转移活性的蛋白质的基因。
2．权利要求1记载的基因，是编码具有序列7～10或12中任一个序列记载的氨基酸序列的具有类黄酮5位糖转移活性的蛋白质、或对这些氨基酸序列添加、缺失一个或多个氨基酸和/或具有通过其它氨基酸替代修饰的氨基酸序列、而且维持类黄酮5位糖转移活性的蛋白质的基因。
3．权利要求1记载的基因，是编码对序列7～10或12中任一个序列记载的氨基酸序列具有30％以上同源性的氨基酸序列、而且具有类黄酮5位糖转移活性的蛋白质的基因。
4．权利要求1记载的基因，是编码对序列7～10或12中任一个序列记载的氨基酸序列具有50％以上同源性的氨基酸序列、而且具有类黄酮5位糖转移活性的蛋白质的基因。
5．权利要求1记载的基因，是编码对序列7～10或12中任一个序列记载的氨基酸序列的碱基序列的一部分或全部、在5×SSC、50℃的条件下能杂化、而且具有类黄酮5位糖转移活性的蛋白质的基因。
6．含有权利要求1～5任一项记载的基因的载体。
7．利用权利要求6记载的载体转化的宿主。
8．通过权利要求1～5任一项记载的基因编码的蛋白质。
9．一种蛋白质的制造方法，其特征在于培养、或培育权利要求7记载的宿主、然后从该宿主提取具有类黄酮5位糖转移活性的蛋白质。
10．权利要求1～5任一项记载的基因导入的植物或具有与该植物具有相同性质的其后代或它们的组织。
11．权利要求10记载的植物或具有与该植物具有相同性质的其后代的切花。
全文摘要
提供编码具有对序列号:7～10或12中任一个序列记载的氨基酸序列的具有类黄酮5位糖转移活性的蛋白质的基因、和具有对这些氨基酸序列进行修饰的氨基酸序列、而且维持类黄酮5位糖转移活性的蛋白质的基因、以及使用该基因的上述蛋白质的制造方法。该基因可以用于植物的颜色的人工改良等方面。
文档编号C12N15/54GK1237206SQ9880121
公开日1999年12月1日 申请日期1998年7月16日 优先权日1997年7月25日
发明者水谷正子, 田中良和, 久住高章, 齐藤和季, 山崎真巳, 巩志忠 申请人:三得利株式会社