CN101268094A - 编码杀虫蛋白的核苷酸序列 - Google Patents
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Abstract
本发明提供编码表现鳞翅目抑制活性的杀虫蛋白的核苷酸序列,以及在本文中称作Cry1A.105杀虫剂的新杀虫蛋白,表达该杀虫剂的转基因植物,和检测生物样品中该核苷酸序列或杀虫剂的存在的方法。
Description
发明背景
本发明提供用于植物的新编码序列。该编码序列编码针对广泛鳞翅目物种作物害虫毒性的嵌合杀虫蛋白。
天然存在的苏云金芽孢杆菌(B.thuringiensis)分离物的商业制剂已长久用于对农业昆虫害虫的生物控制。根据常规农业实践将从苏云金芽孢杆菌物种发酵获得的Bt孢子和晶体浓缩并配制用于叶施用。
已知Cry1晶体蛋白家族成员表现出对抗鳞翅目昆虫幼虫的生物活性,并可用作控制鳞翅目昆虫害虫的试剂。Cry1δ-内毒素的前体形式由两个约相同大小的部分组成。前体蛋白的羧基末端部分或毒素原部分稳定晶体形成,且不表现杀虫活性。前体蛋白氨基末端这一半含有Cry1蛋白的毒素部分,且基于Cry1家族成员中保守或基本保守序列的比对,其可进一步再分为三个结构域。这三个亚结构域以Cry1A δ-内毒素的三维晶体学结构模型为基础,其中如从蛋白毒素部分氨基末端所测量的,三个亚结构域分别称作结构域I、结构域II和结构域III。结构域I含有活性毒素部分的大约第一个三分之一,且已显示对于通道形成是关键的(Thompson等人,1995)。结构域II和III各含活性毒素部分的大约中部和羧基末端部分。依赖于所检查的昆虫和δ-内毒素,结构域II和III均与受体结合和昆虫物种特异性牵连(Thompson等人,1995)。
从本领域中已知的众多天然杀虫晶体蛋白结构域结构重配而任意产生性质增强的嵌合蛋白的可能性是渺茫的。这是蛋白质结构、折叠、寡聚化和活化的复杂本质的结果,所述活化包括对嵌合前体(如果以这种形式表达的话)的正确蛋白酶解加工以释放杀虫毒素部分。只有通过各亲本蛋白内针对包括入嵌合结构的具体靶区作出仔细的选择才能构建出与嵌合体所来源的亲本蛋白相比表现有改进的杀虫活性的功能性杀虫毒素。经验已显示,毒素结构域的重装配,即由任意两个或更多个彼此不同的毒素的结构域I、II和III组成的嵌合毒素的装配,导致构建出晶体形成有缺陷和/或完全缺乏针对优选靶昆虫害虫物种的任何可检测的杀虫活性的蛋白。在某些情况下,嵌合毒素将表现出良好的晶体形成特性,但仍不表现可检测的杀虫活性。只有通过尝试与错误才能配制有效的杀虫嵌合体,且尽管那样,本领域技术人员仍不必定最后得到表现与该嵌合体组分所来源的任何单个亲本毒素蛋白相比相同或改进的杀虫活性的嵌合体。
文献报道了从两个或更多个Bt杀虫晶体蛋白前体构建或装配嵌合蛋白的例子,但是并非全部都表现与嵌合体所来源的前体蛋白相比等同或改进的杀虫或晶体形成特性(Bosch等人(WO95/06730);Thompson等人(WO95/30753);Thompson等人(WO95/30752);Malvar等人(WO98/22595);Gilroy等人(美国专利号5,128,130);Gilroy(美国专利号5,055,294);Lee等人(1992)Gene 267:3115-3121;Honee等人(1991)Mol.Microbiol.5:2799-2806;Schnepf等人(1990)J.Biol.Chem.265:20923-20930;Perlak等人(1990)Bio/Technol.8:939-9943;Perlak等人(1993)Plant Mol.Biol.22:313-321)。
已经证明苏云金芽孢杆菌δ内毒素在转基因玉米植物中的表达是控制农业重要昆虫害虫的有效方法(Perlak等人1990;1993)。表达苏云金芽孢杆菌δ内毒素的转基因作物能够使种植者显著减少与施用局部应用的化学杀虫剂有关的时间和成本。尤其有利的是编码苏云金芽孢杆菌δ内毒素的转基因的使用。在严重昆虫压力的地区中表达苏云金芽孢杆菌δ内毒素的作物植物表现出比其它方面类似的非转基因商业植物品种更好的改进产量。然而,据预测,昆虫将进化出针对转基因植物中表达的苏云金芽孢杆菌δ内毒素的抗性。这样的抗性,若广泛流传的话,无疑将限制含有编码这样的苏云金芽孢杆菌δ内毒素基因的种质的商业价值。提高转基因杀虫剂对抗靶害虫的有效性并同时减弱杀虫剂抗性害虫的发展的一种可能途径可以是确保转基因作物表达高水平的苏云金芽孢杆菌δ内毒素(McGaughey和Whalon 1993;Roush1994)。此外,具有有效对抗多组昆虫害虫且通过不同作用模式显示其作用的杀虫基因库可以抵御任何抗性的发展。两种或更多种对相同昆虫物种具有毒性的杀虫组合物在植物中的表达,各杀虫剂以足够高以有效延迟抗性开始的水平表达,可是实现控制抗性发展的另一途径。这样的可用于这样的组合中的杀虫剂的例子包括但不限于Bt毒素、致病杆菌属物种(Xenorhabdus sp.)或光杆状菌属物种(Photorhabdus sp.)杀虫蛋白、脱变态反应和去糖基化patatin蛋白和/或permuteins、植物凝集素等。实现在相同植物中共表达多种杀虫活性蛋白,和/或那些杀虫蛋白的高表达水平而不产生所不期望的植物形态学效果,是难以捉摸的。
从苏云金芽孢杆菌物种中已鉴定的超过二百五十种个别的杀虫蛋白中只有少数已被测试在植物中的表达。几种Cry1’s、Cry3’s、Cry2Aa和Cry2Ab、二元毒素Cry33/34和Cry23/37、以及Cry9已在植物中成功表达。Cry1蛋白代表已在植物中表达但未以高水平表达的最大类蛋白。为避免不期望的植物毒性作用,必须将Cry2Ab靶向叶绿体。大部分种植重组植物的土地表达Cry1A蛋白。所靶向的昆虫害虫物种对Cry1A蛋白的抗性开始的可能性大大高于若抗性管理等位基因也与cry1等位基因一起表达,或者若cry1等位基因以高水平表达的可能性。因此,需要开发出在植物中表达的替代毒素基因作为那些目前在第一和第二代转基因昆虫抗性植物中使用的毒素基因的补充或替代。
发明概述
本发明提供在植物中表达的分离的核苷酸序列,其编码表现鳞翅目昆虫抑制特性的杀虫蛋白。SEQ ID NO:1是这样的核苷酸序列的例子,其由cry1A.105基因组成且编码昆虫抑制性Cry1A.105蛋白。SEQID NO:1与SEQ ID NO:3类似,二者均编码Cry1A.105蛋白。SEQ IDNO:1优选用于双子叶细胞中,而SEQ ID NO:3优选用于单子叶细胞中。SEQ ID NO:4由SEQ ID NO:3编码,且与SEQ ID NO:2的氨基酸序列相同。该分离的核苷酸序列意欲包括表现与SEQ ID NO:1所示序列至少约88%至约90%或更大的核苷酸序列同一性,或在严格杂交条件下与SEQ ID NO:1杂交的序列。该分离的核苷酸序列还意欲包括表现与SEQ ID NO:3所示序列至少约90%核苷酸序列同一性,或在严格杂交条件下与SEQ ID NO:3杂交的序列。
本发明还提供表现针对鳞翅目昆虫物种的抑制活性的分离和纯化的杀虫蛋白。该杀虫蛋白在本文中至少被称为Cry1A.105毒素部分,且表现SEQ ID NO:2所示氨基酸序列。由SEQ ID NO:2中所示的约1177个氨基酸组成的全长前体蛋白也称作杀虫Cry1A.105蛋白,然而任何表现杀虫生物活性的前体蛋白片段也称作杀虫Cry1A.105蛋白,且至少包括对应于SEQ ID NO:2中所示约氨基酸1至约氨基酸612的氨基酸序列部分的Cry1A.105杀虫蛋白,以及还可能包括自约氨基酸2至约氨基酸610的部分。由杀虫有效量的杀虫蛋白组成的任何组合物也意欲包括在本发明范围内。
本发明还提供用于在宿主细胞中表达如SEQ ID NO:2所示的杀虫蛋白的表达盒。该表达盒优选包含在目的宿主细胞中具有功能的启动子,其与编码Cry1A.105蛋白的杀虫部分的核苷酸序列相连并调节其表达。本文中提供如SEQ ID NO:5和SEQ ID NO:7所示的示例性表达盒,其意欲分别用于双子叶植物细胞或单子叶植物细胞。启动子与编码序列可操作性相连并在宿主细胞中共同发挥功能。该表达盒可预定用于任何宿主细胞,但优选用于细菌细胞、真菌细胞、哺乳动物细胞或植物细胞。细菌细胞优选选自芽孢杆菌属(Bacillu)物种细胞、肠杆菌科(Enterobacteriacae)物种细胞、假单胞菌属(Pseudomona)物种细胞、梭菌属(Clostridium)物种细胞、和根瘤菌属(Rhizobium)物种细胞、和土壤杆菌属(Agrobacterium)物种细胞。如果宿主细胞是植物细胞,则优选其是选自作物物种植物细胞的细胞,优选为双子叶植物或单子叶植物细胞。双子叶植物细胞的例子是苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓(blueberry)、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘(citrus tree)、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘(orange)、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜。单子叶植物细胞的例子是玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草(St.Augustine)、狗芽根、剪股颖)和大麦。意欲在植物细胞中使用的表达盒通常包含可操作地连接的序列,所述序列调节比如Cry1A.105杀虫蛋白这样的目的物质的表达水平和效率。这样的序列可能是表达增强子序列、非翻译前导序列、内含子序列、叶绿体靶向肽编码序列和转录终止和多腺苷酸化序列。
该表达盒优选整合进用于稳定Cry1A.105编码序列在宿主细胞中的维持的载体。载体可以是许多本领域已知的结构,但通常是在整合进宿主细胞之前即已在其中构建或插入了表达盒的质粒或复制子。载体意欲包括但不限于质粒、粘粒、杆状病毒穿梭载体、噬粒、YAC、BAC、自杀载体、插入序列、转座子、或甚至与表达盒相连的或其中插入表达盒的线性核苷酸序列。
对鳞翅目昆虫侵袭有抗性的转基因植物是本发明的实施方案。这样的植物包含编码SEQ ID NO:2所示至少约氨基酸2至约氨基酸612的Cry1A.105杀虫蛋白的核苷酸序列。该转基因植物对于控制由比如卷叶蛾(leaf roller)、地老虎(cutworm)、黏虫(armyworm)、钻蛀虫(borer)、避债虫(bagworm)和任何草料进食者(forage feeder)这样的昆虫引起的鳞翅目昆虫侵袭中有效。优选的害虫是草地夜蛾、玉米螟、棉铃叶蛾(corn earworms)(棉铃虫(cotton bollworms))、西南玉米杆草螟和小地老虎。本发明意欲包括由本发明的转基因植物产生的子代和种子或果实或产物,只要编码Cry1A.105杀虫部分的本发明核苷酸序列维持在植物、其子代、种子等的细胞的可遗传和/或质体基因组中。
本发明还提供一种或多种通过在昆虫害虫食物中提供包含杀虫有效量杀虫Cry1A.105蛋白的组合物来控制鳞翅目昆虫侵袭植物的方法。一种这样的组合物可是已源于或源于用编码SEQ ID NO:2所示Cry1A.105氨基酸序列杀虫部分的核酸序列转化的植物细胞的植物细胞。从已被转化以包含表达盒的植物细胞产生的转基因植物将是提供昆虫食物中的杀虫组合物的一种方法,所述表达盒的示例为SEQ IDNO:5和SEQ ID NO:7所示,其包含编码Cry1A.105杀虫氨基酸序列的序列。另一种方法将是在细菌或真菌细胞中产生杀虫有效量的Cry1A.105蛋白并在一种或多种对Cry1A.105蛋白易感的靶昆虫害虫的食物中提供细菌或真菌细胞或纯化量的Cry1A.105蛋白。
提供了鉴定生物样品中编码Cry1A.105氨基酸序列的核苷酸序列的方法。该方法由下述组成,将待测试其中存在Cry1A.105编码序列的样品与特异性与Cry1A.105编码序列结合的多核苷酸探针接触。具体地,该探针序列在严格杂交条件下与Cry1A.105编码序列结合或杂交。对反应混合物中的结合的检测对于Cry1A.105编码序列的存在有诊断价值。
还提供了鉴定样品中Cry1A.105蛋白杀虫片段的方法。该方法由下述组成,将待测试其中存在Cry1A.105杀虫片段的样品与特异性结合杀虫片段的抗体接触。对反应混合物中的结合的检测对于样品中Cry1A.105蛋白的存在有诊断价值。
还提供了嵌合或杂合杀虫蛋白。这样的杂合体由两个或更多个不同的杀虫蛋白组成,各杀虫蛋白表现出针对相同昆虫物种至少一个成员的杀虫活性。杂合杀虫蛋白由各不同杀虫蛋白的部分组成。构建杂合体中所使用的杀虫蛋白部分由至少约50至至少约200个邻接氨基酸组成,所述邻接氨基酸选自组成不同杀虫蛋白之中的任一的邻接氨基酸。SEQ ID NO:2中约第2位氨基酸至约第612位氨基酸所示的Cry1A.105杀虫蛋白意欲包括在,从其中可以选择出用于构建杂合杀虫蛋白的部分的不同杀虫蛋白组内。
本发明的各种优点和特点是显而易见的,通过参考下述详细说明、实施例以及所附的权利要求书,可以更加清楚地理解本发明的本质。
序列简述
SEQ ID NO:1是优选在双子叶植物细胞中表达Cry1A.105杀虫蛋白的合成序列。
SEQ ID NO:2是从SEQ ID NO:1所示核苷酸序列编码得到的Cry1A.105蛋白。
SEQ ID NO:3是优选在单子叶植物细胞中表达Cry1A.105杀虫蛋白的合成序列。
SEQ ID NO:4是从SEQ ID NO:3所示核苷酸序列编码得到的Cry1A.105蛋白。
SEQ ID NO:5代表由在植物细胞中,且优选在双子叶植物细胞中,发挥表达Cry1A.105杀虫蛋白功能的表达盒组成的核苷酸序列。
SEQ ID NO:6代表由SEQ ID NO:5所示的表达盒内的部分编码的Cry1A.105杀虫蛋白。
SEQ ID NO:7代表由在植物细胞中,且优选在单子叶植物细胞中,发挥表达Cry 1A.105杀虫蛋白功能的表达盒组成的核苷酸序列。
SEQ ID NO:8代表由SEQ ID NO:7所示的表达盒内的部分编码的Cry 1A.105杀虫蛋白。
发明详述
根据本发明,本发明人已经构建了编码本文中鉴定为Cry1A.105蛋白的新杀虫蛋白的核苷酸序列。已鉴定,SEQ ID NO:2中所示Cry1A.105氨基酸序列表现以下特性:其提供高于对鳞翅目昆虫物种有毒性的天然存在Bt杀虫蛋白的益处。具体地,Cry1A.105蛋白在单子叶和双子叶植物中均能以高水平表达,而不出现由于与天然存在的Cry1蛋白在植物中表达时所观察到的作用相比表达水平的提高而表现植物毒性作用的大多数转基因事件。此外,Cry1A.105蛋白在苏云金芽孢杆菌中表达时形成稳定的晶体,这可能是由于与嵌合Cry1A.105蛋白的毒素部分相连的Cry1Ac毒素原部分的稳定作用。此外,Cry1A.105杀虫蛋白表现出用迄今为止已鉴定的其它天然存在的Cry1蛋白未观察到的针对鳞翅目物种的一定范围的杀虫生物活性。因此,Cry1A.105蛋白在转基因植物中的表达导致形态学正常的表达更高水平Cry1毒素类似物的转基因事件数目提高,对于被选择用于商业开发的任何事件,所述类似物表现对鳞翅目昆虫害虫物种的广范围控制。这样的事件应当导致产生延迟对Cry1A毒素类似物抗性开始的优势,且当其与第二种毒素组合时,预计发展出对任一毒素的抗性的任何可能性都是非常渺茫的,所述第二种毒素对一种或多种昆虫害虫物种(Cry1A类似物对其也具有毒性)有毒性且以不同于Cry1A类似物的作用模式发挥作用。
本发明已构建了至少两种不同的用于植物的核苷酸序列,各核苷酸序列编码相同的Cry1A.105杀虫蛋白。Cry1A.105蛋白的杀虫部分的第一个(或氨基末端)约三分之二由源自Cry1Ab氨基酸序列的氨基酸序列组成。该序列与毒素部分的羧基末端以及毒素原结构域的部分相连,所述毒素原结构域具有源自从Ecogen Bt aizawai菌株EG6346获得的杀虫Cry1蛋白的氨基酸序列(Chambers等人,1991,J.Bacteriol.173:3966-3976)。Cry1A.105毒素部分然后与基本上为Cry1Ac毒素原肽序列的部分相连。本发明人证实,该结构提供了独特的氨基酸序列,其与该嵌合体所来源的蛋白所表现的特性相比表现出惊人的改进的杀虫特性。此外,Cry1A.105前体蛋白表现出优越的晶体形成特性,而且在具体靶向的鳞翅目昆虫害虫的肠中有效溶解和加工成活性毒素形式。
已经使用美国专利号5,500,365和5,689,052中所述方法构建了本文中所包含的核苷酸序列,具体是通过避免在编码序列中的已经被观察到对于在植物细胞中表达异源基因序列有问题的特定不利序列。编码Cry1A.105蛋白毒素部分的部分或多或少由SEQ ID NO:1和SEQ IDNO:3约第1位至约第1830位所示的核苷酸组成。构建了用于双子叶植物物种,且具体是用于棉花植物的SEQ ID NO:1所示序列。构建了用于在单子叶植物中,且具体是在玉黍蜀或玉米植物物种中表达的示于SEQ ID NO:3的序列。
本发明的核苷酸序列彼此表现约94.3%的总体同一性,且从约第1330位核苷酸至约第3534位核苷酸彼此相同。编码Cry1A.105蛋白毒素部分的这些核苷酸序列的每一个的部分在约第1位核苷酸至约第1830位核苷酸彼此表现约88.9%同一性。编码Cry1A.105蛋白前两个结构域结构的这些核苷酸序列的部分更加多样,且彼此表现仅约84.7%同一性。
本发明人已经用这些序列构建了转基因植物事件。
将SEQ ID NO:1导入含有表达盒的质粒载体,所述表达盒由与碧冬茄(Petunia hybrida)Hsp70非翻译前导序列(Ph.Hsp70,又名DnaK)、鼠耳芥(Arabidopsis thaliana)核酮糖二磷酸羧化酶小亚基叶绿体靶向肽编码序列、和豌豆(Pisum sativum)E9核酮糖二磷酸羧化酶小亚基基因转录终止和多腺苷酸化序列可操作地连接的增强的玄参花叶病毒(Figwort Mosaic Virus)启动子(eFMV)序列组成。将SEQ ID NO:1所示Cry1A.105编码序列以与靶向肽编码序列3’末端编码序列符合读框地插入该表达盒中,并使其与靶向肽编码序列3’末端编码序列直接接近且位于E9终止序列上游。所得表达盒的核苷酸序列示于SEQ IDNO:5。切除含有与包含植物可表达GUS标记的第二个表达盒连接的Cry1A.105表达盒的载体部分,并用生物射弹方法将其用于产生转基因棉花事件。在生物测定中测试转基因事件对抗几种不同鳞翅目害虫物种的杀虫活性,且确定转基因事件比以前存在的仅包含Cry1Ac或Cry1Ac和Cry2Ab蛋白的组合的昆虫抗性棉花植物表现显著更好的昆虫控制特性。此外,一些Cry1A.105转基因棉花事件在整个种植季,甚至在棉铃中,表现的Cry1A.105蛋白累积水平超过10~20ppm,且对植物或生殖组织不表现任何植物毒性作用。这与从前已经测试过的其它Cry1蛋白形成对照,无论是否靶向叶绿体,所述其它Cry1蛋白通常仅能达到低于约10ppm的累积水平。在棉花中测试其它Cry1类型蛋白时也观察了植物毒性作用,尤其是在Cry1累积水平达到或超过约10ppm的时候。
将SEQ ID NO:3导入含有表达盒的质粒载体,所述表达盒由与普通小麦(Triticum aestivum)主要叶绿素a/b结合蛋白基因非翻译前导序列和稻(Oryza sativa)肌动蛋白内含子序列,和普通小麦hsp17基因转录终止和多腺苷酸化序列可操作地连接的增强的花椰菜花叶病毒启动子(eCaMV)序列组成。将示于SEQ ID NO:3的Cry1A.105编码序列插入该表达盒中,使其与内含子序列3’直接接近且位于终止序列上游。所得表达盒的核苷酸序列示于SEQ ID NO:7。该载体还包含用于选择用Cry1A.105表达盒转化的事件的草甘膦除草剂选择标记。在生物测定中针对几种鳞翅目害虫物种测试用Cry1A.105表达盒转化后选择的玉黍蜀事件,并确定它们表现用比如Cry1Ab这样的其它Bt杀虫蛋白转化的事件中不普遍的广范围杀虫活性。表达杀虫水平的Cry1A.105的事件所表现的草地夜蛾和小地老虎的活性,与表达Cry1Ab的事件针对棉铃叶蛾和玉米螟的杀虫活性相比与之相当或更强的Cry1A.105杀虫活性偶联,为Cry1A.105事件提供了更广谱的杀虫活性。
本发明的核苷酸序列是示例性的。其它的核苷酸序列能够在植物细胞中表达Cry1A.105杀虫蛋白片段,而且其它在其它类型的宿主细胞中很好表达的核苷酸序列也能够被设计。不限制本发明公开内容的范围,用于表达Cry1A.105杀虫片段的核苷酸序列意欲与本文所例举的核苷酸序列表现至少约85%、或至少约90%、或至少约95%、或至少约99%或更大的核苷酸序列同一性。意欲用于在除植物细胞之外的宿主细胞中表达Cry1A.105杀虫片段的其它核苷酸序列可以与例举的核苷酸序列具有任何百分比的同一性或相似性。由于遗传密码的冗余,核苷酸序列可以发生变化,且由此有可能合成任何数目的编码SEQ IDNO:2所示氨基酸序列任意部分的核酸序列,且所有这些序列都意欲在本发明范围内。任何至少编码Cry1.105蛋白杀虫片段的分离和纯化的核酸序列,以及可以通过抗体、核酸探针或一对或多对被设计用于产生由这样的序列组成的扩增子的引物来检测其中的核酸的任何组合物,都意欲包括在本公开内容范围内。
本文例举的且在玉黍蜀中表达的核酸序列仅由Cry1A.105前体蛋白编码序列组成,而在棉花中表达的序列由叶绿体靶向的Cry1A.105前体蛋白编码序列组成。已经证明Cry1蛋白在植物中的表达是有问题的。不知任何特定的Cry1蛋白是否将在任何特定的植物中良好表达,因此需要尝试与错误试验。一些在玉米中表达的Cry1蛋白将导致产生植物毒性作用,且因此将该蛋白靶向叶绿体有时减轻这样的作用。在表达Cry1蛋白的棉花植物中也观察到类似的情况。本文的例子无意于教导如果定位于胞质空间,则Cry1A.105表达只可能在玉米中,以及类似地,无意于教导如果定位于质体,Cry1A.105表达只可能在棉花中。这些实施例意欲教导,任一蛋白质定位方法都与该蛋白一起发挥功能以实现形态学正常的植物,所述植物表现高水平Cry1A.105蛋白表达和累积,以及对选自叶蛾属(Anticarsia)、尺叶蛾属(Pseudoplusia)、Rachiplusia、Helicoverpa、实叶蛾属(Heliothis)、灰翅叶蛾属(Spodoptera)、叶小卷娥属(Epinotia)和Armigera这些属的广范围鳞翅目昆虫植物害虫表现商业水平的抗性。据信,任意质体靶向肽编码序列将有效地发挥将前体Cry1A.105蛋白导向质体/叶绿体的作用。
非翻译前导序列、内含子和3’转录终止以及多腺苷酸化序列是本领域已知的,且本领域技术人员能理解,在特定的情况下,通过将这些序列整合进表达盒中可增强或者稳定表达。许多这样的序列是本领域已知的,且意欲将它们包括在本发明的公开内容范围内。类似地,对所连接的序列发挥功能以实现调节的表达的启动子是本领域已知的,且也意欲包括在本发明的公开内容范围内。可选择启动子用于在许多参数组合中驱动所连接序列的表达,包括但不限于表达的时间控制、表达的空间或组织特异性控制、以及控制需要在特定植物细胞或组织中累积的特定基因产物的量。
含有Cry1A.105氨基酸序列杀虫片段的分离和纯化的蛋白也意欲包括在本发明范围内。变体也意欲包括在本发明范围内,只要影响该变异的一种或多种氨基酸取代对于被取代的氨基酸总体上是保守的,且该(这些)取代不导致杀虫生物活性或物种特异性范围的降低。Cry1A.105蛋白杀虫片段欲指,SEQ ID NO:2约第1位氨基酸至约第650位氨基酸、或约第2位氨基酸至约第612位氨基酸、或约第5位氨基酸至约第610位氨基酸、或约第10位氨基酸至约第600位氨基酸所示氨基酸序列的部分。作为选择,Cry1A.105蛋白的杀虫片段欲由约550至约650个邻接氨基酸组成,所述邻接氨基酸选自SEQ ID NO:2所示第1位至约第650位氨基酸残基。由第1位至约第3534位残基组成的全长前体蛋白,表现优良的晶体形成特性,并由双子叶和单子叶植物物种良好耐受。该前体蛋白在结晶形式下还表现出良好的稳定性,以及在碱性pH下还表现良好的溶解度,具体是在约8.0至约12.0、或约8.5至约11.5、或约pH 9.0至约pH 11.0的碱性pH下。
本发明的蛋白可纯化并以杀虫有效量单独用于许多意欲用作鳞翅目害虫控制试剂的组合物中,或可以以杀虫有效量与许多不同于Cry1A.105蛋白的其它杀虫剂组合使用。这样的其它的杀虫剂意欲包括但不限于其它Bt Cry或其它无论对于鳞翅目物种是否具有毒性的杀虫组合物,包括化学杀虫剂、杀真菌剂或抑真菌剂、抗生素、抗细菌剂、制菌剂、和杀线虫剂或制线虫剂(nematostatic agent)。这样的包括Cry1A.105以及许多其它杀虫剂的杀虫剂组合可通过转基因细胞生产,或用纯化或基本纯化的杀虫剂配制成如下形式的杀虫剂组合物,所述形式由灰尘、颗粒材料、油悬浮液、水悬浮液、油和水乳状液的混合物、或可湿性粉末组成,然后再于用于叶施用的农业可接受载体中提供。也可以将这些组合物配制成种子处理,抑或与欲包含在种子处理中的组合物中的Cry1A.105一起,抑或作为应用于来自已被转化以表达杀虫有效量Cry1A.105的转基因植物的种子的组合物,从而将包含杀虫剂的种子处理组合物连同从所述种子生长的植物的细胞一起提供给靶鳞翅目害虫,所述细胞产生杀虫有效量的Cry1A.105蛋白。各自对相同的昆虫物种有毒性但通过不同的作用模式表现其毒性作用的杀虫蛋白组合对于控制鳞翅目物种或者延迟对任一单种杀虫剂的抗性开始是尤其有用的杀虫剂组合,在未开始抗性前所述任一单种杀虫剂有效对抗特定的鳞翅目物种。这样的蛋白的示例性组合是,本发明的Cry1A.105蛋白,即第一杀虫蛋白,与至少第二种不同于第一蛋白的杀虫蛋白偶联。这样的不同杀虫蛋白包括但不限于其它鳞翅目Bt.结晶蛋白(其它Cry1’s、Cry2’s、Cry5’s、Cry9’s)、VIP蛋白、称作TIC蛋白的鳞翅目杀虫蛋白以及由细菌的致病杆菌属和光杆状菌属物种产生的杀虫蛋白。向昆虫害虫的食物中提供一种或多种杀虫蛋白以及设计用于实现dsDNA介导的对昆虫存活所必需的一种或多种基因的基因抑制的试剂的组合,对于控制鳞翅目物种或者延迟对任一单种杀虫剂的抗性的开始是尤其有用的杀虫剂组合,在未开始抗性前所述任一单种杀虫剂有效对抗特定的鳞翅目物种。
用本发明的核苷酸序列转化的植物也是本发明提供的另一实施方案。将DNA稳定导入植物细胞的方法是本领域已知的,且包括但不限于真空过滤、土壤杆菌或根瘤菌介导的转化、电穿孔和各种冲击法。导入植物中的DNA通常靶向插入核染色体中,尽管也可以实现插入叶绿体或质体DNA中。导入植物中的DNA通常与为鉴定或选择已被目的DNA稳定转化的一种或多种细胞提供工具的序列连接或结合,所述序列包括但不限于可计分标记,比如荧光或光发射基因和编码在适当底物存在下赋予被转化的一种或多种细胞以比色特征的色素或酶的基因,或者通过包括允许对被转化细胞和组织进行阳性选择的选择标记,从而为被转化细胞提供生长优势并基本上使未转化细胞或组织变为静止或死亡。这样的选择标记包括但不限于编码草铵膦、bar、氨甲蝶呤抗性、新霉素磷酸转移酶、草甘膦不敏感的EPSPS酶、草甘膦氧化还原酶(GOX)酶、大肠杆菌(E.coli)phnO或其等价物等的基因。
本发明范围内还包括载体和其它类型的序列,它们被设计用于在实验室中操纵时维持、操纵和/或引导示例核苷酸序列,或者设计用于导入宿主细胞中,这些载体和其它类型的序列意欲包括但不限于噬菌体、质粒、杆状病毒穿梭载体、yacmids、粘粒等。
转化的植物也在本发明范围内。本发明公开内容尤其使被转化以含有编码至少Cry1A.105蛋白杀虫片段的核苷酸序列的植物成为可能。单子叶和双子叶植物都预计在本发明的范围内。单子叶植物意欲包括但不限于玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草、狗芽根、剪股颖)和大麦,且双子叶植物意欲包括至少苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜。这些植物的产物以及由这些植物产生的种子和组织明确包括在本发明内,只要该种子、组织或产物包含编码Cry1A.105蛋白杀虫片段的转基因。
本发明提供了在生物样品中检测Cry1A.105蛋白或编码Cry1A.105蛋白杀虫片段的核苷酸序列的方法。Cry1A.105可用于免疫动物以严生特异于Cry1A.105表位的抗体。Cry1A.105特异性抗体可用于检测生物样品中Cry1A.105的存在。检测抗体与抗原结合的方法是本领域已知的。检测生物样品中抗体与Cry1A.105表位的结合对样品中蛋白的存在有诊断价值。
也可以检测编码Cry1A.105杀虫片段的核苷酸序列。可以使用合成的核苷酸探针与靶序列,即编码Cry1A.105杀虫片段的核苷酸序列结合。检测探针与靶序列结合的方法是本领域已知的。检测探针与靶Cry1A.105编码序列的结合对样品中编码序列的存在有诊断价值。
合成的核苷酸引物可用于热扩增反应以从被怀疑包含编码Cry1A.105蛋白杀虫片段的核苷酸序列的生物样品中产生扩增子。在这样的热扩增反应中产生的扩增子的存在对样品中该核苷酸序列的存在有诊断价值。作为对检测生物样品中本发明Cry1A.105编码序列的存在有诊断价值的探针特别有用的序列是对应于或完全互补于(1)SEQID NO:1或SEQ ID NO:3所示第1401-1420位的核苷酸,或(2)SEQ IDNO:1或SEQ ID NO:3所示第1821-1840位核苷酸的序列。这些序列对应于(1)跨越编码介于不同杀虫蛋白部分的结构域II和结构域III之间的接头的序列的20个核苷酸,所述结构域用于构建本发明蛋白的杀虫部分,和(2)跨越编码介于不同蛋白编码部分的结构域III和毒素原编码部分之间的接头的序列的20个核苷酸,所述结构域和部分用于构建前毒素原Cry1Ab.105蛋白的编码序列。作为这些DNA部分(1401-1420或1821-1840)任一个的核苷酸序列,或与之互补的核苷酸序列可用作检测生物样品中这些编码序列的存在的探针。对这样的结合的检测对生物样品中这样的编码序列的存在有诊断价值。如本领域技术人员所将认识到的这些DNA部分每一侧侧接的其它序列可用作从这样的生物样品中扩增各种大小的扩增子部分的引物,而且这样的扩增子对样品中这样的编码序列的存在有诊断价值。例如,对应于SEQID NO:1中第1201-1220位所示核苷酸序列的第一引物序列可用作其中第二引物序列对应于SEQ ID NO:1中第1581-1600位所示核苷酸序列的反向互补体的热扩增反应中的正向引物。当这样的引物共同用于对包含SEQ ID NO:1的生物样品的热扩增反应中时,其将导致合成对应于SEQ ID NO:1第1201至第1600位核苷酸的扩增子,即400个核苷酸的扩增子,所述扩增子会包含SEQ ID NO:1所示第1401-1420位的20个核苷酸的部分,且由此将对这样的样品中Cry1A.105编码序列的存在有诊断价值。
提供了在样品中检测Cry1A.105存在或检测编码Cry1A.105的核苷酸序列的存在的试剂盒。连同该试剂盒还提供与实施检测目的试剂的方法所需的所有试剂和对照样品,以及使用说明书。
以下实施例描述本发明的优选实施方案。对于本领域技术人员来说,考虑本文所公开的本发明说明书或实践后,权利要求范围内的其它实施方案也是显而易见的。说明书以及实施例,仅欲被认为是示例性的,且实施例之后所附权利要求书指明了本发明的范围和精神。
实施例
实施例1.
该实施例阐述编码杀虫Cry1A.105蛋白的合成的核苷酸序列。
构建用于双子叶植物中的如SEQ ID NO:1所示的编码Cry1A.105杀虫蛋白的核苷酸序列。氨基酸序列翻译示于SEQ ID NO:2。毒素编码部分或多或少由约第1位至约第1830位核苷酸组成。
构建用于在单子叶植物中表达的如SEQ ID NO:3所示的编码Cry1A.105氨基酸序列的核苷酸序列。氨基酸序列翻译示于SEQ IDNO:4。毒素编码部分或多或少由约第1位至约第1830位核苷酸组成。
SEQ ID NO:1和SEQ ID NO:3所示核苷酸序列彼此基本等同。SEQ ID NO:1和SEQ ID NO:3表现约94.3%的总体同一性。这两条编码序列从约第1330位核苷酸至第3534位核苷酸等同。各序列的毒素编码部分由约第1位的核苷酸至第1830位的核苷酸组成,且这些部分彼此表现约88.9%的同一性。两条序列间的基本差别在于约第1位核苷酸至约第1329位核苷酸,或编码Cry1A.105蛋白毒素部分的约第一个三分之二的部分。这两条序列贯穿该部分表现约84.7%的同一性。
根据《国际承认用于专利程序的微生物保存布达佩斯条约)》,于2005年8月31日将用名称为pMON70522的质粒转化的大肠杆菌菌株(TOP10,Invitrogen,Inc.)保藏于位于1815 North University Street,in Peoria,Illinois 61604 U.S.A.的国际保藏单位农业研究机构保藏中心(Agriculture Research Culture Collection)(NRRL),并命名为NRRLB-30873,所述名称为pMON70522的质粒包含β-内酰胺酶选择标记和SEQ ID NO:3所示编码Cry1A.105的序列。
实施例2
该实施例阐述表达Cry1A.105蛋白的转基因棉花植物。
将Delta和Pineland DP50棉花种子表面灭菌,并过夜萌发。分离分生组织外植体,且通过显微解剖除去初生叶。将解剖的外植体放入靶向培养基(targeting medium)中,从而使分生组织定向为与粒子递送(particle delivery)方向垂直。转化载体,pMON47740,含有带有SEQ ID NO:9所示核苷酸序列的表达盒。从该质粒中切除KpnI片段,用HPLC分离,并用于枪(gun)转化棉花分生组织外植体,所述KpnI片段包含在e35S启动子控制下的GUS标记基因和在eFMV启动子控制下的叶绿体靶向的Cry1A.105编码序列。将包含Cry1A.105表达盒和GUS标记两者的纯化的DNA沉淀在显微金珠上,并在Mylar片(sheet)上包被成薄层。在部分真空下,通过放电式粒子递送(electricdischarge particle delivery)将DNA加速进分生组织。轰击后,将外植体去靶向(de-target)到不含选择剂的无激素培养基中。对来自再生小植物的叶组织取样,并测定GUS标记的表达。将表现高水平GUS表达的转基因植物送到温室中作进一步筛选。再次测试这些植物中GUS的表达,并剪除植物中阴性的部分。重复进行取样和剪除GUS-阴性组织的循环,直到取自各植物的所有部分都是GUS标记阳性的。之后在标准温室条件下维持这些植物直到收获种子。
用生物测定测试从F1 GUS阳性转基因棉花植物获得的组织针对棉铃虫(CBW)和草地夜蛾(FAW)的杀虫活性。将以前产生的表达杀虫水平的Cry1Ac或Cry1Ac与Cry2Ab的组合的同基因棉花植物用作阳性对照,且将非转基因同系(isoline)用作阴性对照。
将CBW棉蕾测定(square assays)用作确定转基因棉花植物杀虫活性的一种方法(Adamczyck等人,(2001)J.Econ.Entomol.94:284-290;Kranthi等人(2005)Current Science 89:291-298)。收集叶组织棉蕾(火柴头大小或更大)并单独置于测定孔中。各棉蕾用一个三龄CBW幼虫侵袭。侵袭后五天记录存活昆虫的数目。
还采用CBW棉铃测定(boll assays)确定了从转基因植物收集的棉铃组织的杀虫活性。从各事件收集了8个硬绿色棉铃(开花后)并置于单独的杯中,并用三龄CBW幼虫侵袭。侵袭后五天记录存活昆虫的数目。
进行叶测定来确定转基因叶组织对FAW的杀虫活性。从棉花植物末端取新叶。收集两个直径各为约3/4″的叶孔(punch)并将其置于16个单独的测定孔的各孔中。各孔用单个二龄或三龄FAW幼虫侵袭。侵袭后五天记录存活昆虫的数目。
生物测定结果示于表1。结果显示,表达Cry1A.105的转基因棉花事件比表达Cry1Ac或Cry1Ac与Cry2Ab的组合的转基因事件对FAW和CBW均表现更强的杀虫活性。
表1.用转基因棉花植物组织对FAW和CBW的生物测定结果。
还在类似的生物测定中测试了烟夜蛾和棉铃叶蛾。在各情况中,Cry1A.105植物也表现针对这些害虫的杀虫活性。
实施例3
本实施例阐述表达Cry1A.105蛋白的转基因玉米植物。
从用载体pMON40232转化的细胞再生转基因玉米植物。pMON40232包含具有SEQ ID NO:7所示核苷酸序列的表达盒,所述表达盒包含可操作地连接的增强的CAMV 35S启动子、小麦CAB前导序列、稻肌动蛋白1内含子、Cry1A.105编码序列和小麦hsp17基因3’转录终止和多腺苷酸化序列。编码鼠耳芥(Arabidopsis thaliana)EPSPS叶绿体靶向序列(At.EPSES-CTP2)的核苷酸序列位于Cry1A.105编码序列上游且与其符合读框。pMON40232包含编码对除草剂草甘膦不敏感的EPSPS的重组基因,以用于选择转基因事件。将从用pMON40232转化的组织得到的转基因事件命名为LAJ 105。筛选转基因事件中任何载体主链的不存在、单个简单插入序列的存在、以及包含编码Cry1A.105蛋白的核苷酸序列的表达盒的完整性。
对满足事件筛选限制的事件作生物测定。在该生物测定中,将LAJ 105转基因玉米植物与同基因LH 198阴性对照和表达Cry1Ab蛋白杀虫部分的阳性对照MON810品种作比较。从10个单独的Cry1A.105转基因事件以及对照的每一个获取五个各直径约1厘米的叶盘。将叶盘置于填充有琼脂的孔中以保持植物材料肿胀。之后将叶盘饲喂给FAW、小地老虎(BCW)、玉米螟(ECB)、棉铃叶蛾(CEW)和西南玉米杆草螟(SWCB)新生幼虫。各孔应用一只新生FAW幼虫、一只CEW幼虫、两只新生BCW、两只新生SWCB幼虫或四只新生ECB幼虫。四天后用叶损害率(LDR)等级0-11来评估饲喂损害,0表示没有可见的饲喂损害,11表示至少50%的叶盘被吃掉,且该等级上0至11的各点表示在观察中所观察到的叶盘饲喂损害的5%增长。
生物测定结果表示,表达Cry1A.105蛋白的事件对FAW、ECB和CEW表现的杀虫活性比Cry1Ab对照对相同害虫幼虫所表现的LDR更强。这三种害虫在Cry1A.105事件上的LDR小于1,而Cry1Ab对照表现的LDR范围在约8至约10。对于Cry1A.105事件和Cry1Ab对照,测试针对SWCB的活性时,其LDR始终为1至2,这表示Cry1A.105蛋白对于SWCB不比Cry1Ab具备更高的毒性。该生物测定的结果支持了表明Cry1Ab对于控制BCW无效这一从前的结果。Cry1A.105事件对抗BCW并不比Cry1Ab对照更有效。因此,以Cry1A.105蛋白在植物中(in planta)的表达水平,这些植物在控制包括但不限于叶蛾属、尺叶蛾属、Rachiplusia、实叶蛾属、Helicoverpa、灰翅叶蛾属、叶小卷娥属和Armigera属中的其它鳞翅目属植物害虫中是有效的。
序列表
<110>Monsanto Technology LLC
Bogdanova,Natalia N.
Corbin,David R.
Malvar,Thomas M.
Perlak,Frederick J.
Roberts,James K.
Romano,Charles P.
<120>编码杀虫蛋白的核苷酸序列
<130>38-21(52905)A
<150>60/713,144
<151>2005-08-31
<160>8
<170>PatentIn version 3.3
<210>1
<211>3534
<212>DNA
<213>人工的
<220>
<223>编码Cry1A.105氨基酸序列的合成的核苷酸序列
<220>
<221>CDS
<222>(1)..(3534)
<400>1
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Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
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Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
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Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
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Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
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Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
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Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
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Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
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agc ttc aga gag tgg gaa gcc gat cct act aac cca gct ctc cgc gag 384
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Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
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Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
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530 535 540
agg atc ttt gct ggt cag ttc aac aag act atg gat acc ggt gac cct 1680
Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro
545 550 555 560
ttg aca ttc caa tct ttt agc tac gca act atc aac aca gct ttt aca 1728
Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr
565 570 575
ttc cca atg agc cag agt agc ttc aca gta ggt gct gac act ttc agc 1776
Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser
580 585 590
tca ggg aat gaa gtt tac atc gac agg ttt gaa ttg att cca gtt act 1824
Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr
595 600 605
gca acc ctc gag gct gag tac aac ctt gag aga gcc cag aag gct gtg 1872
Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val
610 615 620
aac gcc ctc ttt acc tcc acc aat cag ctt ggc ttg aaa act aac gtt 1920
Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val
625 630 635 640
act gac tat cac att gac caa gtg tcc aac ttg gtc acc tac ctt agc 1968
Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser
645 650 655
gat gag ttc tgc ctc gac gag aag cgt gaa ctc tcc gag aaa gtt aaa 2016
Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys
660 665 670
cac gcc aag cgt ctc agc gac gag agg aat ctc ttg caa gac tcc aac 2064
His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn
675 680 685
ttc aaa gac atc aac agg cag cca gaa cgt ggt tgg ggt gga agc acc 2112
Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr
690 695 700
ggg atc acc atc caa gga ggc gac gat gtg ttc aag gag aac tac gtc 2160
Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val
705 710 715 720
acc ctc tcc gga act ttc gac gag tgc tac cct acc tac ttg tac cag 2208
Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln
725 730 735
aag atc gat gag tcc aaa ctc aaa gcc ttc acc agg tat caa ctt aga 2256
Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg
740 745 750
ggc tac atc gaa gac agc caa gac ctt gaa atc tac tcg atc agg tac 2304
Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg Tyr
755 760 765
aat gcc aag cac gag acc gtg aat gtc cca ggt act ggt tcc ctc tgg 2352
Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp
770 775 780
cca ctt tct gcc caa tct ccc att ggg aag tgt gga gag cct aac aga 2400
Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg
785 790 795 800
tgc gct cca cac ctt gag tgg aat cct gac ttg gac tgc tcc tgc agg 2448
Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg
805 810 815
gat ggc gag aag tgt gcc cac cat tct cat cac ttc tcc ttg gac atc 2496
Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile
820 825 830
gat gtg gga tgt act gac ctg aat gag gac ctc gga gtc tgg gtc atc 2544
Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile
835 840 845
ttc aag atc aag acc caagac gga cac gca agactt ggc aac ctt gag 2592
Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu
850 855 860
ttt ctc gaa gag aaa cca ttg gtc ggt gaa gct ctc gct cgt gtg aag 2640
Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys
865 870 875 880
aga gca gag aag aag tgg agg gac aaa cgt gag aaa ctc gaa tgg gaa 2688
Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu
885 890 895
act aac atc gtt tac aag gag gcc aaa gag tcc gtg gat gct ttg ttc 2736
Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe
900 905 910
gtg aac tcc caa tat gat cag ttg caa gcc gac acc aac atc gcc atg 2784
Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met
915 920 925
atc cac gcc gca gac aaa cgt gtg cac agc att cgt gag gct tac ttg 2832
Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu
930 935 940
cct gag ttg tcc gtg atc cct ggt gtg aac gct gcc atc ttc gag gaa 2880
Pro Glu Leu Ser Val Ile Pro Gly Val Asn AlaAla Ile Phe Glu Glu
945 950 955 960
ctt gag gga cgt atc ttt acc gca ttc tcc ttg tac gat gcc aga aac 2928
Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn
965 970 975
gtc atc aag aac ggt gac ttc aac aat ggc ctc agc tgc tgg aat gtg 2976
Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val
980 985 990
aaa ggt cat gtg gac gtg gag gaa cag aac aat cag cgt tcc gtc ctg 3024
Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu
995 1000 1005
gtt gtg cct gag tgg gaa gct gaa gtg tcc caa gag gtt aga gtc 3069
Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val
1010 1015 1020
tgt cca ggt aga ggc tac att ctc cgt gtg acc gct tac aag gag 3114
Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu
1025 1030 1035
gga tac ggt gag ggt tgc gtg acc atc cac gag atc gag aac aac 3159
Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn
1040 1045 1050
acc gac gag ctt aag ttc tcc aac tgc gtc gag gaa gaa atc tat 3204
Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr
1055 1060 1065
ccc aac aac acc gtt act tgc aac gac tac act gtg aat cag gaa 3249
Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu
1070 1075 1080
gag tac gga ggt gcc tac act agc cgt aac aga ggt tac aac gaa 3294
Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu
1085 1090 1095
gct cct tcc gtt cct gct gac tat gcc tcc gtg tac gag gag aaa 3339
Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys
1100 1105 1110
tcc tac aca gat ggc aga cgt gag aac cct tgc gag ttc aac aga 3384
Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg
1115 1120 1125
ggt tac agg gac tac aca cca ctt cca gtt ggc tat gtt acc aag 3429
Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys
1130 1135 1140
gag ctt gag tac ttt cct gag acc gac aaa gtg tgg atc gag atc 3474
Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile
1145 1150 1155
ggt gaa acc gag gga acc ttc atc gtg gac agc gtg gag ctt ctc 3519
Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu
1160 1165 1170
ttg atg gag gaa taa 3534
Leu Met Glu Glu
1175
<210>2
<211>1177
<212>PRT
<213>人工的
<220>
<223>合成的构建体
<400>2
Met Asp Asn Asn Pro Asn Iie Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255
Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270
Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285
Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr
290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
305 310 315 320
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335
Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350
Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg
355 360 365
Arg Pro Phe Asn Ile GlyIle Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
385 390 395 400
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415
Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430
Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
450 455 460
Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His
465 470 475 480
Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly
485 490 495
Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile
500 505 510
Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg
515 520 525
Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu
530 535 540
Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro
545 550 555 560
Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr
565 570 575
Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser
580 585 590
Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr
595 600 605
Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val
610 615 620
Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val
625 630 635 640
Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser
645 650 655
Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys
660 665 670
His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn
675 680 685
Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr
690 695 700
Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val
705 710 715 720
Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln
725 730 735
Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg
740 745 750
Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg Tyr
755 760 765
Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp
770 775 780
Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg
785 790 795 800
Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg
805 810 815
Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile
820 825 830
Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile
835 840 845
Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu
850 855 860
Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys
865 870 875 880
Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu
885 890 895
Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe
900 905 910
Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met
915 920 925
Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu
930 935 940
Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu
945 950 955 960
Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn
965 970 975
Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val
980 985 990
Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu
995 1000 1005
Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val
1010 1015 1020
Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu
1025 1030 1035
Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn
1040 1045 1050
Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr
1055 1060 1065
Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu
1070 1075 1080
Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu
1085 1090 1095
Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys
1100 1105 1110
Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg
1115 1120 1125
Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys
1130 1135 1140
Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile
1145 1150 1155
Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu
1160 1165 1170
Leu Met Glu Glu
1175
<210>3
<211>3534
<212>DNA
<213>人工的
<220>
<223>编码Cry1A.105氨基酸序列的合成的核苷酸序列
<220>
<221>CDS
<222>(1)..(3534)
<400>3
atg gac aac aac cca aac atc aac gag tgc atc ccg tac aac tgc ctc 48
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
agc aac cct gag gtc gag gtg ctc ggc ggt gag cgc atc gag acc ggt 96
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
tac acc ccc atc gac atc tcc ctc tcc ctc acg cag ttc ctg ctc agc 144
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
gag ttc gtg cca ggc gct ggc ttc gtc ctg ggc ctc gtg gac atc atc 192
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
tgg ggc atc ttt ggc ccc tcc cag tgg gac gcc ttc ctg gtg caa atc 240
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
gag cag ctc atc aac cag agg atc gag gag ttc gcc agg aac cag gcc 288
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
atc agc cgc ctg gag ggc ctc agc aac ctc tac caa atc tac gct gag 336
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
agc ttc cgc gag tgg gag gcc gac ccc act aac cca gct ctc cgc gag 384
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
gag atg cgc atc cag ttc aac gac atg aac agc gcc ctg acc acc gcc 432
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
atc cca ctc ttc gcc gtc cag aac tac caa gtc ccg ctc ctg tcc gtg 480
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
tac gtc cag gcc gcc aac ctg cac ctc agc gtg ctg agg gac gtc agc 528
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
gtg ttt ggc cag agg tgg ggc ttc gac gcc gcc acc atc aac agc cgc 576
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
tac aac gac ctc acc agg ctg atc ggc aac tac acc gac cac gct gtc 624
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
cgc tgg tac aac act ggc ctg gag cgc gtc tgg ggc cct gat tct aga 672
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
gac tgg att cgc tac aac cag ttc agg cgc gag ctg acc ctc acc gtc 720
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
ctg gac att gtg tcc ctc ttc ccg aac tac gac tcc cgc acc tac ccg 768
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255
atc cgc acc gtg tcc caa ctg acc cgc gaa atc tac acc aac ccc gtc 816
Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270
ctg gag aac ttc gac ggt agc ttc agg ggc agc gcc cag ggc atc gag 864
Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285
ggc tcc atc agg agc cca cac ctg atg gac atc ctc aac agc atc act 912
Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr
290 295 300
atc tac acc gat gcc cac cgc ggc gag tac tac tgg tcc ggc cac cag 960
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
305 310 315 320
atc atg gcc tcc ccg gtc ggc ttc agc ggc ccc gag ttt acc ttt cct 1008
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335
ctc tac ggc acg atg ggc aac gcc gct cca caa caa cgc atc gtc gct 1056
Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350
cag ctg ggc cag ggc gtc tac cgc acc ctg agc tcc acc ctg tac cgc 1104
Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg
355 360 365
agg ccc ttc aac atc ggt atc aac aac cag cag ctg tcc gtc ctg gat 1152
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380
ggc act gag ttc gcc tac ggc acc tcc tcc aac ctg ccc tcc gct gtc 1200
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
385 390 395 400
tac cgc aag agc ggc acg gtg gat tcc ctg gac gag atc cca cca cag 1248
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415
aac aac aat gtg ccc ccc agg cag ggt ttt tcc cac agg ctc agc cac 1296
Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430
gtg tcc atg ttc cgc tcc ggc ttc agc aac tcg tcc gtg agc atc atc 1344
Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445
aga gct cct atg ttc tct tgg ata cac cgt agt gct gag ttc aac aac 1392
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
450 455 460
atc att gca tcc gac agc att act caa ata ccc ttg gtg aaa gca cat 1440
Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His
465 470 475 480
aca ctt cag tca ggt act act gtt gtc aga ggt cca ggg ttt aca gga 1488
Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly
485 490 495
gga gac att ctt cgt cgc aca agt gga gga ccc ttt gct tac act att 1536
Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile
500 505 510
gtt aac atc aat ggc caa ttg ccc caa agg tat cgt gca aga atc cgc 1584
Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg
515 520 525
tat gcc tct act aca aat ctc agg atc tac gtg act gtt gca ggt gaa 1632
Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu
530 535 540
agg atc ttt gct ggt cag ttc aac aag act atg gat acc ggt gac cct 1680
Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro
545 550 555 560
ttg aca ttc caa tct ttt agc tac gca act atc aac aca gct ttt aca 1728
Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr
565 570 575
ttc cca atg agc cag agt agc ttc aca gta ggt gct gac act ttc agc 1776
Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser
580 585 590
tca ggg aat gaa gtt tac atc gac agg ttt gaa ttg att cca gtt act 1824
Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr
595 600 605
gca acc ctc gag gct gag tac aac ctt gag aga gcc cag aag gct gtg 1872
Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val
610 615 620
aac gcc ctc ttt acc tcc acc aat cag ctt ggc ttg aaa act aac gtt 1920
Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val
625 630 635 640
act gac tat cac att gac caa gtg tcc aac ttg gtc acc tac ctt agc 1968
Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser
645 650 655
gat gag ttc tgc ctc gac gag aag cgt gaa ctc tcc gag aaa gtt aaa 2016
Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys
660 665 670
cac gcc aag cgt ctc agc gac gag agg aat ctc ttg caa gac tcc aac 2064
His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn
675 680 685
ttc aaa gac atc aac agg cag cca gaa cgt ggt tgg ggt gga agc acc 2112
Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr
690 695 700
ggg atc acc atc caa gga ggc gac gat gtg ttc aag gag aac tac gtc 2160
Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val
705 710 715 720
acc ctc tcc gga act ttc gac gag tgc tac cct acc tac ttg tac cag 2208
Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln
725 730 735
aag atc gat gag tcc aaa ctc aaa gcc ttc acc agg tat caa ctt aga 2256
Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg
740 745 750
ggc tac atc gaa gac agc caa gac ctt gaa atc tac tcg atc agg tac 2304
Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg Tyr
755 760 765
aat gcc aag cac gag acc gtg aat gtc cca ggt act ggt tcc ctc tgg 2352
Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp
770 775 780
cca ctt tct gcc caa tct ccc att ggg aag tgt gga gag cct aac aga 2400
Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg
785 790 795 800
tgc gct cca cac ctt gag tgg aat cct gac ttg gac tgc tcc tgc agg 2448
Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg
805 810 815
gat ggc gag aag tgt gcc cac cat tct cat cac ttc tcc ttg gac atc 2496
Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile
820 825 830
gat gtg gga tgt act gac ctg aat gag gac ctc gga gtc tgg gtc atc 2544
Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile
835 840 845
ttc aag atc aag acc caa gac gga cac gca aga ctt ggc aac ctt gag 2592
Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu
850 855 860
ttt ctc gaa gag aaa cca ttg gtc ggt gaa gct ctc gct cgt gtg aag 2640
Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys
865 870 875 880
aga gca gag aag aag tgg agg gac aaa cgt gag aaa ctc gaa tgg gaa 2688
Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu
885 890 895
act aac atc gtt tac aag gag gcc aaa gag tcc gtg gat gct ttg ttc 2736
Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe
900 905 910
gtg aac tcc caa tat gat cag ttg caa gcc gac acc aac atc gcc atg 2784
Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met
915 920 925
atc cac gcc gca gac aaa cgt gtg cac agc att cgt gag gct tac ttg 2832
Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu
930 935 940
cct gag ttg tcc gtg atc cct ggt gtg aac gct gcc atc ttc gag gaa 2880
Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu
945 950 955 960
ctt gag gga cgt atc ttt acc gca ttc tcc ttg tac gat gcc aga aac 2928
Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn
965 970 975
gtc atc aag aac ggt gac ttc aac aat ggc ctc agc tgc tgg aat gtg 2976
Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val
980 985 990
aaa ggt cat gtg gac gtg gag gaa cag aac aat cag cgt tcc gtc ctg 3024
Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu
995 1000 1005
gtt gtg cct gag tgg gaa gct gaa gtg tcc caa gag gtt aga gtc 3069
Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val
1010 1015 1020
tgt cca ggt aga ggc tac att ctc cgt gtg acc gct tac aag gag 3114
Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu
1025 1030 1035
gga tac ggt gag ggt tgc gtg acc atc cac gag atc gag aac aac 3159
Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn
1040 1045 1050
acc gac gag ctt aag ttc tcc aac tgc gtc gag gaa gaa atc tat 3204
Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr
1055 1060 1065
ccc aac aac acc gtt act tgc aac gac tac act gtg aat cag gaa 3249
Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu
1070 1075 1080
gag tac gga ggt gcc tac act agc cgt aac aga ggt tac aac gaa 3294
Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu
1085 1090 1095
gct cct tcc gtt cct gct gac tat gcc tcc gtg tac gag gag aaa 3339
Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys
1100 1105 1110
tcc tac aca gat ggc aga cgt gag aac cct tgc gag ttc aac aga 3384
Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg
1115 1120 1125
ggt tac agg gac tac aca cca ctt cca gtt ggc tat gtt acc aag 3429
Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys
1130 1135 1140
gag ctt gag tac ttt cct gag acc gac aaa gtg tgg atc gag atc 3474
Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile
1145 1150 1155
ggt gaa acc gag gga acc ttc atc gtg gac agc gtg gag ctt ctc 3519
Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu
1160 1165 1170
ttg atg gag gaa taa 3534
Leu Met Glu Glu
1175
<210>4
<211>1177
<212>PRT
<213>人工的
<220>
<223>合成的构建体
<400>4
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly
20 25 30
Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser
35 40 45
Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile
50 55 60
Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
65 70 75 80
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu
100 105 110
Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg
180 185 190
Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val
195 200 205
Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg
210 215 220
Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
225 230 235 240
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro
245 250 255
Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val
260 265 270
Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu
275 280 285
Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr
290 295 300
Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
305 310 315 320
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
325 330 335
Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala
340 345 350
Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg
355 360 365
Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp
370 375 380
Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
385 390 395 400
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln
405 410 415
Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His
420 425 430
Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile
435 440 445
Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn
450 455 460
Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His
465 470 475 480
Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly
485 490 495
Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile
500 505 510
Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg
515 520 525
Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu
530 535 540
Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro
545 550 555 560
Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr
565 570 575
Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser
580 585 590
Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr
595 600 605
Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val
610 615 620
Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val
625 630 635 640
Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser
645 650 655
Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys
660 665 670
His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn
675 680 685
Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr
690 695 700
Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val
705 710 715 720
Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln
725 730 735
Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg
740 745 750
Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg Tyr
755 760 765
Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp
770 775 780
Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg
785 790 795 800
Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg
805 810 815
Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile
820 825 830
Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile
835 840 845
Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu
850 855 860
Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys
865 870 875 880
Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu
885 890 895
Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe
900 905 910
Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met
915 920 925
Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu
930 935 940
Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu
945 950 955 960
Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn
965 970 975
Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val
980 985 990
Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu
995 1000 1005
Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val
1010 1015 1020
Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu
1025 1030 1035
Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn
1040 1045 1050
Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr
1055 1060 1065
Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu
1070 1075 1080
Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu
1085 1090 1095
Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys
1100 1105 1110
Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg
1115 1120 1125
Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys
1130 1135 1140
Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile
1145 1150 1155
Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu
1160 1165 1170
Leu Met Glu Glu
1175
<210>5
<211>5480
<212>DNA
<213>人工的
<220>
<223>编码CrylA.105氨基酸序列的表达盒
<220>
<221>启动子
<222>(1)..(981)
<223>FMV.e35S启动子
<220>
<221>5’UTR
<222>(985)..(1080)
<220>
<221>转运_肽
<222>(1090)..(1263)
<223>叶绿体靶向肽序列
<220>
<221>CDS
<222>(1267)..(4797)
<223>CrylA.105的编码序列
<220>
<221>终止子
<222>(4833)..(5480)
<223>Ps.E9rbcS转录终止和多腺苷酸化序列
<400>5
aattctcagt ccaaagcctc aacaaggtca gggtacagag tctccaaacc attagccaaa 60
agctacagga gatcaatgaa gaatcttcaa tcaaagtaaa ctactgttcc agcacatgca 120
tcatggtcag taagtttcag aaaaagacat ccaccgaaga cttaaagtta gtgggcatct 180
ttgaaagtaa tcttgtcaac atcgagcagc tggcttgtgg ggaccagaca aaaaaggaat 240
ggtgcagaat tgttaggcgc acctaccaaa agcatctttg cctttattgc aaagataaag 300
cagattcctc tagtacaagt ggggaacaaa ataacgtgga aaagagctgt cctgacagcc 360
cactcactaa tgcgtatgac gaacgcagtg acgaccacaa aagaattagc ttgagctcag 420
gatttagcag cattccagat tgggttcaat caacaaggta cgagccatat cactttattc 480
aaattggtat cgccaaaacc aagaaggaac tcccatcctc aaaggtttgt aaggaagaat 540
tctcagtcca aagcctcaac aaggtcaggg tacagagtct ccaaaccatt agccaaaagc 600
tacaggagat caatgaagaa tcttcaatca aagtaaacta ctgttccagc acatgcatca 660
tggtcagtaa gtttcagaaa aagacatcca ccgaagactt aaagttagtg ggcatctttg 720
aaagtaatct tgtcaacatc gagcagctgg cttgtgggga ccagacaaaa aaggaatggt 780
gcagaattgt taggcgcacc taccaaaagc atctttgcct ttattgcaaa gataaagcag 840
attcctctag tacaagtggg gaacaaaata acgtggaaaa gagctgtcct gacagcccac 900
tcactaatgc gtatgacgaa cgcagtgacg accacaaaag aattccctct atataagaag 960
gcattcattc ccatttgaag gacacagaaa aatttgctac attgtttcac aaacttcaaa 1020
tattattcat ttatttgtca gctttcaaac tctttgtttc ttgtttgttg attgagaata 1080
tttaaaacaa tggcttcctc tatgctctct tccgctacta tggttgcctc tccggctcag 1140
gccactatgg tcgctccttt caacggactt aagtcctccg ctgccttccc agccacccgc 1200
aaggctaaca acgacattac ttccatcaca agcaacggcg gaagagttaactgcatgcag 1260
gcc atg gac aac aac cca aac atc aac gaa tgc att cca tac aac tgc 1308
Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys
1 5 10
ttg agt aac cca gaa gtt gaa gta ctt ggt gga gaa cgc att gaa acc 1356
Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr
15 20 25 30
ggt tac act ccc atc gac atc tcc ttg tcc ttg aca cag ttt ctg ctc 1404
Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu
35 40 45
agc gag ttc gtg cca ggt gct ggg ttc gtt ctc gga cta gtt gac atc 1452
Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile
50 55 60
atc tgg ggt atc ttt ggt cca tct caa tgg gat gca ttc ctg gtg caa 1500
Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln
65 70 75
att gag cag ttg atc aac cag agg atc gaa gag ttc gcc agg aac cag 1548
Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln
80 85 90
gcc atc tct agg ttg gaa gga ttg agc aat ctc tac caa atc tat gca 1596
Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala
95 100 105 110
gag agc ttc aga gag tgg gaa gcc gat cct act aac cca gct ctc cgc 1644
Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg
115 120 125
gag gaa atg cgt att caa ttc aac gac atg aac agc gcc ttg acc aca 1692
Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr
130 135 140
gct atc cca ttg ttc gca gtc cag aac tac caa gtt cct ctc ttg tcc 1740
Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser
145 150 155
gtg tac gtt caa gca gct aat ctt cac ctc agc gtg ctt cga gac gtt 1788
Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val
160 165 170
agc gtg ttt ggg caa agg tgg gga ttc gat gct gca acc atc aat agc 1836
Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser
175 180 185 190
cgt tac aac gac ctt act agg ctg att gga aac tac acc gac cac gct 1884
Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala
195 200 205
gtt cgt tgg tac aac act ggc ttg gag cgt gtc tgg ggt cct gat tct 1932
Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser
210 215 220
aga gat tgg att aga tac aac cag ttc agg aga gaa ttg acc ctc aca 1980
Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr
225 230 235
gtt ttg gac att gtg tct ctc ttc ccg aac tat gac tcc aga acc tac 2028
Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr
240 245 250
cct atc cgt aca gtg tcc caa ctt acc aga gaa atc tat act aac cca 2076
Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro
255 260 265 270
gtt ctt gag aac ttc gac ggt agc ttc cgt ggt tct gcc caa ggt atc 2124
Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile
275 280 285
gaa ggc tcc atc agg agc cca cac ttg atg gac atc ttg aac agc ata 2172
Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile
290 295 300
act atc tac acc gat gct cac aga gga gag tat tac tgg tct gga cac 2220
Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His
305 310 315
cag atc atg gcc tct cca gtt gga ttc agc ggg ccc gag ttt acc ttt 2268
Gln Ile Met Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Thr Phe
320 325 330
cct ctc tat gga act atg gga aac gcc gct cca caa caa cgt atc gtt 2316
Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val
335 340 345 350
gct caa cta ggt cag ggt gtc tac aga acc ttg tct tcc acc ttg tac 2364
Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr
355 360 365
aga aga ccc ttc aat atc ggt atc aac aac cag caa ctt tcc gtt ctt 2412
Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu
370 375 380
gac gga aca gag ttc gcc tat gga acc tct tct aac ttg cca tcc gct 2460
Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala
385 390 395
gtt tac aga aag agc gga acc gtt gat tcc ttg gac gaa atc cca cca 2508
Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro
400 405 410
cag aac aac aat gtg cca ccc agg caa gga ttc tcc cac agg ttg agc 2556
Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser
415 420 425 430
cac gtg tcc atg ttc cgt tcc gga ttc agc aac agt tcc gtg agc atc 2604
His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile
435 440 445
atc aga gct cct atg ttc tct tgg ata cac cgt agt gct gag ttc aac 2652
Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn
450 455 460
aac atc att gca tcc gac agc att act caa ata ccc ttg gtg aaa gca 2700
Asn Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala
465 470 475
cat aca ctt cag tca ggt act act gtt gtc aga ggt cca ggg ttt aca 2748
His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr
480 485 490
gga gga gac att ctt cgt cgc aca agt gga gga ccc ttt gct tac act 2796
Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr
495 500 505 510
att gtt aac atc aat ggc caa ttg ccc caa agg tat cgt gca aga atc 2844
Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile
515 520 525
cgc tat gcc tct act aca aat ctc agg atc tac gtg act gtt gca ggt 2892
Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly
530 535 540
gaa agg atc ttt gct ggt cag ttc aac aag act atg gat acc ggt gac 2940
Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp
545 550 555
cct ttg aca ttc caa tct ttt agc tac gca act atc aac aca gct ttt 2988
Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe
560 565 570
aca ttc cca atg agc cag agt agc ttc aca gta ggt gct gac act ttc 3036
Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe
575 580 585 590
agc tca ggg aat gaa gtt tac atc gac agg ttt gaa ttg att cca gtt 3084
Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val
595 600 605
act gca acc ctc gag gct gag tac aac ctt gag aga gcc cag aag gct 3132
Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala
610 615 620
gtg aac gcc ctc ttt acc tcc acc aat cag ctt ggc ttg aaa act aac 3180
Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn
625 630 635
gtt act gac tat cac att gac caa gtg tcc aac ttg gtc acc tac ctt 3228
Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu
640 645 650
agc gat gag ttc tgc ctc gac gag aag cgt gaa ctc tcc gag aaa gtt 3276
Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val
655 660 665 670
aaa cac gcc aag cgt ctc agc gac gag agg aat ctc ttg caa gac tcc 3324
Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser
675 680 685
aac ttc aaa gac atc aac agg cag cca gaa cgt ggt tgg ggt gga agc 3372
Asn Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser
690 695 700
acc ggg atc acc atc caa gga ggc gac gat gtg ttc aag gag aac tac 3420
Thr Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr
705 710 715
gtc acc ctc tcc gga act ttc gac gag tgc tac cct acc tac ttg tac 3468
Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr
720 725 730
cag aag atc gat gag tcc aaa ctc aaa gcc ttc acc agg tat caa ctt 3516
Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu
735 740 745 750
aga ggc tac atc gaa gac agc caa gac ctt gaa atc tac tcg atc agg 3564
Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg
755 760 765
tac aat gcc aag cac gag acc gtg aat gtc cca ggt act ggt tcc ctc 3612
Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu
770 775 780
tgg cca ctt tct gcc caa tct ccc att ggg aag tgt gga gag cct aac 3660
Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn
785 790 795
aga tgc gct cca cac ctt gag tgg aat cct gac ttg gac tgc tcc tgc 3708
Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys
800 805 810
agg gat ggc gag aag tgt gcc cac cat tct cat cac ttc tcc ttg gac 3756
Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp
815 820 825 830
atc gat gtg gga tgt act gac ctg aat gag gac ctc gga gtc tgg gtc 3804
Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val
835 840 845
atc ttc aag atc aag acc caa gac gga cac gca aga ctt ggc aac ctt 3852
Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu
850 855 860
gag ttt ctc gaa gag aaa cca ttg gtc ggt gaa gct ctc gct cgt gtg 3900
Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val
865 870 875
aag aga gca gag aag aag tgg agg gac aaa cgt gag aaa ctc gaa tgg 3948
Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp
880 885 890
gaa act aac atc gtt tac aag gag gcc aaa gag tcc gtg gat gct ttg 3996
Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu
895 900 905 910
ttc gtg aac tcc caa tat gat cag ttg caa gcc gac acc aac atc gcc 4044
Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala
915 920 925
atg atc cac gcc gca gac aaa cgt gtg cac agc att cgt gag gct tac 4092
Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr
930 935 940
ttg cct gag ttg tcc gtg atc cct ggt gtg aac gct gcc atc ttc gag 4140
Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu
945 950 955
gaa ctt gag gga cgt atc ttt acc gca ttc tcc ttg tac gat gcc aga 4188
Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg
960 965 970
aac gtc atc aag aac ggt gac ttc aac aat ggc ctc agc tgc tgg aat 4236
Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn
975 980 985 990
gtg aaa ggt cat gtg gac gtg gag gaa cag aac aat cag cgt tcc gtc 4284
Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val
995 1000 1005
ctg gtt gtg cct gag tgg gaa gct gaa gtg tcc caa gag gtt aga 4329
Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg
1010 1015 1020
gtc tgt cca ggt aga ggc tac att ctc cgt gtg acc gct tac aag 4374
Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys
1025 1030 1035
gag gga tac ggt gag ggt tgc gtg acc atc cac gag atc gag aac 4419
Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn
1040 1045 1050
aac acc gac gag ctt aag ttc tcc aac tgc gtc gag gaa gaa atc 4464
Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile
1055 1060 1065
tat ccc aac aac acc gtt act tgc aac gac tac act gtg aat cag 4509
Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln
1070 1075 1080
gaa gag tac gga ggt gcc tac act agc cgt aac aga ggt tac aac 4554
Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn
1085 1090 1095
gaa gct cct tcc gtt cct gct gac tat gcc tcc gtg tac gag gag 4599
Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu
1100 1105 1110
aaa tcc tac aca gat ggc aga cgt gag aac cct tgc gag ttc aac 4644
Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn
1115 1120 1125
aga ggt tac agg gac tac aca cca ctt cca gtt ggc tat gtt acc 4689
Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr
1130 1135 1140
aag gag ctt gag tac ttt cct gag acc gac aaa gtg tgg atc gag 4734
Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu
1145 1150 1155
atc ggt gaa acc gag gga acc ttc atc gtg gac agc gtg gag ctt 4779
Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu
1160 1165 1170
ctc ttg atg gag gaa taa tgagatccac gatatcctgc aggaattggc 4827
Leu Leu Met Glu Glu
1175
cggccagctt tcgttcgtat catcggtttc gacaacgttc gtcaagttca atgcatcagt 4887
ttcattgcgc acacaccaga atcctactga gtttgagtat tatggcattg ggaaaactgt 4947
ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat 5007
cgaactgtga aatggaaatg gatggagaag agttaatgaa tgatatggtc cttttgttca 5067
ttctcaaatt aatattattt gttttttctc ttatttgttg tgtgttgaat ttgaaattat 5127
aagagatatg caaacatttt gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac 5187
cgaagttaat atgaggagta aaacacttgt agttgtacca ttatgcttat tcactaggca 5247
acaaatatat tttcagacct agaaaagctg caaatgttac tgaatacaag tatgtcctct 5307
tgtgttttag acatttatgg actttccttt atgtaatttt ccagaatcct tgtcagattc 5367
taatcattgc tttataatta tagttatact catggatttg tagttgagta tgaaaatatt 5427
ttttaatgca ttttatgact tgccaattga ttgacaacat gcatcaatcg acc 5480
<210>6
<211>1176
<212>PRT
<213>人工的
<220>
<223>合成的构建体
<400>6
Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser
1 5 10 15
Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr
20 25 30
Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser Glu
35 40 45
Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile Trp
50 55 60
Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile Glu
65 70 75 80
Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile
85 90 95
Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser
100 105 110
Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu Glu
115 120 125
Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala Ile
130 135 140
Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val Tyr
145 150 155 160
Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val
165 170 175
Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr
180 185 190
Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val Arg
195 200 205
Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg Asp
210 215 220
Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val Leu
225 230 235 240
Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile
245 250 255
Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu
260 265 270
Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu Gly
275 280 285
Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr Ile
290 295 300
Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln Ile
305 310 315 320
Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu
325 330 335
Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln
340 345 350
Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg Arg
355 360 365
Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp Gly
370 375 380
Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val Tyr
385 390 395 400
Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn
405 410 415
Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val
420 425 430
Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile Arg
435 440 445
Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn Ile
450 455 460
Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu Val Lys Ala His Thr
465 470 475 480
Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro Gly Phe Thr Gly Gly
485 490 495
Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe Ala Tyr Thr Ile Val
500 505 510
Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr
515 520 525
Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr Val Ala Gly Glu Arg
530 535 540
Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp Thr Gly Asp Pro Leu
545 550 555 560
Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe
565 570 575
Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala Asp Thr Phe Ser Ser
580 585 590
Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu Ile Pro Val Thr Ala
595 600 605
Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala Val Asn
610 615 620
Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn Val Thr
625 630 635 640
Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu Ser Asp
645 650 655
Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val Lys His
660 665 670
Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser Asn Phe
675 680 685
Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser Thr Gly
690 695 700
Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr
705 710 715 720
Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys
725 730 735
Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu Arg Gly
740 745 750
Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg Tyr Asn
755 760 765
Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro
770 775 780
Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn Arg Cys
785 790 795 800
Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp
805 810 815
Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp Ile Asp
820 825 830
Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe
835 840 845
Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe
850 855 860
Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg
865 870 875 880
Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr
885 890 895
Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val
900 905 910
Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala Met Ile
915 920 925
His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Leu Pro
930 935 940
Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Leu
945 950 955 960
Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg Asn Val
965 970 975
Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn Val Lys
980 985 990
Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val Leu Val
995 1000 1005
Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val Cys
1010 1015 1020
Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly
1025 1030 1035
Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr
1040 1045 1050
Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro
1055 1060 1065
Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu
1070 1075 1080
Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala
1085 1090 1095
Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser
1100 1105 1110
Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn Arg Gly
1115 1120 1125
Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr Lys Glu
1130 1135 1140
Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly
1145 1150 1155
Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu
1160 1165 1170
Met Glu Glu
1175
<210>7
<211>4990
<212>DNA
<213>人工的
<220>
<223>编码Cry1A.105氨基酸序列的表达盒
<220>
<221>启动子
<222>(1)..(614)
<223>CAMV e35s启动子
<220>
<221>5’UTR
<222>(650)..(710)
<220>
<221>内含子
<222>(727)..(1206)
<223>稻肌动蛋白内含子RACT1
<220>
<221>CDS
<222>(1216)..(4752)
<223>Cry1A.105的编码序列
<220>
<221>终止子
<222>(4781)..(4990)
<223>Ta.Hsp17转录终止和多腺苷酸化序列
<400>7
ggtccgatgt gagacttttc aacaaagggt aatatccgga aacctcctcg gattccattg 60
cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct cctacaaatg 120
ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca gtggtcccaa 180
agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc 240
aaagcaagtg gattgatgtg atggtccgat gtgagacttt tcaacaaagg gtaatatccg 300
gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag atagtggaaa 360
aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc gttgaagatg 420
cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc gtggaaaaag 480
aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgatatctcc actgacgtaa 540
gggatgacgc acaatcccac tatccttcgc aagacccttc ctctatataa ggaagttcat 600
ttcatttgga gaggacacgc tgacaagctg actctagcag atcctctaga accatcttcc 660
acacactcaa gccacactat tggagaacac acagggacaa cacaccataa gatccaaggg 720
aggcctccgc cgccgccggt aaccaccccg cccctctcct ctttctttct ccgttttttt 780
ttccgtctcg gtctcgatct ttggccttgg tagtttgggt gggcgagagg cggcttcgtg 840
cgcgcccaga tcggtgcgcg ggaggggcgg gatctcgcgg ctggggctct cgccggcgtg 900
gatccggccc ggatctcgcg gggaatgggg ctctcggatg tagatctgcg atccgccgtt 960
gttgggggag atgatggggg gtttaaaatt tccgccgtgc taaacaagat caggaagagg 1020
ggaaaagggc actatggttt atatttttat atatttctgc tgcttcgtca ggcttagatg 1080
tgctagatct ttctttcttc tttttgtggg tagaatttga atccctcagc attgttcatc 1140
ggtagttttt cttttcatga tttgtgacaa atgcagcctc gtgcggagct tttttgtagg 1200
tagaagtgat caacc atg gac aac aac cca aac atc aac gag tgc atc ccg 1251
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro
1 5 10
tac aac tgc ctc agc aac cct gag gtc gag gtg ctc ggc ggt gag cgc 1299
Tyr Asn Cys Leu Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg
15 20 25
atc gag acc ggt tac acc ccc atc gac atc tcc ctc tcc ctc acg cag 1347
Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln
30 35 40
ttc ctg ctc agc gag ttc gtg cca ggc gct ggc ttc gtc ctg ggc ctc 1395
Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu
45 50 55 60
gtg gac atc atc tgg ggc atc ttt ggc ccc tcc cag tgg gac gcc ttc 1443
Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe
65 70 75
ctg gtg caa atc gag cag ctc atc aac cag agg atc gag gag ttc gcc 1491
Leu Val Gln Ile Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala
80 85 90
agg aac cag gcc atc agc cgc ctg gag ggc ctc agc aac ctc tac caa 1539
Arg Asn Gln Ala Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln
95 100 105
atc tac gct gag agc ttc cgc gag tgg gag gcc gac ccc act aac cca 1587
Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro
110 115 120
gct ctc cgc gag gag atg cgc atc cag ttc aac gac atg aac agc gcc 1635
Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala
125 130 135 140
ctg acc acc gcc atc cca ctc ttc gcc gtc cag aac tac caa gtc ccg 1683
Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro
145 150 155
ctc ctg tcc gtg tac gtc cag gcc gcc aac ctg cac ctc agc gtg ctg 1731
Leu Leu Ser Val Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu
160 165 170
agg gac gtc agc gtg ttt ggc cag agg tgg ggc ttc gac gcc gcc acc 1779
Arg Asp Val Ser Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr
175 180 185
atc aac agc cgc tac aac gac ctc acc agg ctg atc ggc aac tac acc 1827
Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr
190 195 200
gac cac gct gtc cgc tgg tac aac act ggc ctg gag cgc gtc tgg ggc 1875
Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly
205 210 215 220
cct gat tct aga gac tgg att cgc tac aac cag ttc agg cgc gag ctg 1923
Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu
225 230 235
acc ctc acc gtc ctg gac att gtg tcc ctc ttc ccg aac tac gac tcc 1971
Thr Leu Thr Val Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser
240 245 250
cgc acc tac ccg atc cgc acc gtg tcc caa ctg acc cgc gaa atc tac 2019
Arg Thr Tyr Pro Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr
255 260 265
acc aac ccc gtc ctg gag aac ttc gac ggt agc ttc agg ggc agc gcc 2067
Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala
270 275 280
cag ggc atc gag ggc tcc atc agg agc cca cac ctg atg gac atc ctc 2115
Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu
285 290 295 300
aac agc atc act atc tac acc gat gcc cac cgc ggc gag tac tac tgg 2163
Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp
305 310 315
tcc ggc cac cag atc atg gcc tcc ccg gtc ggc ttc agc ggc ccc gag 2211
Ser Gly His Gln Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu
320 325 330
ttt acc ttt cct ctc tac ggc acg atg ggc aac gcc gct cca caa caa 2259
Phe Thr Phe Pro Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln
335 340 345
cgc atc gtc gct cag ctg ggc cag ggc gtc tac cgc acc ctg agc tcc 2307
Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser
350 355 360
acc ctg tac cgc agg ccc ttc aac atc ggt atc aac aac cag cag ctg 2355
Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu
365 370 375 380
tcc gtc ctg gat ggc act gag ttc gcc tac ggc acc tcc tcc aac ctg 2403
Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu
385 390 395
ccc tcc gct gtc tac cgc aag agc ggc acg gtg gat tcc ctg gac gag 2451
Pro Ser Ala Val Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu
400 405 410
atc cca cca cag aac aac aat gtg ccc ccc agg cag ggt ttt tcc cac 2499
Ile Pro Pro Gln Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His
415 420 425
agg ctc agc cac gtg tcc atg ttc cgc tcc ggc ttc agc aac tcg tcc 2547
Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser
430 435 440
gtg agc atc atc aga gct cct atg ttc tct tgg ata cac cgt agt gct 2595
Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala
445 450 455 460
gag ttc aac aac atc att gca tcc gac agc att act caa ata ccc ttg 2643
Glu Phe Asn Asn Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Leu
465 470 475
gtg aaa gca cat aca ctt cag tca ggt act act gtt gtc aga ggt cca 2691
Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr Val Val Arg Gly Pro
480 485 490
ggg ttt aca gga gga gac att ctt cgt cgc aca agt gga gga ccc ttt 2739
Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr Ser Gly Gly Pro Phe
495 500 505
gct tac act att gtt aac atc aat ggc caa ttg ccc caa agg tat cgt 2787
Ala Tyr Thr Ile Val Asn Ile Asn Gly Gln Leu Pro Gln Arg Tyr Arg
510 515 520
gca aga atc cgc tat gcc tct act aca aat ctc agg atc tac gtg act 2835
Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg Ile Tyr Val Thr
525 530 535 540
gtt gca ggt gaa agg atc ttt gct ggt cag ttc aac aag act atg gat 2883
Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe Asn Lys Thr Met Asp
545 550 555
acc ggt gac cct ttg aca ttc caa tct ttt agc tac gca act atc aac 2931
Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser Tyr Ala Thr Ile Asn
560 565 570
aca gct ttt aca ttc cca atg agc cag agt agc ttc aca gta ggt gct 2979
Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser Phe Thr Val Gly Ala
575 580 585
gac act ttc agc tca ggg aat gaa gtt tac atc gac agg ttt gaa ttg 3027
Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile Asp Arg Phe Glu Leu
590 595 600
att cca gtt act gca acc ctc gag gct gag tac aac ctt gag aga gcc 3075
Ile Pro Val Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala
605 610 615 620
cag aag gct gtg aac gcc ctc ttt acc tcc acc aat cag ctt ggc ttg 3123
Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu
625 630 635
aaa act aac gtt act gac tat cac att gac caa gtg tcc aac ttg gtc 3171
Lys Thr Asn Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val
640 645 650
acc tac ctt agc gat gag ttc tgc ctc gac gag aag cgt gaa ctc tcc 3219
Thr Tyr Leu Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser
655 660 665
gag aaa gtt aaa cac gcc aag cgt ctc agc gac gag agg aat ctc ttg 3267
Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu
670 675 680
caa gac tcc aac ttc aaa gac atc aac agg cag cca gaa cgt ggt tgg 3315
Gln Asp Ser Asn Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp
685 690 695 700
ggt gga agc acc ggg atc acc atc caa gga ggc gac gat gtg ttc aag 3363
Gly Gly Ser Thr Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys
705 710 715
gag aac tac gtc acc ctc tcc gga act ttc gac gag tgc tac cct acc 3411
Glu Asn Tyr Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr
720 725 730
tac ttg tac cag aag atc gat gag tcc aaa ctc aaa gcc ttc acc agg 3459
Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg
735 740 745
tat caa ctt aga ggc tac atc gaa gac agc caa gac ctt gaa atc tac 3507
Tyr Gln Leu Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr
750 755 760
tcg atc agg tac aat gcc aag cac gag acc gtg aat gtc cca ggt act 3555
Ser Ile Arg Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr
765 770 775 780
ggt tcc ctc tgg cca ctt tct gcc caa tct ccc att ggg aag tgt gga 3603
Gly Ser Leu Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly
785 790 795
gag cct aac aga tgc gct cca cac ctt gag tgg aat cct gac ttg gac 3651
Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp
800 805 810
tgc tcc tgc agg gat ggc gag aag tgt gcc cac cat tct cat cac ttc 3699
Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe
815 820 825
tcc ttg gac atc gat gtg gga tgt act gac ctg aat gag gac ctc gga 3747
Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly
830 835 840
gtc tgg gtc atc ttc aag atc aag acc caa gac gga cac gca aga ctt 3795
Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu
845 850 855 860
ggc aac ctt gag ttt ctc gaa gag aaa cca ttg gtc ggt gaa gct ctc 3843
Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu
865 870 875
gct cgt gtg aag aga gca gag aag aag tgg agg gac aaa cgt gag aaa 3891
Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys
880 885 890
ctc gaa tgg gaa act aac atc gtt tac aag gag gcc aaa gag tcc gtg 3939
Leu Glu Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val
895 900 905
gat gct ttg ttc gtg aac tcc caa tat gat cag ttg caa gcc gac acc 3987
Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr
910 915 920
aac atc gcc atg atc cac gcc gca gac aaa cgt gtg cac agc att cgt 4035
Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg
925 930 935 940
gag gct tac ttg cct gag ttg tcc gtg atc cct ggt gtg aac gct gcc 4083
Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala
945 950 955
atc ttc gag gaa ctt gag gga cgt atc ttt acc gca ttc tcc ttg tac 4131
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Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val
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Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser
1155 1160 1165
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Val Glu Leu Leu Leu Met Glu Glu
1170 1175
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taaattgttc tgattttgag tgcaaaaaaa aaggaatt 4990
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Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu
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Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala
130 135 140
Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
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Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro
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1175
权利要求书(按照条约第19条的修改)
根据“专利合作条约第19条”的声明
申请人在此考虑到2007年3月6日发出的国际检索报告和书面意见提供对权利要求书的修改。和本声明一致,对权利要求1-6、9、12、14、17-26、28、32和34进行了修改,且提供了6页替换页,所述替换页根据它们所替换的权利要求页进行编页,连续地从第19至第24页。所述替换页含有权利要求书修改,但不含显示修改内容的标记。
对权利要求书的修改由下述组成:
在权利要求1-7、12、14、18-21、23-26和32中,参考检索报告和书面意见中进一步的意见,将“核苷酸序列”修改为“多核苷酸”。
在权利要求9、14、19-23、28和34中,参考检索报告和书面意见中进一步的意见No.1.2,将蛋白的基本特征与在SEQ ID NO:2中示出的各自氨基酸序列相关联,且对于权利要求14,引入“在…中表达”的内容以处理所述意见。
对权利要求17的修改仅仅澄清棉铃叶蛾和棉铃虫特性。
考虑到检索报告和书面意见中处理提及“核酸序列”的问题的评论,对权利要求21的修改区别了对使用词语“多核苷酸”的不同的参考。“多核苷酸探针”的基础可见原说明书第7页第18-24行。
没有添加新权利要求。
对权利要求书的修改可以根据原说明书或权利要求书直接且毫无疑义地确定,且这些修改没有添加新内容。
1. 分离的多核苷酸,其编码含有SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列的杀虫蛋白。
2. 权利要求1的分离的多核苷酸,其选自SEQ ID NO:1和SEQ IDNO:3。
3. 权利要求2的分离的多核苷酸,其用于在作物植物中表达所述杀虫蛋白。
4. 权利要求3的分离的多核苷酸,其中所述作物植物选自单子叶作物植物和双子叶作物植物。
5. 权利要求4的分离的多核苷酸,其中所述单子叶植物选自玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草、狗芽根、剪股颖)和大麦。
6. 权利要求4的分离的多核苷酸,其中所述双子叶植物选自苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜。
7. 分离和纯化的杀虫蛋白,其含有SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列。
8. 一种组合物,其含有杀虫有效量的权利要求7的分离和纯化的蛋白。
9. 一种用于在宿主细胞中表达具有SEQ ID NO:2所示氨基酸序列的杀虫蛋白的表达盒,其中所述表达盒含有可操作地连接的在所述宿主细胞中发挥功能的启动子序列和编码所述蛋白的多核苷酸。
10. 权利要求9的表达盒,其中所述宿主细胞选自细菌细胞、真菌细胞、哺乳动物细胞和植物细胞。
11. 权利要求10的表达盒,其中
(a)所述细菌细胞选自芽孢杆菌属物种细胞、肠杆菌科物种细胞、假单胞菌属物种细胞、梭菌属物种细胞、和根瘤菌属物种细胞、和土壤杆菌属物种细胞;和
(b)所述植物细胞选自双子叶植物和单子叶植物,所述双子叶植物进一步选自苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜,且所述单子叶植物进一步选自玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草、狗芽根、剪股颖)和大麦。
12. 权利要求9的表达盒,其中所述宿主细胞是植物细胞,且所述表达盒还含有选自以下的可操作地连接的多核苷酸:表达增强子序列、非翻译前导序列、内含子序列、叶绿体靶向肽编码序列和转录终止和多腺苷酸化序列。
13. 含有权利要求9至权利要求12之中任一项的表达盒的载体。
14. 抵抗鳞翅目昆虫侵袭的转基因植物或植物细胞,其含有编码显示SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列的蛋白的多核苷酸,其中所述蛋白当在所述植物或植物细胞中表达时表现杀虫活性。
15. 权利要求14的的转基因植物或植物细胞,其中所述转基因植物选自双子叶植物和单子叶植物,所述双子叶植物进一步选自苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜,且所述单子叶植物进一步选自玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草、狗芽根、剪股颖)和大麦。
16. 对抗鳞翅目昆虫侵袭的权利要求15的转基因植物或植物细胞,其中所述鳞翅目昆虫选自卷叶蛾、地老虎、黏虫、钻蛀虫、避债虫和草料进食者。
17. 权利要求16的转基因植物或植物细胞,其中所述鳞翅目昆虫进一步选自草地夜蛾、玉米螟、棉铃叶蛾或棉铃虫、西南玉米杆草螟和小地老虎。
18. 权利要求15的转基因植物或植物细胞的子代或种子,其中所述子代或种子含有所述多核苷酸。
19. 通过在昆虫食物中提供一种或多种植物细胞来控制鳞翅目昆虫对植物的侵袭的方法,所述植物细胞是用含有可操作地连接的植物功能性启动子和编码显示SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列的蛋白的多核苷酸的核酸序列转化的,所述蛋白表现鳞翅目杀虫活性。
20. 权利要求12的表达盒,其含有选自SEQ ID NO:5和SEQ IDNO:7的多核苷酸。
21. 鉴定生物样品中编码显示SEQ ID NO:2中自约第10位氨基酸至约第600位氨基酸所示氨基酸序列的Cry1A.105蛋白的多核苷酸的方法,所述方法包括将所述样品与在严格杂交条件下与该多核苷酸杂交的多核苷酸探针接触,并检测所述多核苷酸探针与所述多核苷酸的结合,其中所述结合对所述样品中所述多核苷酸的存在具有诊断价值。
22. 鉴定样品中显示SEQ ID NO:2中自约第10位氨基酸至约第600位氨基酸所示氨基酸序列的Cry1A.105蛋白的方法,所述方法包括将所述样品与特异性结合所述蛋白的抗体接触,并检测结合,其中所述结合对所述样品中所述蛋白的存在具有诊断价值。
23. 分离的多核苷酸,其编码显示SEQ ID NO:2中自约第1位氨基酸至约第612位氨基酸所示氨基酸序列的杀虫蛋白。
24. 分离的多核苷酸,其编码SEQ ID NO:2中自约第1位氨基酸至约第610位氨基酸所示的杀虫蛋白。
25. 权利要求23的分离的多核苷酸,其与SEQ ID NO:1所示核苷酸序列表现至少约90%同一性。
26. 权利要求23的分离的多核苷酸,其与SEQ ID NO:3所示核苷酸序列表现至少约90%同一性。
27. 杂合杀虫蛋白,其含有包含选自SEQ ID NO:2约第10位氨基酸至约第600位氨基酸所示氨基酸部分的约500至约600个邻接氨基酸的氨基酸部分。
28. 含有杀虫有效量的显示SEQ ID NO:2中自约第10位氨基酸至约第600位氨基酸所示氨基酸序列的Cry1A.105蛋白的组合物,其中所述组合物选自植物细胞、细菌细胞、真菌细胞、胶体、乳状液、种子涂层、诱饵和粉末。
29. 权利要求28的组合物,其中所述Cry1A.105蛋白以百万分之约0.5至约200(PPM)的量存在。
30. 权利要求29的组合物,其中所述Cry1A.105蛋白以约0.5至约20PPM的量存在。
31. 权利要求29的组合物,其中所述组合物是植物细胞或一组植物细胞。
32. 权利要求31的组合物,其中编码杀虫有效量的所述Cry1A.105蛋白的多核苷酸以使用探针可检测的量存在,所示探针是选自SEQ IDNO:1第1401-1420位核苷酸和SEQ ID NO:1第1821-1840位核苷酸的序列或与之互补。
33. 权利要求31的组合物,其中当所述杀虫有效量的所述蛋白在所述害虫的食物中提供时足以控制鳞翅目属植物害虫,所述害虫选自叶蛾属、尺叶蛾属、Rachiplusia、实叶蛾属、Helicoverpa、灰翅叶蛾属、叶小卷娥属和Armigera。
34. 保护田间作物避免鳞翅目昆虫侵袭的方法,该方法包括种植在鳞翅目昆虫食物中含有杀虫有效量Cry1A.105蛋白杀虫剂的转基因作物植物,以抑制昆虫在所述转基因作物植物上存活,其中所述杀虫剂包含SEQ ID NO:2中自约第10位氨基酸至约第600位氨基酸所示的氨基酸序列。
35. 权利要求34的方法,其中所述转基因作物植物还含有对与Cry1A.105所针对的相同昆虫物种有毒性的其它杀虫剂,且其选自芽孢杆菌属毒素、致病杆菌属毒素、光杆状菌属毒素和特异于抑制所述昆虫物种中一种或多种必需基因的dsRNA。
36. 权利要求34或35的方法,其中所述作物的产量相比于缺乏所述一种或多种杀虫剂的同基因作物的产量有所提高。
Claims (36)
1. 分离的核苷酸序列,其编码含有SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列的杀虫蛋白。
2. 权利要求1的分离的核苷酸序列,其选自SEQ ID NO:1和SEQID NO:3。
3. 权利要求2的分离的核苷酸序列,其用于在作物植物中表达所述杀虫蛋白。
4. 权利要求3的分离的核苷酸序列,其中所述作物植物选自单子叶作物植物和双子叶作物植物。
5. 权利要求4的分离的核苷酸序列,其中所述单子叶植物选自玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草、狗芽根、剪股颖)和大麦。
6. 权利要求4的分离的核苷酸序列,其中所述双子叶植物选自苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜。
7. 分离和纯化的杀虫蛋白,其含有SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列。
8. 一种组合物,其含有杀虫有效量的权利要求7的分离和纯化的蛋白。
9. 一种用于在宿主细胞中表达SEQ ID NO:2所示杀虫蛋白的表达盒,其中所述表达盒含有可操作地连接的在所述宿主细胞中发挥功能的启动子序列和编码所述蛋白的核苷酸序列。
10. 权利要求9的表达盒,其中所述宿主细胞选自细菌细胞、真菌细胞、哺乳动物细胞和植物细胞。
11. 权利要求10的表达盒,其中
(a)所述细菌细胞选自芽孢杆菌属物种细胞、肠杆菌科物种细胞、假单胞菌属物种细胞、梭菌属物种细胞、和根瘤菌属物种细胞、和土壤杆菌属物种细胞;和
(b)所述植物细胞选自双子叶植物和单子叶植物,所述双子叶植物进一步选自苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜,且所述单子叶植物进一步选自玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草、狗芽根、剪股颖)和大麦。
12. 权利要求9的表达盒,其中所述宿主细胞是植物细胞,且所述表达盒还含有选自以下的可操作地连接的序列:表达增强子序列、非翻译前导序列、内含子序列、叶绿体靶向肽编码序列和转录终止和多腺苷酸化序列。
13. 含有权利要求9至权利要求12之中任一项的表达盒的载体。
14. 抵抗鳞翅目昆虫侵袭的转基因植物或植物细胞,其含有编码SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列的核苷酸序列,其中所述氨基酸序列表现杀虫活性。
15. 权利要求14的的转基因植物或植物细胞,其中所述转基因植物选自双子叶植物和单子叶植物,所述双子叶植物进一步选自苜蓿、苹果、杏、芦笋、豆、浆果、黑莓、蓝莓、低芥酸菜子、胡萝卜、花椰菜、芹菜、樱桃、鹰嘴豆、柑橘、棉花、豇豆、蔓越橘、黄瓜、葫芦、茄子、果树、葡萄、柠檬、莴苣、亚麻子、瓜、芥子、产干果的树、秋葵、橘、豌豆、桃、花生、梨、李、马铃薯、大豆、南瓜、草莓、甜菜、向日葵、甘薯、烟草、西红柿、芜菁和蔬菜,且所述单子叶植物进一步选自玉米、小麦、燕麦、稻、高粱、买罗高粱、荞麦、黑麦、草(羊茅、梯牧草、雀麦草、鸭茅、圣奥古斯丁草、狗芽根、剪股颖)和大麦。
16. 对抗鳞翅目昆虫侵袭的权利要求15的转基因植物或植物细胞,其中所述鳞翅目昆虫选自卷叶蛾、地老虎、黏虫、钻蛀虫、避债虫和草料进食者。
17. 权利要求16的转基因植物或植物细胞,其中所述鳞翅目昆虫进一步选自草地夜蛾、玉米螟、棉铃叶蛾(与棉铃虫相同)、西南玉米杆草螟和小地老虎。
18. 权利要求15的转基因植物或植物细胞的子代或种子,其中所述子代或种子含有所述核苷酸序列。
19. 通过在昆虫食物中提供一种或多种植物细胞来控制鳞翅目昆虫对植物的侵袭的方法,所述植物细胞是用含有可操作地连接的植物功能性启动子和编码SEQ ID NO:2中自第10位氨基酸至第600位氨基酸所示氨基酸序列的核苷酸序列的核酸序列转化的,所述氨基酸序列表现鳞翅目杀虫活性。
20. 权利要求12的表达盒,其含有选自SEQ ID NO:5和SEQ IDNO:7的多核苷酸序列。
21. 鉴定生物样品中编码Cry1A.105氨基酸序列的核苷酸序列的方法,所述方法包括将所述样品与在严格杂交条件下与该核苷酸序列杂交的多核苷酸接触,并检测所述多核苷酸与所述核苷酸序列的结合,其中所述结合对所述样品中所述核苷酸序列具有诊断价值。
22. 鉴定样品中Cry1A.105蛋白的方法,所述方法包括将所述样品与特异性结合所述蛋白的抗体接触,并检测结合,其中所述结合对所述样品中所述蛋白的存在具有诊断价值。
23. 分离的核苷酸序列,其编码SEQ ID NO:2中自约第1位氨基酸至约第612位氨基酸所示的杀虫蛋白。
24. 分离的核苷酸序列,其编码SEQ ID NO:2中自约第1位氨基酸至约第610位氨基酸所示的杀虫蛋白。
25. 权利要求23的分离的核苷酸序列,其与SEQ ID NO:1所示核苷酸序列表现至少约90%同一性。
26. 权利要求23的分离的核苷酸序列,其与SEQ ID NO:3所示核苷酸序列表现至少约90%同一性。
27. 杂合杀虫蛋白,其含有包含选自SEQ ID NO:2约第10位氨基酸至约第600位氨基酸所示氨基酸部分的约500至约600个邻接氨基酸的氨基酸部分。
28. 含有杀虫有效量Cry1A.105蛋白的组合物,其中所述组合物选自植物细胞、细菌细胞、真菌细胞、胶体、乳状液、种子涂层、诱饵和粉末。
29. 权利要求28的组合物,其中所述Cry1A.105蛋白以百万分之约0.5至约200(PPM)的量存在。
30. 权利要求29的组合物,其中所述Cry1A.105蛋白以约0.5至约20PPM的量存在。
31. 权利要求29的组合物,其中所述组合物是植物细胞或一组植物细胞。
32. 权利要求31的组合物,其中编码杀虫有效量的所述Cry1A.105蛋白的核苷酸序列以使用探针可检测的量存在,所示探针是选自SEQID NO:1第1401-1420位核苷酸和SEQ ID NO:1第1821-1840位核苷酸的序列或与之互补。
33. 权利要求31的组合物,其中当所述杀虫有效量的所述蛋白在所述害虫的食物中提供时足以控制鳞翅目属植物害虫,所述害虫选自叶蛾属、尺叶蛾属、Rachiplusia、实叶蛾属、Helicoverpa、灰翅叶蛾属、叶小卷娥属和Armigera。
34. 保护田间作物避免鳞翅目昆虫侵袭的方法,该方法包括种植在鳞翅目昆虫食物中含有杀虫有效量Cry1A.105蛋白杀虫剂的转基因作物植物,以抑制昆虫在所述转基因作物植物上存活。
35. 权利要求34的方法,其中所述转基因作物植物还含有对与Cry1A.105所针对的相同昆虫物种有毒性的其它杀虫剂,且其选自芽孢杆菌属毒素、致病杆菌属毒素、光杆状菌属毒素和特异于抑制所述昆虫物种中一种或多种必需基因的dsRNA。
36. 权利要求34或35的方法,其中所述作物的产量相比于缺乏所述一种或多种杀虫剂的同基因作物的产量有所提高。
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