CN106455545B - 可用于控制昆虫有害生物的营养生长杀虫蛋白 - Google Patents
可用于控制昆虫有害生物的营养生长杀虫蛋白 Download PDFInfo
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Abstract
公开了DIG‑657营养生长杀虫毒素,编码此类毒素的多核苷酸,此类毒素用于控制有害生物的用途,以及生成此类毒素的转基因植物。本发明包括DIG‑657变体,片段和类似物。
Description
对相关申请的交叉引用
本申请要求2014年6月20日提交的美国临时申请62/014,916的优先权和权益。在此通过提及将本申请的全部内容并入本申请中。
发明领域
本发明一般涉及分子生物学领域。更具体地,本发明关注自在苏云金芽孢杆菌中找到的新型营养生长杀虫蛋白毒素开发的新型杀虫蛋白毒素及其用于控制昆虫的用途。
发明背景
昆虫和其它害虫在作物损失和控制这些害虫的费用方面每年导致农民花费数十亿美元。除了田间作物的损失外,昆虫害虫也对蔬菜和水果种植者,观赏花卉的生产者和家庭园艺师造成负担。在农业生产环境中由昆虫害虫引起的损失包括作物产率的降低,作物质量的降低和收获成本的增加。
昆虫害虫主要通过化学杀虫剂(pesticides)的密集应用来控制,所述化学杀虫剂通过抑制昆虫生长,防止昆虫进食或繁殖或导致死亡而发挥作用。因此,可以达到良好的昆虫控制,但是这些化学品有时也可以影响其它有益昆虫。源自广泛使用化学杀虫剂的另一个问题是昆虫抗性群体的出现。这已经通过各种抗性管理实践得到部分缓解,但是对替代的害虫控制剂的需求日益增加。生物害虫控制剂,诸如表达杀虫毒素如δ-内毒素的苏云金芽孢杆菌(Bacillus thuringiensis)(Bt)菌株也已经应用于作物植物产生了令人满意的结果,提供了对化学杀虫剂的备选或补充。已经分离了编码这些δ-内毒素中某一些的基因,并且已经显示它们在异源宿主中的表达提供了用于控制经济上重要的昆虫害虫的另一种工具。特别地,在转基因植物中杀虫毒素(诸如苏云金芽孢杆菌δ-内毒素)的表达已经提供了针对选定的昆虫害虫的有效保护,并且表达此类毒素的转基因植物已经产业化,为农民减少化学昆虫控制剂的应用创造了条件。
土壤微生物苏云金芽孢杆菌是一种革兰氏阳性的孢子形成细菌,其特征在于伴孢结晶蛋白包含体。苏云金芽孢杆菌仍然是用于开发植物整合杀虫剂的新型杀虫蛋白的主要来源。使用细菌分离株的各种菌株,我们发明了新的Bt毒素,其对商业上重要的昆虫害虫有活性。在北美玉米昆虫抗性市场中,草地贪夜蛾(Spodoptera frugiperda)(秋粘虫(fallarmyworm)“FAW”),欧洲玉米螟(Ostrinia nubialis Hübner)(欧洲玉米螟“ECB”)和谷实夜蛾(Helicoverpa zea Boddie)(玉米穗虫“CEW”)是最关键的害虫,尽管其它地区中存在其它关键的昆虫害虫(例如棉铃虫(Helicoverpa armigera)(棉铃虫(cotton bollworm)“CBW”或玉米穗虫“CEW”)),和其他次要但重要的昆虫害虫物种。Bt毒素占生物杀虫剂市场的90%以上,为已经开发的转基因作物提供昆虫摄食抗性的基因几乎全部来源于Bt毒素。Bt细菌生成杀虫δ-内毒素,包括晶体(Cry),细胞毒素(Cyt)和营养生长杀虫蛋白(VIP)毒素,这取决于它们的基因和蛋白质结构。Cry毒素在孢子形成期间作为不溶性晶体蛋白生成。VIP毒素则在Bt细菌生长的营养生长阶段中作为可溶性蛋白质生成。VIP蛋白在其结构上不同于Cry蛋白,但是与Cry毒素一样拥有作用于昆虫中肠中的细胞的成孔剂(poreformer)的性质(Yu,C.-G.,et al.,1997 Appl.Environ.Microbiol.63:532-536,Lee,M.K.,et al.,2003,Appl.Environ.Microbiol.69:4648-4657,Shotkoski,F.,et al.,2003,Proc.Beltwide Cotton Conf,89-93)。我们在此描述了新VIP毒素的发明,这些毒素具有广谱杀虫活性,包括针对ECB的杀虫活性,其与目前已知的VIP蛋白(Yu,C.-G.,et al.,1997Appl.Environ.Microbiol.63:532-536)相比是独特的。
专利文献WO2013/134523,WO 94/21795,WO 96/10083,5,877,012,6,107,279,6,137,033,和6,291,156,以及Estruch et al.(1996,Proc.Natl.Acad.Sci.93:5389-5394)和Yu et al.(1997,Appl.Environ.Microbiol.63:532-536)描述了称为VIP3的一类杀虫蛋白。VIP3基因编码大约88kDa的蛋白质,由芽孢杆菌(Bacillus)在其生长的营养阶段期间生成并分泌。据报道,这些毒素不同于形成结晶的δ-内毒素。这些文献特别提及称作VIP1A(a),VIP1A(b),VIP2A(a),VIP2A(b),VIP3A(a),和VIP3A(b)的毒素。关于VIP蛋白的作用机制和截短的讨论,还参见Lee et al.,AEMvol.69,no.8(August 2003),4648–4657页。
VIP3A蛋白对广谱的鳞翅目害虫(包括FAW,CEW,小地老虎(Agrotis ipsilonHufnagel)(黑切根虫(black cutworm)“BCW”)和烟芽夜蛾(Heliothis virescensFabricius)(烟草夜蛾“TBW”))有杀虫活性。新近,已经发现VIP蛋白质对半翅目昆虫害虫的某些物种有毒性(Nanasaheb,P.et al,Toxins(Basel)vol.4,no.6(2012年6月),第405-429页,Sattar S.and Maiti M.K.,J.Microbiol.Biotechnol.2011,21:937–946)。因此,VIP类蛋白质展现出独特的杀虫活性谱。其它公开,WO 98/18932,WO 98/33991,WO 98/00546和WO99/57282,现在也鉴定了VIP3蛋白质类别的同源物。
为了控制昆虫害虫而持续使用化学和生物剂可增加昆虫形成对此类控制措施的抗性的几率。还有,生物控制剂的高选择性通常导致每种药剂仅控制几种特定的昆虫害虫。尽管ECB抗性转基因玉米取得了成功,抗性昆虫群体形成的可能性威胁到Cry蛋白在ECB控制中的长期可持续性,并且因此需要发现和开发新的Cry或其它类型的生物控制剂来控制ECB和其它害虫。昆虫对Bt Cry蛋白的抗性可以通过几种机制形成(Heckel等人,2007,Pigott和Ellar,2007)。已经在昆虫内鉴定了Cry蛋白的多种受体蛋白类别,并且在每种受体类别内存在多个实例。对特定Cry蛋白的抗性可以,例如,通过受体蛋白的钙粘蛋白域的毒素结合部分内的突变而形成。另一种抗性手段可以由加工前毒素的蛋白酶介导。因此,鳞翅目物种中对Cry毒素的抗性具有复杂的遗传基础,具有至少四种不同的主要抗性基因。DBM(菱纹背蛾)(Tabashnik,1994),粉纹夜蛾(Trichoplusia ni Hübner)(卷心菜尺蠖“CL”;Janmaat和Myers2003,2005)和CEW(Tabashnik et al.,2008)等物种的鳞翅目昆虫已经在田间形成对Cry蛋白的抗性。因此,开发和部署高效力植物掺入的新型杀虫蛋白(诸如本文公开的那些)是有用且必需的。
因此,仍然需要发现对农民提供经济利益、且环境上可接受的新型有效害虫控制剂。特别需要针对广谱的经济上重要的昆虫害虫,能有效地控制对现有昆虫控制剂有抗性或者可能产生抗性的昆虫群体,并且与目前的控制剂相比具有相等或更高的效力的控制剂。
发明概述
本发明提供了杀虫VIP毒素,包括本文中称为DIG-657的蛋白毒素以及DIG-657的变体,编码这些毒素的核酸,使用毒素控制害虫的方法,在转基因宿主细胞中生成毒素的方法和表达毒素的转基因植物。本发明还提供了包含编码选自下组的杀虫蛋白的核酸序列的核酸构建体:DIG-657,DIG-657变体和DIG-657片段。公开了选自下组的分离的杀虫蛋白:DIG-657,DIG-657变体,DIG-657片段和包含SEQ ID NO:2的残基206至803的杀虫蛋白。还公开了包含编码选自下组的蛋白质的核酸序列的植物,植物部分和种子:DIG-657,DIG-657变体和DIG-657片段。还公开了用于控制昆虫害虫群体的方法,包括使所述害虫群体中的个体与杀虫有效量的包含SEQ ID NO:2的残基206至803的多肽接触。
在一个实施方案中,本发明提供了分离的DIG-657昆虫毒素多肽,其包含选自下组的核心毒素区段:(a)包含SEQ ID NO:2的残基206至803的氨基酸序列的多肽;(b)包含与SEQ ID NO:2的残基206至803的氨基酸序列具有至少90%序列同一性的氨基酸序列的多肽;(c)包含SEQ ID NO:2的残基206至803的氨基酸序列,但具有多至20个对由SEQ ID NO:2编码的毒素的表达或活性没有不利影响的氨基酸取代,缺失或修饰的多肽;或其杀虫活性片段。
在另一个实施方案中,本发明提供了分离的DIG-657昆虫毒素多肽,其包含选自下组的DIG-657核心毒素区段:(a)包含SEQ ID NO:2的残基1至803的氨基酸序列的多肽;(b)包含与SEQ ID NO:2的残基1至803的氨基酸序列具有至少95%或96%或97%或98%或99%序列同一性的氨基酸序列的多肽;(c)包含SEQ ID NO:2的残基1至803的氨基酸序列,但具有多至20个对由SEQ ID NO:1编码的毒素的表达或活性没有不利影响的氨基酸取代,缺失或修饰的多肽;或其杀虫活性片段;(d)包含与SEQ ID NO:2的残基206至803的氨基酸序列具有至少93%或94%或95%或96%或97%或98%或99%序列同一性的氨基酸序列的多肽,对表达或毒素活性没有不利影响。
在另一个实施方案中,本发明提供了包含DIG-657昆虫毒素的能育植物。本发明还提供了生成昆虫抗性或昆虫耐受性转基因植物的方法,包括将本发明的核酸分子导入转基因植物中,其中核酸分子在转基因植物中能够以有效量表达以控制昆虫。
在另一个实施方案中,本发明提供了控制害虫群体的方法,包括使所述群体与杀虫有效量的DIG-657昆虫毒素接触。
在另一个实施方案中,本发明提供了编码本发明的DIG-657毒素的分离的核酸分子。鉴于DIG-657毒素的氨基酸序列,可以通过使用意图宿主植物优选的密码子逆翻译氨基酸序列,然后使用备选密码子精修序列以除去可能造成引起问题的序列,并且提供周期性终止密码子以消除非编码阅读框中的长可读框序列。
在另一个实施方案中,本发明提供了DNA构建体,其包含与启动子可操作连接的编码DIG-657昆虫毒素的核苷酸序列,所述启动子不源自Bt,并且能够在植物中驱动表达。本发明还提供了包含稳定整合入其基因组中的DNA构建体的转基因植物和保护植物免受害虫为害的方法,包括将所述构建体导入所述植物中。
在又一个实施方案中,本发明提供了生成昆虫抗性或昆虫耐受性植物的方法,包括用非转基因植物与转基因植物育种,所述转基因植物包含稳定整合到该植物的基因组中的能够表达DIG-657毒素的外来DNA构建体,并且通过分析来自所述转基因植物的所述外来DNA构建体的至少一部分来选择后代。
附图简述
图1是纯化的DIG-657的SDS-PAGE的图像。第1道,分子量标志物;第2道,3μg纯化的全长DIG-657;第3道,用胰蛋白酶(1:10DIG-657wt/胰蛋白酶wt)处理纯化的全长蛋白质1小时后DIG-657的反应产物。
序列描述
SEQ ID NO:1编码全长DIG-657昆虫毒素的DNA序列。
SEQ ID NO:2推导的DIG-657蛋白质序列。
SEQ ID NO:3编码DIG-657变体1的玉米优化的DNA序列。
SEQ ID NO:4具有针对在荧光假单胞菌(Pseudomonas fluorescens)中表达优化的密码子的DIG-657变体2。
SEQ ID NO:5DIG-657v4,玉米优化的高GC含量。
SEQ ID NO:6DIG-657v5玉米优化的高GC含量,从DIG-657的N端截短205AA。
SEQ ID NO:7SEQ ID NO:6的推导的蛋白质序列。
SEQ ID NO:8全长DIG-657大豆最优选的密码子优化形式。
SEQ ID NO:9截短的DIG-657的大豆最优选的密码子优化形式。
SEQ ID NO:10编码与全长DIG-657的大豆最优选密码子优化形式融合的编码叶绿体转运肽4(Trap4)的DNA。
SEQ ID NO:11TraP4DIG-657全长大豆最优选密码子的推导的蛋白质序列。
SEQ ID NO:12与截短的DIG-657的大豆最优选的密码子优化形式融合的Trap 4。
SEQ ID NO:13Trap4DIG-657截短形式的推导的蛋白质序列。
发明详述
本发明提供了对多种昆虫目、优选鳞翅目昆虫具有功能活性和效果的杀虫蛋白毒素,以及用于递送毒素的方法。“功能活性”(或“对…有活性”)的意思是:蛋白质发挥口服活性毒素或昆虫控制剂的功能,蛋白质具有毒性作用,或能够破坏或阻止昆虫的生长或进食。当通过转基因植物表达、配制的蛋白质组合物、可喷雾的蛋白质组合物、诱饵基质、或其它递送系统递送的有效量的本主题发明毒素与昆虫接触时,通常导致昆虫死亡,昆虫的生长或增殖被抑制,或昆虫被阻止以给昆虫提供该毒素的来源(优选转基因植物)为食。本发明的各种功能性蛋白质也可以一起或单独起作用以增强或改善一种或多种其它毒素蛋白的活性。术语“毒性的”,“毒性”或“毒素”意在表示主题毒素具有如本文中定义的功能活性。
对于进食昆虫的完全致死性是优选的,但不是实现功能活性所必需的。如果昆虫回避毒素或停止进食,那么回避在某些应用中将是有用的,即使效应是亚致死的,或致死是延迟的或间接的。例如,如果期望昆虫抗性转基因植物,则昆虫不愿对植物为食与对昆虫的致死毒性同样有用,因为最终目标是避免昆虫诱导的植物损害。
在Bt菌株PS46L中发现并分离了编码DIG-657的核酸。申请人用“分离的”,来意指核酸分子已经从其天然环境中取出并且已经被人工置于不同的环境中。由于遗传密码的冗余性,多种不同的DNA序列可以编码本文中公开的氨基酸序列。一旦知晓了氨基酸序列,创建这些编码相同或基本相同毒素的备选DNA序列完全在本领域技术人员的技能范围内。测定了DIG-657的全长编码区的核酸序列(SEQ ID NO:1),并且从核酸序列推导出了DIG-657的全长蛋白序列(SEQ ID NO:2)。针对GenomeQuest数据库“GQ-Pat Platinum protein”和“GQ-Pat GoldPlus protein”以及GenBank非冗余蛋白质数据库中查询DIG-657蛋白质序列。最接近的已知同源物是XMI335(94%同一性,WO2013134523-002)和Vip3Ba(74%同一性,登录号AAV70653,Rang等人)。
在本文中也描述了DIG-657毒素的昆虫活性变体,并且统称为DIG-657昆虫毒素或变体,并且包括片段和截短形式。可以通过特定DIG-命名法来标识DIG-657的单个变体。DIG-657毒素是用于控制鳞翅目害虫的理想候选者。
DIG-657及其变体毒素的一个令人惊奇的性质是,发现它们对于抗Cry1F和Cry1A毒素的ECB和DBM群体具有活性。因而,DIG-657毒素是控制和预防抗性鳞翅目害虫群体的理想候选者。
本发明的DIG-657毒素对鳞翅目昆虫,优选对DBM,ECB,FAW,CEW,CBW和TBW有活性。预期对于CBW、CL、甜菜夜蛾(Spodoptera exigua)(甜菜粘虫(beet armyworm)“BAW”),棉红铃虫(Pectinophora gossypiella)(粉红棉铃虫(pink bollworm)),Cochyles hospesWalsingham(带状向日葵蛾(banded sunflower moth))和Homoeosoma electellum(向日葵头蛾(sunflower head moth))也有杀虫活性。
证明在荧光假单胞菌中生成的DIG-657的杀虫活性对鳞翅目物种有活性,所述鳞翅目物种包括ECB、cry1F抗性ECB(rECB)、DBM、cry1A抗性DBM(rDBM)、CEW、BCW和TBW。还测试了DIG-657蛋白对CBW、FAW和Cry1F抗性FAW(rFAW)的活性。
此类针对玉米和大豆二者的昆虫害虫的生物活性谱是非常有利的。在生物测定中测试了胰蛋白酶截短的毒素(SEQ ID NO:7)对ECB的作用,显示其与全长DIG-657的活性近似相等,表明该蛋白质N末端的前205个氨基酸不是生物活性所需要的。
全长DIG-657具有完整的N-末端,当用胰蛋白酶(1:10胰蛋白酶/DIG-657)处理时,蛋白质被切割成两条带,一条为约65kDa,另一条为约18kDa(图1)。此外,还有一条小的残余全长DIG-657。对DIG-657的65kDa切割产物的N-端氨基酸分析表明胰蛋白酶切割的位点是(205K/S206)。
编码DIG-657、其变体、截短和片段的核苷酸序列可以通过常规手段合成并克隆到标准质粒载体中,或者可以通过对含有该核苷酸序列的其它构建体的标准分子生物学操作获得。可以鉴定DIG-657编码区内部的独特限制性位点,然后可以合成包含DIG-657编码区的这些限制性位点之间的序列的DNA片段,每种这样的片段编码一种特定的缺失、插入或其它DIG-657变异。可以将这些编码修饰的DIG-657片段的DNA片段在适当的限制位点与其它DIG-657编码区片段或其它Cry或VIP编码区片段连接,以获得编码期望的全长DIG-657蛋白、缺失或变体DIG-657蛋白的编码区。例如,可以在第一种DIG-657编码区的起始处鉴定适当的限制性识别位点,并在该DIG-657编码区内部鉴定第二限制性位点。在这些限制性位点切割此第一DIG-657编码区将产生包含第一DIG-657编码区的一部分的DNA片段。可以使用第二个DNA片段,其侧翼有另一种DIG-657编码区或其它VIP3编码区所特有的、类似定位的相容的限制性位点,与第一DNA限制性片段组合,来构建变体。
抗毒素抗体.可以使用本领域中公知的标准规程制备针对本文中公开的毒素或这些毒素的片段的抗体。此类抗体可用于检测植物组织中DIG-657毒素和多种其它物质的存在。此类抗体和抗血清可用于各种检测本发明所请求保护的DIG-657毒素及其变体或片段的方法。众所周知,用报告基团标记的抗体可以用于鉴定多种环境中抗原的存在。用放射性同位素标记的抗体已经用于放射性免疫测定中,以较大的精确性和灵敏性鉴定多种生物流体中抗原的存在。新近,酶标记的抗体已经在ELISA测定法中用作放射性标记抗体的替代物。此外,对本发明的Bt杀虫毒素有免疫反应性的抗体可以与固定化物质如聚苯乙烯孔或颗粒结合,并且用于免疫测定法中以测定Bt毒素是否存在于测试样品中。抗DIG-657抗体也可用于从重组生产系统或天然来源分离大量的DIG-657毒素。
DIG-657的转基因表达.可以以多种方式“应用”或提供主题蛋白质毒素以接触靶标昆虫。例如,DIG-657毒素可以用作转基因植物中的植物整合的保护剂(由植物生成和存在于植物中),并且是本领域中公知的。毒素基因的表达还可以实现植物的特定组织(例如根,叶等)的选择性。这可以通过使用本领域中公知的组织特异性启动子来实现。
本发明的优选实施方案是用编码主题杀虫蛋白或其变体的基因转化植物。经转化的植物由于在转化植物的细胞中存在控制量的主题杀虫蛋白或其变体而对昆虫靶标害虫的攻击有抗性。通过将编码DIG-657毒素的遗传物质掺入并表达到由特定昆虫害虫食用的植物的基因组中,成虫或幼虫在食用该食物植物后将死亡。已经转化了许多属于单子叶和双子叶分类的成员。转基因农业作物以及水果和蔬菜具有商业意义。此类作物包括但不限于玉米,稻,大豆,芸苔,向日葵,苜蓿,高粱,小麦,棉,花生,番茄,马铃薯等。有许多公知的技术可用于将外来遗传物质导入单子叶或双子叶植物细胞中,以及用于获得稳定保持和表达导入的基因的能育植物。
在一个优选的实施方案中,DIG-657或变体经由包含表达本发明的毒素的核酸序列的转基因植物经口递送。本发明提供了生成昆虫抗性转基因植物的方法,包括将本发明的核酸分子导入植物中,其中所述毒素在转基因植物中能够以有效控制昆虫的量表达。在非限制性例子中,基本克隆策略可以是将全长或修饰的DIG-657编码序列亚克隆到植物表达质粒中的NcoI和SacI限制性位点处。所得的植物表达盒含有在植物表达元件(例如植物可表达启动子,3'末端转录终止和聚腺苷酸添加决定簇等)控制下的合适的DIG-657编码区,利用例如技术或标准限制性酶片段克隆程序将其亚克隆到二元载体质粒中。如果利用技术,那么可以例如使用LR ClonaseTM(Invitrogen,Carlsbad,CA)将全长和经修饰的基因植物表达盒重组到二元植物转化质粒中。当质粒存在于大肠杆菌和土壤杆菌细胞中时,采用含有赋予对抗生素壮观霉素的抗性的细菌基因的二元植物转化载体是方便的。采用含有在期望的宿主植物中有功能的植物可表达选择标志物基因的二元载体质粒也是方便的。植物可表达的选择标志物基因的例子包括但不限于编码转座子Tn5的氨基糖苷磷酸转移酶基因(aphII)(其赋予对抗生素卡那霉素,新霉素和G418的抗性)的那些,以及那些编码对草甘膦(glyphosate);潮霉素;甲氨蝶呤;膦丝菌素(双丙氨膦(bialaphos)),咪唑啉酮,磺酰脲和三唑并嘧啶除草剂,如氯磺隆(chlorosulfuron),溴苯腈(bromoxynil),达拉泮(dalapon)等。
或者,通过土壤杆菌操作领域的技术人员公知的标准分子生物学方法,对由候选土壤杆菌分离株制备的质粒DNA进行限制性消化物指纹定位,进行含有DIG-657基因插入物的二元植物转化载体的质粒结构。
经由土壤杆菌介导的转化方法获得转化的植物领域的技术人员将理解,还可以使用Z707S以外的其它土壤杆菌菌株,并且菌株的选择可以取决于待转化的宿主植物物种的身份。
转基因拟南芥(Arabidopsis)的昆虫生物测定法.可以使用表达经修饰的DIG-657蛋白的转基因拟南芥品系在人工食料重叠测定法中证明针对敏感昆虫物种的活性。从转基因和非转基因拟南芥品系提取的蛋白质可以通过适当的方法来量化,并调整样品体积以使蛋白质浓度标准化。然后如下所述对人工食料进行生物测定法。测定法中应包括非转基因拟南芥和/或缓冲液和水作为背景检查处理。
转基因玉米的生物测定法.植物细胞中生成的DIG-657毒素和变体的生物活性亦可通过常规生物测定方法证明(参见例如Huang等人,2006)。可以通过在受控饲养环境中将源自生成DIG-657毒素的植物的各种植物组织或组织块喂给靶标昆虫来测试功效。或者,可以从源自生成DIG-657毒素的植物的各种植物组织制备蛋白质提取物,并将提取的蛋白质掺入人工食料生物测定法中。应当理解,此类喂养测定法的结果要与类似进行的生物测定法进行比较,所述类似进行的生物测定法采用来自不生成DIG-657蛋白或变体的宿主植物的合适对照组织或其它对照样品。
DIG-657毒素和杀虫活性变体.除了SEQ ID NO:2的全长DIG-657毒素外,本发明涵盖SEQ ID NO:2,SEQ ID NO:11或SEQ ID NO:13的杀虫活性变体。通过术语“变体”,申请人意欲包括某些缺失,取代和插入突变体。DIG-657片段是在SEQ ID NO:2中发现的任何小于全长DIG-657氨基酸序列且具有杀虫性质的蛋白质序列。DIG-657变体片段也作为本发明的一部分涵盖在内,并且定义为DIG-657的含有本文中所述的某些缺失、取代和插入突变体并且具有杀虫活性的片段。在描述包括在本发明中包括的DIG-657毒素的变体之前,简要回顾DIG-657蛋白毒素的构造是有用的。
通过进行有限数目的氨基酸缺失、代或添加创建的DIG-657变体.SEQ ID NO:2的氨基酸序列的氨基酸缺失、取代和添加可以以顺序的方式容易地进行,并且可以通过生物测定法测试此类变化对杀虫活性的影响。只要改变的数目是数目有限的,此类测试不会涉及过度的实验。本发明还包括核心毒素区段的杀虫活性变体(SEQ ID NO:2的氨基酸206-803,或其中已经进行多达10个,多达15个或多达20个独立的氨基酸添加,缺失或取代)。
可以通过进行随机突变制备变体,或者可以设计变体。在设计的突变体的情况下,当在毒素的关键区域(负责生物活性、或参与决定三维构造并且最终负责生物活性)中维持氨基酸同一性时,有高概率生成与天然毒素具有相似活性的变体。如果取代是保守的,保留活性的概率也高。氨基酸可以置于以下类别中:非极性,不带电荷的极性,碱性和酸性。其中一个类别的氨基酸被另一个相同类型的氨基酸替换的保守取代最不可能实质上改变该变体的生物活性。表1提供属于每个类别的氨基酸的例子的列表。
表1
本发明的杀害虫(pesticide)蛋白由与SEQ ID NO:1的核苷酸序列充分相同的核苷酸序列编码,或者所述杀虫蛋白与SEQ ID NO:2所示的氨基酸序列充分相同。“足够相同”是指使用本文中所述的对比程序之一使用标准参数与参照序列进行比较时,具有至少约60%或65%序列同一性,约70%或75%序列同一性,约80%或85%序列同一性,约90%,91%,92%,93%,94%,95%,96%,97%,98%,99%或更大的序列同一性的氨基酸或核苷酸序列。本领域技术人员将认识到,可以通过考虑密码子简并性,氨基酸相似性,阅读框定位等适当地调整这些值以确定由两个核苷酸序列编码的蛋白质的相应同一性。
为了确定两个氨基酸序列或两个核酸的同一性百分比,为了最佳比较目的将各序列比对。两个序列之间的百分比同一性是序列共有的相同位置的数目(即同一性百分比=相同位置的数目/位置的总数(例如,重叠位置)x 100)的函数。在一个实施方案中,两个序列长度相同。在另一个实施方案中,计算整个参照序列的同一性百分比。可以使用类似于下文描述的技术在允许或不允许空位的情况下确定两个序列之间的百分比同一性。在计算同一性百分比时,通常会计算精确匹配。空位(比对中在一个序列中存在残基但在另一个序列中不存在残基的位置)被认为是具有不相同残基的位置。
两个序列之间的同一性百分比的测定可以使用数学算法来完成。用于比较两个序列的数学算法的非限制性例子是Karlin和Altschul(1990)Proc.Nat’l.Acad.Sci.USA 87:2264,如在Karlin和Altschul(1993)Proc.Nat’l.Acad.Sci.USA 90:5873-5877中修改的。此类算法整合在Altschul等人(1990)J.Mol.Bioi.215:403的BLASTN和BLASTX程序中。可以用BLASTN程序,得分=100,字长=12进行BLAST核苷酸搜索,来获得与本发明的杀虫样核酸分子同源的核苷酸序列。可以用BLASTX程序,得分=50,字长=3进行BLAST蛋白质搜索,来获得与本发明的杀虫蛋白质分子同源的氨基酸序列。为了获得带空位的比对用于比较目的,可以利用Gapped BLAST(在BLAST 2.0中),如Altschul等人(1997)Nucleic AcidsRes.25:3389中描述的。或者,可以使用PSI Blast进行迭代搜索,检测分子之间的远距离关系。参见Altschul等人(1997)同上。当利用BLAST,Gapped BLAST和PSI-Blast程序时,可以使用相应程序(例如,BLASTX和BLASTN)的默认参数。也可以通过检视人工比对。
用于比较序列的数学算法的另一个非限制性例子是ClustalW算法(Higgins等人(1994)Nucleic Acids Res.22:4673-4680)。ClustalW比较序列并比对氨基酸或DNA序列整体,因此可以提供关于氨基酸序列整体的序列保守性的数据。在几种商品化的DNA/氨基酸分析软件包,如Vector NTI程序套件(Invitrogen Corporation,Carlsbad,CA)的ALIGNX模块中使用ClustalW算法。在用ClustalW比对氨基酸序列后,可以评估氨基酸同一性百分比。可用于分析ClustalW比对的软件程序的一个非限制性例子是GENEDOCTM。GENEDOCTM(KarlNicholas)允许评估多个蛋白质之间的氨基酸(或DNA)相似性和同一性。用于比较序列的数学算法的另一个非限制性例子是Myers和Miller(1988)CABIOS 4(1):11-17的算法。将此类算法被纳入ALIGN程序(版本2.0)中,该程序是GCG Wisconsin Genetics软件包第10版(可从Accelrys,Inc.,San Diego,CA,USA获得)的一部分。当利用ALIGN程序比较氨基酸序列时,可以使用PAM120权重残基表,空位长度罚分12和空位罚分4。除非另有说明,序列同一性或相似性会使用GAP第10版(其使用Needleman和Wunsch(1970)J.Mol.Biol.48(3):443-453的算法)来确定,使用以下参数:核苷酸序列的%同一性和%相似性(使用GAP权重50和长度权重3以及nwsgapdna.cmp评分矩阵);氨基酸序列的%同一性或%相似性(使用GAP权重8和长度权重2,以及BLOSUM62评分程序)。也可以使用等同程序。“等同程序”意指任何这样的序列比较程序,对于任何两个讨论的序列,与由GAP第10版产生的相应比对相比,该程序产生具有相同核苷酸残基匹配和相同百分比序列同一性的比对。
蛋白酶敏感性变体.可以将VIP3蛋白(包括DIG-657)从大小约88kDa蛋白水解截短成大小约66kDa的产物。66kDa蛋白包含氨基酸残基206-803。昆虫肠蛋白酶通常发挥功能帮助昆虫从食料蛋白质获得需要的氨基酸。了解最充分的昆虫消化蛋白酶是丝氨酸蛋白酶,其似乎是最常见的类型(Englemann和Geraerts(1980),特别是在鳞翅目昆虫中。鞘翅目昆虫的肠比鳞翅目的肠更为中性至酸性。大多数鞘翅目幼虫和成虫,例如科罗拉多马铃薯甲虫(Colorado potato beetle,CPB),具有微酸性中肠,并且半胱氨酸蛋白酶提供大部分蛋白水解活性(Wolfson和Murdock,1990)。更精确地,Thie和Houseman(1990)鉴定和鉴定了CPB中的半胱氨酸蛋白酶,组织蛋白酶B样和组织蛋白酶H样,以及天冬氨酰蛋白酶,组织蛋白酶D样。Gillikin等人(1992)表征了WCR幼虫肠中的蛋白水解活性,并且主要发现是半胱氨酸蛋白酶。美国专利No.7,230,167公开了归因于组织蛋白酶G的蛋白酶活性存在于WCR中。昆虫肠蛋白酶的多样性和不同的活性水平可能影响昆虫对特定Bt毒素的敏感性。
在本发明的另一个实施方案中,可以在期望的位置处工程化改造蛋白酶切割位点,以影响某些昆虫害虫的易感幼虫的中肠内的蛋白质加工。可以通过诸如化学基因合成或剪接重叠PCR(Horton等人,1989)的方法引入这些蛋白酶切割位点。例如,丝氨酸蛋白酶识别序列可以任选地插入Cry蛋白结构中的特定位点以影响在易感幼虫的中肠内的期望的缺失点处的蛋白质加工。可以以此类方式利用的丝氨酸蛋白酶包括鳞翅目中肠丝氨酸蛋白酶,如胰蛋白酶或胰蛋白酶样酶,胰凝乳蛋白酶,弹性蛋白酶等(Christeller等,1992)。此外,可以工程改造凭经验鉴定的缺失位点以实现蛋白质激活,经验鉴定是通过对用未分级的幼虫中肠蛋白酶制剂产生的Cry蛋白消化产物进行测序,或者通过结合至刷状缘膜囊。通过基因缺失或通过引入蛋白酶切割位点产生的经修饰的Cry或VIP3蛋白对鳞翅目害虫,包括ECB,CEW,CBW,BCW,FAW,BAW,巨座玉米螟(Diatraea grandiosella),小蔗螟(Diatraeasaccharalis),豆白隆切根虫(Loxagrotis albicosta)和其它目标害虫具有改善的活性。
通过在期望的加工位点处构建合适的识别序列,可以进一步利用鳞翅目和鞘翅目丝氨酸蛋白酶如胰蛋白酶,胰凝乳蛋白酶和组织蛋白酶G样蛋白酶,鳞翅目和鞘翅目半胱氨酸蛋白酶如组织蛋白酶(B样,L样,O样和K样蛋白酶)(Koiwa等(2000)和Bown等人(2004)),鳞翅目和鞘翅目金属蛋白酶如ADAM10(Ochoa-Campuzano等人,(2007))和鳞翅目和鞘翅目天冬氨酸蛋白酶如组织蛋白酶D样和E样,胃蛋白酶,胞质蛋白酶和凝乳酶,以影响某些昆虫害虫的易感幼虫的中肠内的Cry蛋白加工,并且还可能提供对非易感性昆虫害虫的活性。
为了允许或消除昆虫、植物或微生物蛋白酶对较大变体蛋白的蛋白水解切割而在编码序列中的适当位置引入或消除蛋白酶加工位点,由此产生的DIG-657变体在本发明的范围内。此类操作的最终结果理解为具有与完整(全长)毒素蛋白相同或更好活性的毒素片段分子的产生。
喷撒施用是另一个实例,并且在本领域中也是已知的。主题蛋白质可以适当地配制用于期望的最终用途,然后喷撒(或以其它方式施用)到植物上和/或植物周围和/或待保护的植物附近——在发现侵袭前,在发现靶昆虫后,在之前和之后两者,等等。例如,也可以使用诱饵颗粒,并且是本领域中已知的。
当昆虫与有效量的通过转基因植物表达、配制的蛋白质组合物、可喷雾蛋白质组合物,诱饵基质、或其它递送系统递送的毒素接触时,结果通常是昆虫的死亡,或昆虫不以向昆虫提供毒素的来源为食。
使用合适的微生物宿主,例如,假单胞菌属时,可以将微生物使用于害虫的环境,在那里它们将增殖并被摄取。结果导致对害虫的控制。或者,可以在延长毒素活性并稳定细胞的条件下处理含有毒素基因的微生物。然后可以将保持毒性活性的处理过的细胞施用于靶害虫的环境。
寡核苷酸探针的开发.另一种用于鉴定本发明的毒素和基因的方法是通过使用寡核苷酸探针。这些探针是可检测的核苷酸序列。这些序列可以凭借适当的放射性标记物而被检测到,或者可以如例如美国专利号6,268,132中所述使其具有内在荧光。如本领域中众所周知的,如果探针分子和核酸样品通过在两个分子之间形成强碱基配对键而杂交,则可以合理地推定探针和样品具有实质的序列同源性。优选地,通过本领域熟知的技术在严格条件下进行杂交,如例如在Keller和Manak(1993)中所述。探针的检测提供了以已知方式测定是否发生杂交的方法。此类探针分析提供了鉴定本发明的毒素编码基因的快速方法。根据本发明用作探针的核苷酸区段可以使用DNA合成仪和标准规程合成。这些核苷酸序列也可以用作PCR引物来扩增本发明的基因。
核酸杂交.如分子生物学领域的技术人员公知的,两种核酸的相似性可以通过它们杂交的趋势来表征。如本文中使用的,术语“严格条件”或“严格杂交条件”意指探针会以比与其它序列大得可检测的程度(例如超过背景的至少2倍)与其靶序列杂交(退火)。严格条件是序列依赖性的,并且在不同情况下将是不同的。通过控制杂交和/或清洗条件的严格性,可以鉴定与探针100%互补的靶序列(同源探测)。或者,可以调整严格性条件以允许序列中的一些错配,使得较低程度的相似性被检测到(异源探测)。通常,探针长度小于约1000个核苷酸,优选长度小于500个核苷酸。
通常,严格条件会是那些条件,其中在pH 7.0至pH 8.3下盐浓度小于约1.5M Na离子,通常约0.01至1.0M Na离子浓度(或其它盐),并且温度至少为为约30℃(对于短探针(例如10至50个核苷酸))和至少约60℃(对于长探针(例如,大于50个核苷酸))。严格条件也可以通过添加去稳定剂如甲酰胺来实现。示例性的低严格性条件包括在37℃在30%至35%甲酰胺,1M NaCl,1%SDS(十二烷基硫酸钠)的缓冲溶液的情况下杂交和在1X至2X SSC(20XSSC=3.0M NaCl/0.3M柠檬酸三钠)在50℃至55℃清洗。示例性中度严格性条件包括在37℃在40%至45%甲酰胺,1.0M NaCl,1%SDS中杂交和在55℃至60℃在0.5X至1X SSC中清洗。示例性的高严格条件包括在37℃在50%甲酰胺,1M NaCl,1%DS中杂交和在60℃至65℃在0.1X SSC中清洗。任选地,清洗缓冲液可以包含约0.1%至约1%SDS。杂交的持续时间通常小于约24小时,通常约4至12小时。
特异性通常是杂交后清洗的功能,关键因素是最终清洗溶液的离子强度和温度。对于DNA/DNA杂交体,热熔点(Tm)是50%的互补靶序列与完全匹配的探针杂交(在限定的离子强度和pH下)的温度。对于每1%的错配,Tm降低约1℃;因此,可以调节Tm,杂交条件和/或清洗条件以促进期望同一性的序列的退火。例如,如果寻找具有>90%同一性的序列,则T m可以降低10℃。通常,将严格条件选择为比在限定的离子强度和pH下特定序列及其互补序列的Tm低约5℃。然而,高度严格条件可以在比Tm低1℃,2℃,3℃或4℃使用杂交和/或清洗;中等严格条件可以利用比Tm低6℃,7℃,8℃,9℃或10℃的杂交和/或清洗,并且低严格性条件可以利用比Tm低11℃,12℃,13℃,14℃,15℃或20℃的杂交和/或清洗。
Tm(以℃计)可以通过实验确定或可以通过计算取近似值。对于DNA-DNA杂交体,可以从Meinkoth和Wahl(1984)的方程对Tm取近似值:Tm(℃)=81.5℃+16.6(logM)+0.41(%GC)-0.61(%甲酰胺)500/L;其中M是一价阳离子的摩尔浓度,%GC是DNA中鸟苷和胞嘧啶核苷酸的百分比,%甲酰胺是杂交溶液中甲酰胺的百分比,并且L是碱基对中杂交体的长度。或者,Tm由下式(Beltz等,1983)描述:Tm(℃)=81.5℃+16.6(log[Na+])+0.41(%GC)-0.61(%甲酰胺)-600/L,其中[Na+]是钠离子的摩尔浓度,%GC是DNA中鸟苷和胞嘧啶核苷酸的百分比,%甲酰胺是杂交溶液中甲酰胺的百分比,并且L是碱基对中杂交体的长度。
使用等式,杂交和清洗组合物和期望的Tm,普通技术人员将理解,固有描述了杂交和/或清洗溶液的严格性的变化。如果期望的错配程度导致Tm小于45℃(水溶液)或32℃(甲酰胺溶液),则优选提高SSC浓度,使得可以使用更高的温度。关于核酸杂交的广泛指导参见P.Tijssen(1993,ISBN-10:0444898840,ISBN-13:9780444898845Hardcover)的Hybridization with Nucleic Acid Probes Vol.1和Ausubel等人(1995)。也参见Sambrook等人(1989)。
实施例
实施例1
编码DIG-657杀虫毒素的表达质粒的构建和在细菌宿主中的表达。在荧光假单胞菌(Pf)表达质粒的构建中使用标准克隆方法,所述表达质粒经工程化以生成由植物优化的编码区编码的全长DIG-657蛋白。限制性内切核酸酶和T4DNA连接酶从New EnglandBioLabs(NEB;Ipswich,MA)获得,分别用于限制性消化和DNA连接。使用质粒试剂盒(Macherey-NagelInc,Bethlehem,PA)根据用于低拷贝质粒纯化的供应商的说明书进行质粒制备。在琼脂糖Tris-乙酸凝胶电泳后,使用凝胶提取试剂盒(Qiagen,Venio,Limburg)纯化DNA片段。
使用含有用于克隆的特异性限制性位点的基因特异性引物,从亲本Bt菌株PS46L/DBt11889中扩增出编码DIG-657的基因。然后使用常规的连接克隆方法将所得的DIG-657聚合酶链式反应(PCR)产物克隆到荧光假单胞菌表达载体pDOW1169中。
基本克隆策略包括将DIG-657毒素编码序列(CDS)(SEQ ID NO:1)亚克隆到pDOW1169中的SpeI和SalI限制性位点处,由此将其置于来自质粒pKK223-3(PL Pharmacia,Milwaukee,WI)的Ptac启动子和rrnBT1T2终止子的表达控制下。pDOW1169是一种中等拷贝质粒,具有RSF1010复制起点、pyrF基因、和核糖体结合位点,后面是可以引入含有蛋白质编码区的DNA片段的限制酶识别位点(美国申请20080193974)。通过电穿孔将称为pDOW1169的表达质粒转化入DC454(具有突变deltapyrF和lsc::lacIQI的近野生型荧光假单胞菌菌株)或其衍生物中,在SOC-Soy水解产物培养基中恢复,并在选择培养基(缺少尿嘧啶的M9葡萄糖琼脂,Sambrook等人,同上)上铺板。微生物操作的细节可以在Squires等人,(2004),美国专利申请20060008877,美国专利申请20080193974和美国专利申请20080058262中获得,其通过提及并入本文。首先通过小提质粒DNA的限制性消化筛选菌落。用四种限制酶消化含有DIG-657毒素的所选克隆的质粒DNA,并确认序列以进一步验证插入物的存在。
实施例2
在摇瓶中的生长和表达分析.通过摇瓶培养的含有表达构建体(pDOW1169)的荧光假单胞菌菌株来完成用于表征和昆虫生物测定法的DIG-657毒素的生成。将DIG-657培养物的甘油储液(0.5mL)接种到具有9.5%甘油(Teknova目录号3D7426,Hollister,CA)的50mL限定的生产培养基中。在30℃震荡下初始温育24小时后添加异丙基-β-D-1-硫代吡喃半乳糖苷(IPTG),诱导Ptac启动子表达DIG-657毒素基因。在诱导时和诱导后各个时间取样培养物。通过在600nm的光密度(OD600)测量细胞密度。也可以利用适合于荧光假单胞菌生长的其它培养基,例如,如记载于Huang等人(2007)和美国专利申请20060008877。
实施例3
土壤杆菌转化.在二元植物转化和表达质粒的构建中使用标准克隆方法。限制性核酸内切酶和T4DNA连接酶获自NEB。使用质粒制备试剂盒或AX Xtra Midi试剂盒(均来自Macherey-Nagel,Duren,Germany),依照制造商的说明书进行质粒制备。凝胶分离后,使用PCR纯化试剂盒或凝胶提取试剂盒(均来自Qiagen Venio,Limburg)纯化DNA片段。
制备根癌土壤杆菌菌株Z707S(Z707的链霉素抗性衍生物;Hepburn等人,1985)的电感受态细胞,并使用电穿孔转化(Weigel和Glazebrook,2002)。电穿孔后,向小杯添加1mL酵母提取物蛋白胨(YEP)肉汤(10gm/L酵母提取物,10gm/L蛋白胨和5gm/L NaCl),并且将细胞-YEP悬浮液转移至15mL培养管,在28℃在恒定搅拌的水浴中温育4小时。将细胞在含有壮观霉素(200μg/mL)和链霉素(250μg/mL)的YEP+琼脂(25gm/L)上铺板,并将板在28℃温育2-4天。选择分离良好的单菌落,并如上所述在具有壮观霉素和链霉素的新鲜YEP+琼脂板上划线,并在28℃温育1-3天。
从选定的土壤杆菌菌落制备模板质粒DNA,使用载体特异性引物进行PCR分析,来分析二元植物转化载体中DIG-657基因插入物的存在。取15mL含壮观霉素和链霉素的YEP中培养过夜培养物的4mL等分试样的细胞离心沉淀,使用Qiagen(Venlo,Limburg,Netherlands)Mini Preps根据制造商的说明进行提取。包括来自用于土壤杆菌电穿孔转化的二元载体的质粒DNA作为对照。使用来自Invitrogen(Carlsbad,CA)的Taq DNA聚合酶根据制造商的说明以0.5X浓度完成PCR反应。在MJ Research Peltier热循环仪中实施PCR反应,其以下述条件编程:步骤1)94℃3分钟;步骤2)94℃45秒;步骤3)55℃30秒;步骤4)72℃1分钟(每kb预期产物长度);步骤5)29次至步骤2;步骤6)72℃10分钟。循环后反应维持于4℃。通过琼脂糖凝胶电泳(例如0.7%至1%琼脂糖,w/v)分析扩增产物并通过溴化乙锭显现。选择其PCR产物与质粒对照相同的菌落。
实施例4
DIG-657的生成.通过离心分离含有表达构建体(pDOW1169)的摇瓶培养的荧光假单胞菌菌株的细胞,并将所得细胞离心沉淀在-80℃冷冻。使用EasyLyseTM细菌蛋白提取溶液(Biotechnologies,Madison,WI)产生来自冷冻摇瓶细胞离心沉淀样品的可溶性和不溶性级分。将每个细胞离心沉淀悬浮于1mL EasyLyseTM溶液中,并在裂解缓冲液中进一步以1:4稀释,并在室温震荡下温育30分钟。将裂解物在4℃以14,000rpm离心20分钟,并且以可溶性级分回收上清液。然后将离心沉淀(不溶性级分)在等体积的磷酸盐缓冲盐水(PBS;11.9mM Na2HPO4,137mM NaCl,2.7mM KCl,pH7.4)中悬浮。
将样品与含有β-巯基乙醇(Sambrook等人,同上)的2X Laemmli样品缓冲液以1:1混合,并煮沸5分钟,然后加载到CriterionBis-Tris 12%凝胶(Bio-Rad Inc.,Hercules,CA)。在推荐的XT MOPS缓冲液中进行电泳。根据制造商的方案用Bio-Safe考马斯染色(Bio-Rad,Richmond,CA)将凝胶染色,并使用Alpha Innotech成像系统(San Leandro,CA)成像。
实施例5
包含体制备.DIG-657通常位于含有DIG-657基因的荧光假单胞菌细胞的可溶性级分中。在一些情况下,如通过SDS-PAGE和MALDI-MS(基质辅助激光解吸/电离质谱术)所证明的,发现不同比例的DIG-657蛋白位于蛋白包含体(IB)制备物中。为了从此级分中分离此蛋白质,将荧光假单胞菌发酵离心沉淀物在37℃水浴中融化。将细胞在裂解缓冲液(50mMTris,pH 7.5,200mM NaCl,20mM EDTA二钠盐(乙二胺四乙酸),1%Triton X-100和5mM二硫苏糖醇(DTT))中重悬至25%w/v;在即将使用前添加5mL/L细菌蛋白酶抑制剂混合物(P8465Sigma-Aldrich,St.Louis,MO)。用设置到最低的手持式匀浆器(Tissue Tearor,BioSpec Products,Inc.,Bartlesville,OK)悬浮细胞。将溶菌酶(25mg的Sigma,St.Louis,MO,来自鸡蛋白的L7651)添加到细胞悬浮液,用金属刮刀混合,将悬液在室温温育1小时。将悬液在冰上冷却15分钟,然后使用Branson(Danbury,CT)Sonifier 250(两个1分钟时段,50%工作循环,30%输出)进行超声处理。通过显微术检查细胞裂解。如果需要的话,再添加25mg溶菌酶,并重复温育和超声处理步骤。当通过显微术确认细胞裂解时,将裂解物以11,500xg离心25分钟(4℃)以形成IB离心沉淀,并弃去上清液。将IB沉淀用100mL裂解缓冲液重浮,用手持式混合器匀浆化并如上所述离心。通过重悬(在50mL裂解缓冲液中),匀浆化,超声处理和离心重复清洗IB离心沉淀,直到上清液变为无色,并且IB离心沉淀变得坚固且颜色变为灰白。对于最终清洗,将IB离心沉淀重悬于含有2mM EDTA的无菌过滤(0.22μm)蒸馏水中,并离心。将最终离心沉淀重悬于含有2mM EDTA的无菌过滤的蒸馏水中,并以1mL等分试样贮存于-80℃。
如下所述对IB制备物中蛋白质的SDS-PAGE分析和量化:融化IB离心沉淀的1mL等分试样,并用无菌过滤的蒸馏水以1:20稀释。然后,将稀释的样品用4X还原样品缓冲液[250mM Tris,pH6.8,40%甘油(v/v),0.4%溴酚蓝(w/v),8%SDS(w/v)和8%β-巯基乙醇(v/v)]煮沸并上样到4-20%Tris-甘氨酸,12+2孔凝胶(Invitrogen,Carlsbad,CA)上,用1X Tris/甘氨酸/SDS缓冲液(BioRad,Richmond,CA)电泳。以200伏特凝胶电泳60分钟,然后用考马斯蓝(在45%甲醇,10%乙酸中的50%G-250/50%R-250)染色,并用含7%乙酸,5%甲醇的蒸馏水脱色。用牛血清清蛋白(BSA)样品在相同凝胶上电泳以产生标准曲线,将目标条带的密度计值与标准曲线比较来进行目标条带的定量。
实施例6
包含体的溶解.在设置到最高的Eppendorf型5415C微量离心机(约14,000xg)上离心6mL包含体悬浮液(含有32mg/mL DIG-657蛋白质)以沉淀包含体。除去贮存缓冲液上清液,并用50mL锥形管中的25mL 100mM碳酸钠缓冲液(pH 11)替换。使用移液管将包含体重悬,并涡旋振荡以彻底混合。在4℃下将管置于轻轻摇动的平台上过夜以提取靶蛋白。将提取物在4℃以30,000xg离心30分钟,并且使用Amicon Ultra-15再生纤维素离心过滤装置(30,000分子量截留;Millipore,Billerica,MA)将所得上清液浓缩5倍。然后使用一次性PD-10柱(GE Healthcare,Piscataway,NJ)将样品缓冲液变为10mM CAPS[3-(环己氨基)-1-丙磺酸]pH 10。
实施例7
凝胶电泳.将浓缩的提取物在含有5mM二硫苏糖醇作为还原剂的LDS样品缓冲液(Invitrogen,Carlsbad,CA)中以1:50稀释,并在95℃加热4分钟,以准备用于电泳。将样品加载到4-12%凝胶的一式两份泳道中,旁边是0.2至2μg/道的5个BSA标准品(用于生成标准曲线)。使用MOPS SDS运行缓冲液(Invitrogen,Carlsbad,CA),施加200V电压,直到跟踪染料到达凝胶底部。用在45%甲醇,10%乙酸中的0.2%考马斯蓝G-250染色凝胶,并且首先用45%甲醇,10%乙酸短暂脱色,然后用7%乙酸,5%甲醇长时间脱色,直到背景清除。脱色后,用Biorad Fluor-S MultiImager扫描凝胶。使用仪器的Quantity One v.4.5.2软件获得背景扣除的染色蛋白条带的体积,并产生用于计算储备溶液中DIG-657蛋白浓度的BSA标准曲线。
实施例8
DIG-657纯化.如Lee,MK等人(2003)所述,类似于VIP3进行DIG-657的纯化,其中将来源于用DIG-657基因转化的Pf的细胞解冻,在0.5ml蛋白酶抑制剂混合物(Sigma,St.Louis,MO)存在下悬浮于提取缓冲液(10mM Tris-HCl,2mM EDTA,2mM DTT)中,超声处理5次,每次1分钟,在每个超声处理循环之间在冰上静止1分钟。将溶液以20,000xg离心10分钟,收集上清液并施加到MonoQ离子交换柱(1cm直径x10cm长)上。通过在离子交换(MonoQ1010,GE Healthcare,Piscataway,NJ),疏水层析和尺寸排阻层析上顺序进行层析来实现蛋白质纯化。最终的制备物显示了以约90kDa的表观分子量迁移的单条蛋白质条带。
实施例9
样品制备和生物测定法.将DIG-657的纯化制备物适当地在10mM CAPS,pH 10中稀释,并且所有生物测定法均包括由该缓冲液构成的对照处理,充当死亡率和/或生长抑制的背景检查。通过使用牛血清清蛋白(BSA)的凝胶电泳来估计生物测定法缓冲液中的蛋白浓度,以产生凝胶光密度测定法的标准曲线,其使用BioRad(Richmond,CA)成像系统(Fluor-SMultiImager with Quantity One software version 4.5.2)来测量。用基于考马斯蓝的染料染色凝胶基质中的蛋白质,并在读取前脱色。
在用1-2日龄的新生鳞翅目幼虫对人工昆虫食料进行的生物测定法中测试纯化的蛋白质的杀虫活性。从昆虫养殖商(Benzon Research Inc.,Carlisle,PA)维持的群落获得的卵孵化CEW,SBL,DBM,VBC,ECB,FAW和TBW的幼虫。从自专有群落(Dow AgroSciences LLC,Indianapolis,IN)收集的卵孵化rECB和rFAW的幼虫。
在特别为昆虫生物测定法(C-D International,Pitman,NJ)设计的128孔塑料盘中进行生物测定法。每个孔含有2.0mL多物种鳞翅目食料(Southland Products,LakeVillage,AR)。通过移液管将40μL等分试样的蛋白质样品递送到每孔的2.0cm2食料表面(20μL/cm2)。食料浓度作为孔中每平方厘米(cm2)表面积的DIG-657蛋白的量(ng)计算。使用范围为9,000至3ng/cm2的9个剂量浓度,每个剂量测试16只幼虫。将处理的盘保持在通风橱中,直到食料表面上的液体蒸发或吸收到食料中。
在孵化的24-48小时内,用湿润的骆驼毛刷挑出幼虫个体并且将其放置在经处理的食料上,每孔一个幼虫。然后用透明塑料粘合片密封带虫的孔,并通风以允许气体交换(C-D International,Pitman,NJ)。将生物测定盘在受控的环境条件(28℃,约60%相对湿度,16:8[光照:黑暗])下保持5天,之后记录暴露于每种蛋白质样品的昆虫总数、死亡昆虫的数目、和存活的昆虫的重量。计算每种处理的百分死亡率和生长抑制百分比。使用以下的等式计算生长抑制(GI):
GI=[1-(TWIT/TNIT)/(TWIBC/TNIBC)]
其中TWIT是处理中昆虫的总重量,TNIT是处理中昆虫的总数,TWIBC是背景检查(缓冲液对照)中昆虫的总重量,并且TNBBC是背景检查(缓冲液对照)中昆虫的总数。
GI50确定为GI值为50%时食料中DIG-657蛋白的浓度。50%致死浓度(LC50)记录为食料中50%测试昆虫被杀死时的DIG-657蛋白浓度。通过使用经由JMP Pro,第9.0.3版,软件(SAS Institute Inc.,Cary,NC)的非线性逻辑3参数来确定生长抑制浓度-响应曲线。致死浓度-响应曲线通过Probit分析(Finney,1971)合并的死亡率数据分析,并使用POLO-PC(LeOra Software)进行。
表2呈现了DIG-657蛋白对ECB,rECB,TBW,Pseudoplusia includes (Walker)(大豆尺蠖,“SBL”),黎豆夜蛾(Anticarsia gemmatalis)(Hübner)(velvetbean caterpillar,“VBC”),CEW,FAW,rFAW和DBM的生物测定测试的结果。一个出乎意料且令人惊讶的发现是,rECB测试昆虫与ECB昆虫的敏感株系对DIG-657蛋白的作用一样易感(即使不是更易感)。
表2
人工食料重叠生物测定法(ng/cm2)中测试的纯化的DIG-657针对多种昆虫物种的效力。
昆虫 | LC<sub>50</sub>(CI 95%)(ng/cm<sup>2</sup>) | GI<sub>50</sub>(CI 95%)(ng/cm<sup>2</sup>) |
ECB | 281(166-470) | 8.6(6.6-11.1) |
rECB | 37(17-74) | 8.1(4.2-15.9) |
TBW | 608(311-1,432) | 12.9(5.9-28.1) |
SBL | 80(50-120) | 5.4(4.3-6.7) |
VBC | 66(45-95) | 18.9(12.2-29.3) |
CEW | >9,000 | 17.5(10.9-28.2) |
FAW | >9,000 | 563(398-796) |
rFAW | ~9,000 | 2,651(1,030-6,822) |
DBM | 6.3(1.6-11.8) | 3.7(3.0-4.6) |
通过使用新生幼虫的表面污染进行棉铃虫(Helicoverpa armigera)生物测定法。在128个小室的盘中进行毒性测试,每个小室2cm2。用仅缓冲液作为对照,使用25和2500ng/cm2的浓度测定死亡百分比(表3)。使用7种不同浓度的纯化原毒素进行测定LC50值的测试,用仅缓冲液作为对照(表4)。在25℃,16:8光照:黑暗条件条件下7天后评估死亡率和发育停滞。“功能死亡率”是获得的评分死亡和L1发育停滞的幼虫。结果证明了DIG-657对棉铃虫的活性。
表3
用人工食料生物测定法测试的针对棉铃虫的DIG-657的百分比死亡率。
表4
用人工食料生物测定法测试的针对棉铃虫的DIG-657的LC50(ng/cm2)。
*()中的数值是0.95置信限度
实施例10
双子叶植物中DIG-657Bt杀虫蛋白和变体的生成
拟南芥转化.使用花浸渍方法转化拟南芥Col-01(Weigel和Glazebrook,2002)。使用选定的土壤杆菌菌落接种1mL至15mL YEP肉汤培养物(含有适于选择的抗生素)。将培养物在28℃以220rpm恒定搅拌温育过夜。使用每种培养物接种含有合适于选择的抗生素的两个500mL YEP肉汤培养物,并将新培养物在28℃在恒定搅拌下温育过夜。将细胞在室温以约8700xg沉淀10分钟,并且弃去所得的上清液。将细胞离心沉淀轻轻地重悬于500mL浸润培养基中,所述浸润培养基含有:1/2X Murashige和Skoog盐(Sigma-Aldrich)/Gamborg氏B5维生素(Gold BioTechnology,St.Louis,MO),10%(w/v)蔗糖,0.044μM苄基氨基嘌呤(10μL/L的1mg/mL在DMSO中的储液)和300μL/L Silwet L-77。将约1个月龄的植物浸入培养基中15秒,注意确保浸没最新生的花序。然后,将植物侧放并覆盖(透明或不透明)24小时,用水清洗,并竖立放置。在22℃,以16:8光照:黑暗光周期培养植物。浸渍后约4周,收获种子。
拟南芥生长和选择。使新鲜收获的T1种子在有干燥剂的条件下在室温干燥至少7天。将种子悬浮在0.1%琼脂/水(Sigma-Aldrich)溶液中,然后在4℃分层2天。为了准备种植,用细蛭石覆盖在10.5英寸x21英寸的萌发盘(TO Plastics Inc.,Clearwater,MN)中的Sunshine Mix LP5(Sun Gro Horticulture Inc.,Bellevue,WA),用霍格兰(Hoagland)氏溶液(Hoagland和Arnon,1950)地下灌溉,直到湿润,然后排干24小时。将分层的种子播种到蛭石上并用湿度罩(KORD Products,Bramalea,Ontario,Canada)覆盖7天。使种子发芽,并在Conviron(CMP4030或CMP3244型;Controlled Environments Limited,Winnipeg,Manitoba,Canada)中,在长日照条件(16:8光照:黑暗光周期)、120-150μmol/m2sec的光强度、恒定的温度(22℃)和湿度(40-50%)下种植植物。最初用霍格兰氏溶液浇灌植物,随后用去离子水浇灌,以保持土壤润而不湿。
在播种后5-6天除去罩,并用化学选择剂喷雾植物以杀死未转化的种子萌发出的植物。例如,如果二元植物转化载体所提供的植物表达选择标志物基因是pat或bar基因(Wehrmann等人,1996),可以通过用1000X Finale溶液(5.78%草铵膦(glufosinateammonium),Farnam Companies Inc.,Phoenix,AZ.)喷雾来选择经转化的植物。随后以5-7天的间隔进行两次喷雾。在最后一次喷雾后7-10天鉴定存活者(活跃生长的植物),并移植到备有Sunshine Mix LP5的盆中。用湿度罩将移植的植物覆盖3-4天,并在上文提及的生长条件下置于Conviron中。
双子叶植物转化领域的技术人员将理解,当使用其它植物可表达的选择标志物基因(例如除草剂耐受性基因)时,可以使用其他选择转化植物的方法。
实施例11
包含DIG-657的转基因大豆(Glycine max).,按照本领域已知的方式,包括例如通过土壤杆菌介导的转化,产生10至20株携带包含DIG-657的核酸的表达载体的转基因T0大豆植物。将成熟的大豆种子用氯气灭菌过夜,历时16小时。在用氯气灭菌后,将种子置于LAMINARTM流动罩中的敞口容器中以驱除氯气。接着,使用黑箱,在24℃在黑暗中将灭菌的种子用无菌H2O吸水16小时。
分割种子大豆的制备。包含部分胚轴的分割大豆种子规程需要准备大豆种子材料,使用固定到解剖刀上的#10刀片沿着种脐纵向切割大豆种子材料,以分离和除去种皮,并将种子分割成两个子叶段。小心地除去部分胚轴,其中约1/2-1/3的胚轴保持附着于子叶的节端。
接种。然后,将包含部胚轴的一部分的分割大豆种子浸入含有根癌土壤杆菌的溶液中约30分钟,根癌土壤杆菌是包含DIG-657的二元质粒的根癌土壤杆菌(例如菌株EHA101或EHA 105)。在浸没包含胚轴的子叶前,将根癌土壤杆菌溶液稀释至λ=0.6OD650的终浓度。
共培养。接种后,让分割的大豆种子在覆盖有滤纸的培养皿中与根癌土壤杆菌菌株在共培养培养基(Wang,Kan.Agrobacterium Protocols.2.1.New Jersey:HumanaPress,2006.Print.)上共培养5天。
芽诱导。在共培养5天后,在由B5盐,B5维生素,28mg/L亚铁,38mg/L Na2EDTA,30g/L蔗糖,0.6g/L MES,1.11mg/L BAP,100mg/L TimentinTM,200mg/L头孢噻肟和50mg/L万古霉素组成的液体芽诱导(SI)培养基(pH 5.7)中清洗分割的大豆种子。然后,在由B5盐,B5维生素,7g/L Noble琼脂,28mg/L亚铁,38mg/L Na2EDTA,30g/L蔗糖,0.6g/L MES,1.11mg/LBAP,50mg/L TIMENTINTM,200mg/L头孢噻肟,50mg/L万古霉素组成的芽诱导I(SI I)培养基(pH 5.7)上培养分割的大豆种子,使子叶的平坦面向上且将子叶的结节端包埋到培养基中。培养2周后,将来自经转化的分割大豆种子的外植体转移到含有补充有6mg/L草丁膦的SI I培养基的芽诱导II(SI II)培养基。
芽伸长。在SI II培养基上培养2周后,从外植体中取出子叶,并通过在子叶的基部处进行切割来切出含有胚轴的平齐芽垫(flush shoot pad)。将来自子叶的分离的芽垫转移至芽伸长(SE)培养基。SE培养基由MS盐,28mg/L亚铁,38mg/L Na2EDTA,30g/L蔗糖和0.6g/L MES,50mg/L天冬酰胺,100mg/L L-焦谷氨酸,0.1mg/L IAA,0.5mg/L GA3,1mg/L玉米素核苷,50mg/L TIMENTINTM,200mg/L头孢噻肟,50mg/L万古霉素,6mg/L草丁膦,7g/LNoble琼脂(pH 5.7)。每2周将培养物转移到新鲜的SE培养基中。在24℃在CONVIRONTM生长室中以80-90μmol/m2sec的光强度以18小时光周期培养培养物。
生根。通过在子叶芽垫的基部处切割伸长的芽来分离从子叶芽垫形成的伸长的芽,并将伸长的芽浸入1mg/L IBA(吲哚3-丁酸)中1-3分钟促进生根。接着,将伸长的芽转移到植物盘中的生根培养基(MS盐,B5维生素,28mg/L亚铁,38mg/L Na2EDTA,20g/L蔗糖和0.59g/L MES,50mg/L天冬酰胺,100mg/L L-焦谷氨酸7g/L Noble琼脂,pH 5.6)。
培养。在24℃,18小时光周期在CONVIRONTM生长室中培养1-2周后,将已经形成根的芽转移到覆盖的圣代杯中的土壤混合物,并在恒定温度(22℃)和湿度(40-50%)下在长日照条件(16小时光照/8小时黑暗)下以120-150μmol/m2sec的光强度置于CONVIRONTM生长室(CMP4030和CMP3244型,Controlled Environments Limited,Winnipeg,Manitoba,Canada)中用于驯化小植物。将生根的小植物在圣代杯中驯化几周,之后将它们转移到温室中以进一步驯化和建立强健的转基因大豆植物。
将转基因品系的发育和形态学特征与非转化植物进行比较。比较植物根,枝条,叶和繁殖特征。在转基因和非转化植物的根长度和生长模式中没有观察到的差异。植物枝条特征,例如高度,叶数目和大小,开花时间,花大小和外观是相似的。一般来说,当在体外和在温室中的土壤中培养时,在转基因品系与没有DIG蛋白表达的那些品系之间没有可观察到的形态学差异。
实施例12
在单子叶植物中生成DIG-657Bt杀虫蛋白和变体。
土壤杆菌介导的玉米转化.将来自High II或B-104F1杂交的种子(Armstrong等人,1991)种植到5加仑盆中,该盆含有95%Metro-Mix 360无土生长培养基(Sun GroHorticulture,Bellevue,WA)和5%粘土/壤土的混合物。在温室中种植植物,使用高压钠和金属卤化物灯的组合,光周期为16:8小时光照:黑暗。为了获得用于转化的未成熟F2胚,进行受控的近缘授粉。在授粉后8-10天,当胚的大小为约1.0至2.0mm时分离未成熟的胚。
感染和共培养.如下对玉米穗表面灭菌:用洗洁精擦洗,浸入70%乙醇中2分钟,然后浸入20%商业漂白剂(0.1%次氯酸钠)中30分钟,之后用无菌水漂洗。如下制备含有超二元载体的土壤杆菌细胞的悬液:取1-2环在含有100mg/L壮观霉素,10mg/L四环素和250mg/L链霉素的YEP固体培养基上28℃培养2-3天后的细菌,转移到5mL液体感染培养基(LS基本培养基(Linsmaier和Skoog,1965),N6维生素(Chu等,1975),1.5mg/L 2,4-二氯苯氧基乙酸(2,4-D),68.5gm/L蔗糖,36.0gm/L葡萄糖,6mM L-脯氨酸,pH 5.2)中。涡旋振荡溶液直到得到均匀的悬浮液,将浓度调节至最终密度约200Klett单位(使用具有紫色滤器的Klett-Summerson比色计)或550nm的光密度约0.4。将未成熟的胚直接分离到含有2mL感染培养基的微量离心管中。除去培养基并替换为1mL密度200Klett单位的土壤杆菌溶液,并且将土壤杆菌和胚溶液在室温温育5分钟,然后转移至共培养培养基(LS基础培养基,(含有N6维生素,1.5mg/L 2,4-D,30.0gm/L蔗糖,6mM L-脯氨酸,0.85mg/L AgNO3,100μM乙酰丁香酮和3.0gm/L Gellan胶(PhytoTechnology Laboratories.,Lenexa,KS),pH 5.8),25℃黑暗条件下历时5天。
共培养后,将胚转移到选择性培养基,之后在约8周的过程里获得经转化的分离株。为了选择出转化了含有植物可表达pat或bar选择标志物基因的超二元质粒转化的玉米组织,使用含有双丙氨磷(Bialaphos)(Gold BioTechnology,St.Louis,MO)的基于LS的培养基(LS基础培养基,具有1X N6维生素,1.5mg/L 2,4-D,0.5gm/L MES(2-(N-吗啉代)乙磺酸一水合物;PhytoTechnology Laboratories.,Lenexa,KS),30.0gm/L蔗糖,6mM L-脯氨酸,1.0mg/L AgNO3,250mg/L头孢噻肟,2.5gm/L Gellan胶,pH 5.7)。将胚转移至含有3mg/L双丙氨磷的选择培养基,直到获得生胚的分离株。通过每隔2周转移到新鲜选择培养基来积累回收的分离株,用于再生和进一步分析。玉米转化领域的技术人员将理解,转化植物的其它选择方法在使用其它植物可表达选择标志物基因(例如,例如除草剂耐受性基因)时是可用的。
将包含在质粒pDAB114534和pDAB114535中的DIG-657的两种不同构建体(SEQ IDNO:5和SEQ ID NO:6)(表5)转化到玉米中。再生T0植物,并测试其控制目标昆虫害虫的能力。构建体pDAB115782是含有YFP(黄色荧光蛋白)基因的质粒,作为用非杀虫基因转化的植物的阴性对照。
表5
描述含有转化到玉米植物中的不同DIG-657构建体的质粒。
从V5T0转基因植物的V3或V4叶中一式两份采集1.0X0.5平方英寸叶切片。对于每个测试,首先取样野生型植物叶组织,以防止叶边缘上的Bt毒素的交叉污染,然后采样转化的植物。在每个生物测定法盘孔(32孔盘和盖,CD International,Pitman,NJ)中,将1片叶切片置于2%水-琼脂(Fisher Scientific,Fair Lawn,NJ)上,并且对每个昆虫物种、每个事件和每个构建体重复2次。用10个ECB或Diatraea saccharalis(Fabricius)(甘蔗螟(Sugarcane Borer)“SCB”)新生虫(24-48小时龄)侵染每个孔,并用塑料的带孔盖密封以允许空气交换。将盘置于28℃(16:8小时光照:黑暗,40%RH),并且在3天后,对它们的百分比叶损伤分级。当通过使用Pro 9.0.1(2010SAS Institute Inc.,Cary,NC),方差均匀时,用ANOVA和用Tukey-Kramer检验的平均分离分析百分比叶损伤数据。表6中显示了在转基因植物材料上观察到的由每种昆虫物种所致的叶损伤的水平。
表6
由以来自构建体114534,114535或115782的T0玉米叶组织为食的ECB or SCB幼虫引起的平均百分比叶损伤
*标准差(n=10)
与YFP阴性对照构建体(115782)相比,构建体114534和114535两者均导致更小的均值损伤。与表达截短的DIG-657蛋白的构建体114535相比,表达全长DIG-657蛋白的构建体114534表现出更大的昆虫活性。
实施例13
再生和种子生产.为了再生,将培养物转移到“28”诱导培养基(MS盐和维生素,30gm/L蔗糖,5mg/L苄基氨基嘌呤,0.25mg/L 2,4-D,3mg/L双丙氨膦,250mg/L双丙氨磷,250mg/L头孢噻肟,2.5gm/L结冷胶,pH 5.7),在低光照条件(14μEm-2s-1)下1周,然后在高光照条件(约89μEm-2s-1)1周。随后,将组织转移至“36”再生培养基(与缺少植物生长调节剂的诱导培养基相同)。将3-5cm长的小植株转移到含有SHGA培养基(Schenk和Hildebrandt盐和维生素(1972);PhytoTechnology Laboratories.,Lenexa,KS),1.0gm/L肌醇,10gm/L蔗糖和2.0gm/L Gellan胶,pH5.8)的玻璃培养管,以允许芽和根的进一步生长和发育。将植物移植到如本文早先描述的相同的土壤混合物中,并在温室中培养至开花。进行用于种子生产的受控授粉。
实施例14
用于DIG-657 Bt杀虫蛋白的编码序列的植物优化形式的设计.设计并合成具有植物密码子偏好的DNA序列,以在转基因单子叶植物和双子叶植物中产生DIG-657蛋白。从自GenBank中保存的序列获得的706种蛋白质编码序列(CD)计算玉米(玉蜀黍)的密码子选择表。从网站http://www.kazusa.or.jp/codon/的数据下载烟草(烟草(Nicotianatabacum),1268种CD),芸苔(欧洲油菜(Brassica napus),530种CD),棉(陆地棉(Gossypiumhirsutum),197种CD)和大豆(大豆;约1000种CD)的密码子选择表。在省略掉任一植物类型中的使用占某一氨基酸的总密码子使用小于约10%的冗余密码子后,计算出一个偏倚的密码子集,其包括玉米数据集和双子叶数据集两者共有的高使用密码子,这些密码子具有合适的加权平均相对量。为了得到编码DIG-657蛋白的植物优化序列,对实验测定的DIG-657DNA序列进行密码子取代,使得所得的DNA序列具有上述植物优化的密码子偏好表的总体密码子组成。对该序列进一步精修以消除不想要的限制性酶识别位点、潜在的植物内含子剪接位点、长段的A/T或C/G残基、和其它可能干扰植物细胞中的编码区的RNA稳定性,转录,或翻译的基序。进行其它变化以引入期望的限制性酶识别位点,并且消除长的内部可读框(除了+1外的框)。这些变化均在保留植物偏爱的密码子组成的约束下进行。设计出来的序列由商业供应商(DNA2.0,Menlo Park,CA)合成。其他关于生成合成基因的指导可以参见例如WO 97/13402和美国专利号5,380,831。SEQ ID NO:3中给出了一种编码DIG-657变体1的玉米优化的DNA序列。SEQ ID NO:4中给出了一种编码DIG-657变体2的荧光假单胞菌优化的DNA序列。SEQ ID NO:5中给出了一种编码DIG-657的玉米优化的高GC DNA序列。SEQ IDNO:6中给出了一种编码来自N-末端的截短204aa的DIG-657基因的玉米优化的高GC DNA序列。编码全长DIG-657的双子叶优化的DNA序列作为SEQ ID NO:8公开。编码截短的DIG-657的双子叶优化的DNA序列作为SEQ ID NO:9公开。
通过标准方法(Sambrook等人,同上)进行Southern印迹上固定化DNA与放射性标记的基因特异性探针的杂交。用于标记多核苷酸探针的放射性同位素包括32P,33P,14C或3H。将放射性同位素掺入多核苷酸探针分子通过分子生物学领域技术人员公知的几种方法中的任一种完成。(参见例如Sambrook等人,同上)。通常,杂交和随后的清洗在容许检出与要求保护的毒素编码基因具有同源性的靶序列的严格条件下实施。对于双链DNA基因探针,在6X SSPE、5X登哈特氏溶液、0.1%SDS,0.1mg/mL变性DNA中,在比于DNA杂交体的Tm的低20℃至25℃的温度下杂交过夜[20X SSPE为3M NaCl,0.2M NaHPO4,和0.02M EDTA(乙二胺四乙酸钠盐);100X登哈特氏溶液(20gm/L聚乙烯吡咯烷酮,20gm/L Ficoll型400和20gm/LBSA-级份V)]。
清洗通常如下实施:
a.在1X SSPE,0.1%SDS中,室温,15分钟,两次(低严格性清洗)。
b.在0.2X SSPE,0.1%SDS中,比Tm低的20℃的温度,15分钟,一次(中等严格性清洗)。
对于寡核苷酸探针,可以在6X SSPE、5X登哈特氏溶液、0.1%SDS,0.1mg/mL变性DNA中,在比杂交体的Tm低10℃至20℃的温度进行杂交过夜。寡核苷酸探针的Tm可以通过以下公式(Suggs等,1981)求出。
Tm(℃)=2(T/A碱基对的数目)+4(G/C碱基对的数目)
通常如下实施清洗:
a.在1X SSPE,0.1%SDS中,室温,15分钟,两次(低严格性清洗)。。
b.在1X SSPE,0.1%SDS中,在杂交温度下,15分钟,一次(中等严格性清洗)。
可以通过除了放射性标记之外的手段使得用于杂交的探针分子以及探针和靶分子之间形成的杂交分子可检测。这些替代方法意图在本发明的范围内。
本文中已经详细描述了具体实施方案作为示例,但本公开内容的各种修改和备选形式是容易作出的。然而,应当理解,本公开不旨在限于所公开的特定形式。相反,本公开将覆盖落入由所附权利要求书及其法律等同物限定的本公开的范围内的所有修改,等同方案和备选方式。
序列表
<110> 美国陶氏益农公司
Armstrong, Janna M.
Etter, Audrey J.
Frey, Meghan L.
Gandra, Premchand
Letherer, Ted
Lin, Gaofeng
Madduri, Krishna M.
Mowery, Haley R.
Narva, Kenneth E.
Sheets, Joel J.
Tan, Sek Yee
<120> 可用于控制昆虫有害生物的营养生长杀虫蛋白
<130> 74948
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 2412
<212> DNA
<213> Bacillus thuringiensis
<400> 1
atggtacaaa aatggatgca aaggatgata attgtggata ataataaatt aaatgtaaga 60
gctttaccaa gctttattga ttattttaac ggtatttatg gatttgccac tggtatcaaa 120
gatattatgg gaatgatttt taaaacagat acaggtggta gtaatttaac attagatgag 180
attttaaaga atcaaaattt actaaatgat atctcaggta agctcgatgg aattaatgga 240
gatttagggg atcttattgc acaagggaac ttgaattcag aattagctaa ggaattgcta 300
aaaatctcta atgagcagaa tcaaatgtta aatcatgtta atgctcaact taatgcaatc 360
aattcaacac ttaatatata tcttccaaaa attacatcta tgttaaatga ggtgatgaag 420
caaaaccatg ttttaagtct acaaatagaa tttcttagta agcaattgca ggaaatttca 480
gataaacttg atattatcaa cttaaacgta ttgattaact ctacattaac agagattact 540
cctgcttatc aacgtattaa atatgtaaac gaaaaatttg atgaattgac ttctactgta 600
gagaaaaatc caaaatcata tcaagataac gttactaaag aagttattga aaacttaaat 660
gagctaactg agttggcgaa aagtgttacc aaaaatgata tggatagttt tgaattttat 720
cttcaaactt tccatgatgt aatgactgga aataatttat tcggccgctc agcattaaaa 780
actgcttcag aattaattac aaaagaaaat gtcacgacaa ggggaagtga gataggaaaa 840
gtttataatt tcttaattgt tttaacttct ttacaagcaa aagcttttct cactttaact 900
gcatgtcgaa agttattagg tttaacagat atcgattata ctcaaattat gaatcatcat 960
atagatggtc aaaaaagaga atttcgtatt aatattcttc caacactttc taataatttt 1020
tctaatccta gttattcaaa aaatagagga agtgatatcg atgatccaat tgttgtgtta 1080
gaagcagcac ctggatatgc cttaatagga tttgaaattc taaacgatcc acttccaatt 1140
ttaaaaggat atcaggctag gttaaaacca aattatcaag ttgacaggga gtcgatgtca 1200
gaaacgattt atggggacat tcataaatta ttttgcccaa aacagctgga gcaaaaatat 1260
tatattaaag atattgaatt tcctgagggc tatgtaatta ctaaaatcgt ttttgaaaaa 1320
aggctaaatc aattggggta tgaggtaaca gcaaattttt atgacccgtc tacaggaagt 1380
atcgatttaa ataaggttaa agtagaatct tggaaggaaa agtcttgcga ggaggattcc 1440
tgcgaagatg agtatagtat tataaaggcc gaaacggatg gcatttatat gccattaggc 1500
gtagtaagtg agactttttt aacccctatt tatggttttg gattaacagt tgacgaaaaa 1560
aatcaaaaaa taactttaac aggtaaatcc tatttacgtg aatccttact agaaacagac 1620
ttacttaaca atgaaacata tttaattgct tcaccagacg gttatattag tagtattgta 1680
gaaaactgga atataacatc agataatact gggtcttgga gagcaaataa taataatgca 1740
tttgtcgata aggcagatac tataaaagga tcaagttctc tgtatactca taaagatggg 1800
gaattctcgc aatttattgg aaataagcta aaacctaaaa ctaattatgt tattcaatat 1860
gttataaaag gaagacctgc tatttattta aaaaataata aagatacttt atttgaggat 1920
accaaaaata actttagcga ttttcagact gtaactaaaa aattcaattc aggagtaaat 1980
ccttcggaaa tttatttcct ttttaaaaat caaagtgaat acgaagcctg gggaaataac 2040
tttattattt tagaaattaa atcgcttgaa ttcttgccac aaatgctgaa gcctgaggat 2100
tggataccat caggaaatgt gcaaatgaaa gatggaggac gcctagagat tttgggagat 2160
ggttatttta aacaattcat taaattggaa aatgattcaa cctatcatct aagattatct 2220
gttaagggaa caggtagggt atctataatt gatgaatcta aatatttact ttttgtaaat 2280
gttaaggatg aagatcttac tagagttatt aaaaatacct cttcaaaggg tgagtgtttt 2340
atagctcttg agggtactta tgtagaaaat tcaagtacta ttttctctaa tgtatctatt 2400
gttaaagagt ag 2412
<210> 2
<211> 803
<212> PRT
<213> Bacillus thuringiensis
<400> 2
Met Val Gln Lys Trp Met Gln Arg Met Ile Ile Val Asp Asn Asn Lys
1 5 10 15
Leu Asn Val Arg Ala Leu Pro Ser Phe Ile Asp Tyr Phe Asn Gly Ile
20 25 30
Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile Met Gly Met Ile Phe Lys
35 40 45
Thr Asp Thr Gly Gly Ser Asn Leu Thr Leu Asp Glu Ile Leu Lys Asn
50 55 60
Gln Asn Leu Leu Asn Asp Ile Ser Gly Lys Leu Asp Gly Ile Asn Gly
65 70 75 80
Asp Leu Gly Asp Leu Ile Ala Gln Gly Asn Leu Asn Ser Glu Leu Ala
85 90 95
Lys Glu Leu Leu Lys Ile Ser Asn Glu Gln Asn Gln Met Leu Asn His
100 105 110
Val Asn Ala Gln Leu Asn Ala Ile Asn Ser Thr Leu Asn Ile Tyr Leu
115 120 125
Pro Lys Ile Thr Ser Met Leu Asn Glu Val Met Lys Gln Asn His Val
130 135 140
Leu Ser Leu Gln Ile Glu Phe Leu Ser Lys Gln Leu Gln Glu Ile Ser
145 150 155 160
Asp Lys Leu Asp Ile Ile Asn Leu Asn Val Leu Ile Asn Ser Thr Leu
165 170 175
Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile Lys Tyr Val Asn Glu Lys
180 185 190
Phe Asp Glu Leu Thr Ser Thr Val Glu Lys Asn Pro Lys Ser Tyr Gln
195 200 205
Asp Asn Val Thr Lys Glu Val Ile Glu Asn Leu Asn Glu Leu Thr Glu
210 215 220
Leu Ala Lys Ser Val Thr Lys Asn Asp Met Asp Ser Phe Glu Phe Tyr
225 230 235 240
Leu Gln Thr Phe His Asp Val Met Thr Gly Asn Asn Leu Phe Gly Arg
245 250 255
Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile Thr Lys Glu Asn Val Thr
260 265 270
Thr Arg Gly Ser Glu Ile Gly Lys Val Tyr Asn Phe Leu Ile Val Leu
275 280 285
Thr Ser Leu Gln Ala Lys Ala Phe Leu Thr Leu Thr Ala Cys Arg Lys
290 295 300
Leu Leu Gly Leu Thr Asp Ile Asp Tyr Thr Gln Ile Met Asn His His
305 310 315 320
Ile Asp Gly Gln Lys Arg Glu Phe Arg Ile Asn Ile Leu Pro Thr Leu
325 330 335
Ser Asn Asn Phe Ser Asn Pro Ser Tyr Ser Lys Asn Arg Gly Ser Asp
340 345 350
Ile Asp Asp Pro Ile Val Val Leu Glu Ala Ala Pro Gly Tyr Ala Leu
355 360 365
Ile Gly Phe Glu Ile Leu Asn Asp Pro Leu Pro Ile Leu Lys Gly Tyr
370 375 380
Gln Ala Arg Leu Lys Pro Asn Tyr Gln Val Asp Arg Glu Ser Met Ser
385 390 395 400
Glu Thr Ile Tyr Gly Asp Ile His Lys Leu Phe Cys Pro Lys Gln Leu
405 410 415
Glu Gln Lys Tyr Tyr Ile Lys Asp Ile Glu Phe Pro Glu Gly Tyr Val
420 425 430
Ile Thr Lys Ile Val Phe Glu Lys Arg Leu Asn Gln Leu Gly Tyr Glu
435 440 445
Val Thr Ala Asn Phe Tyr Asp Pro Ser Thr Gly Ser Ile Asp Leu Asn
450 455 460
Lys Val Lys Val Glu Ser Trp Lys Glu Lys Ser Cys Glu Glu Asp Ser
465 470 475 480
Cys Glu Asp Glu Tyr Ser Ile Ile Lys Ala Glu Thr Asp Gly Ile Tyr
485 490 495
Met Pro Leu Gly Val Val Ser Glu Thr Phe Leu Thr Pro Ile Tyr Gly
500 505 510
Phe Gly Leu Thr Val Asp Glu Lys Asn Gln Lys Ile Thr Leu Thr Gly
515 520 525
Lys Ser Tyr Leu Arg Glu Ser Leu Leu Glu Thr Asp Leu Leu Asn Asn
530 535 540
Glu Thr Tyr Leu Ile Ala Ser Pro Asp Gly Tyr Ile Ser Ser Ile Val
545 550 555 560
Glu Asn Trp Asn Ile Thr Ser Asp Asn Thr Gly Ser Trp Arg Ala Asn
565 570 575
Asn Asn Asn Ala Phe Val Asp Lys Ala Asp Thr Ile Lys Gly Ser Ser
580 585 590
Ser Leu Tyr Thr His Lys Asp Gly Glu Phe Ser Gln Phe Ile Gly Asn
595 600 605
Lys Leu Lys Pro Lys Thr Asn Tyr Val Ile Gln Tyr Val Ile Lys Gly
610 615 620
Arg Pro Ala Ile Tyr Leu Lys Asn Asn Lys Asp Thr Leu Phe Glu Asp
625 630 635 640
Thr Lys Asn Asn Phe Ser Asp Phe Gln Thr Val Thr Lys Lys Phe Asn
645 650 655
Ser Gly Val Asn Pro Ser Glu Ile Tyr Phe Leu Phe Lys Asn Gln Ser
660 665 670
Glu Tyr Glu Ala Trp Gly Asn Asn Phe Ile Ile Leu Glu Ile Lys Ser
675 680 685
Leu Glu Phe Leu Pro Gln Met Leu Lys Pro Glu Asp Trp Ile Pro Ser
690 695 700
Gly Asn Val Gln Met Lys Asp Gly Gly Arg Leu Glu Ile Leu Gly Asp
705 710 715 720
Gly Tyr Phe Lys Gln Phe Ile Lys Leu Glu Asn Asp Ser Thr Tyr His
725 730 735
Leu Arg Leu Ser Val Lys Gly Thr Gly Arg Val Ser Ile Ile Asp Glu
740 745 750
Ser Lys Tyr Leu Leu Phe Val Asn Val Lys Asp Glu Asp Leu Thr Arg
755 760 765
Val Ile Lys Asn Thr Ser Ser Lys Gly Glu Cys Phe Ile Ala Leu Glu
770 775 780
Gly Thr Tyr Val Glu Asn Ser Ser Thr Ile Phe Ser Asn Val Ser Ile
785 790 795 800
Val Lys Glu
<210> 3
<211> 2412
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 3
atggtgcaga aatggatgca gcggatgata attgtagata acaacaagct caacgttcgt 60
gcattgccgt ctttcataga ctatttcaac ggcatatatg ggttcgccac cggcatcaag 120
gacatcatgg gaatgatctt caagaccgac acgggtggat ctaatctgac cctcgatgag 180
atcttgaaaa atcagaatct gctgaatgac atctcaggga aactagacgg tattaacggc 240
gaccttggcg acttgattgc ccaggggaac ctaaactctg aactcgccaa ggaacttctc 300
aagattagca acgaacaaaa tcagatgctc aaccatgtga atgcacaatt gaatgcgatc 360
aatagtactc tcaacatcta ccttcccaag atcacatcca tgctgaacga ggttatgaag 420
caaaaccatg tgttatcgct gcagattgag ttcctgtcaa agcagttgca agagatttcc 480
gataagcttg acatcatcaa cttaaacgtc ctcatcaatt cgaccctcac cgaaattacc 540
cctgcgtatc agagaattaa gtacgtcaac gaaaagtttg acgagctgac cagtaccgtg 600
gagaaaaacc ccaagtcata tcaggataat gtcactaagg aagttattga aaacctcaat 660
gagcttacgg agctggcgaa aagcgtgacg aaaaacgata tggatagctt cgagttttac 720
ctccaaacct tccatgacgt aatgaccggg aacaacctct tcgggaggtc agcgctcaag 780
acagcctccg agttaatcac gaaggagaac gtcacaaccc ggggttcaga aattgggaag 840
gtatacaact tcctaatcgt gcttacgagc cttcaagcta aagctttcct cacactgacg 900
gcctgccgga aactacttgg gctgaccgac atcgactata ctcagattat gaatcaccac 960
atcgacggtc aaaagagaga gtttcgcatt aacattctcc caacgctcag taacaacttc 1020
tccaatccgt cgtattccaa aaaccgaggc tcagatattg acgatcctat tgtcgtcttg 1080
gaggcagcgc cagggtacgc actcatcggc ttcgaaatct taaacgaccc gctgcccatc 1140
cttaagggtt atcaggccag gctgaagccg aactaccagg tggaccgcga atccatgtcc 1200
gaaacgatct acggcgacat ccacaagctg ttctgtccga agcagctcga acaaaagtac 1260
tacattaaag acatcgagtt cccagaggga tatgtgatta cgaaaattgt gttcgaaaag 1320
aggctcaacc aacttggata tgaggtcaca gctaatttct acgatccttc gactggctct 1380
atcgatttga ataaggtgaa ggttgaatcg tggaaggaga aaagctgtga ggaagatagt 1440
tgcgaggatg aatattcaat cataaaggcg gaaacagatg gaatatacat gccgctcggc 1500
gttgtcagcg agacttttct gacccctatc tacggttttg gtctaacagt cgacgagaaa 1560
aaccagaaga tcactctgac cggtaaaagt taccttcgcg agtcgctgct ggagactgac 1620
ctgctgaaca acgagactta cttgatcgct agcccagacg ggtacattag ctctatcgtc 1680
gaaaactgga atatcacctc tgataatact ggctcgtgga gggccaataa caacaatgcg 1740
tttgtggata aagcagatac aatcaaaggc tcaagcagcc tctacacgca caaggatgga 1800
gagttttccc agtttatagg caacaagttg aagccaaaga cgaattacgt tatacagtac 1860
gtgatcaagg gcaggccagc catatacttg aaaaacaata aggacacgct tttcgaagat 1920
acgaagaata acttctccga tttccaaacc gttacaaaga agttcaatag tggagtgaat 1980
cctagcgaga tctactttct gttcaaaaac caatctgagt acgaggcttg gggcaacaac 2040
ttcataattc tagagataaa gtccctggag tttctgcccc agatgctgaa gcccgaggat 2100
tggatcccct ccggcaatgt acagatgaag gacggcggac gcctagagat tctcggcgac 2160
ggctacttta agcagttcat caagttagag aacgactcga cttatcacct tcgtctctca 2220
gtgaagggta cagggagagt cagcatcatc gacgagtcga agtacctttt gttcgtcaac 2280
gttaaggacg aggacctgac acgagtcata aagaatacca gcagtaaggg cgagtgcttt 2340
atcgctttgg agggcactta tgttgagaac tcctctacca tattctccaa tgtttctata 2400
gtgaaggagt ga 2412
<210> 4
<211> 2412
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 4
atggcccaga agtggatgca acgcatgatc atcgtggata acaacaagct gaacgtccgt 60
gccctgccga gcttcatcga ctacttcaac ggcatctacg gcttcgcgac cggcattaaa 120
gacattatgg gcatgatctt caagaccgac acgggcggta gcaacttgac cctggacgaa 180
attctgaaga atcagaatct gctcaacgat atcagcggca agctcgacgg cattaacggg 240
gatctgggcg acctgatcgc ccaagggaac ctgaacagcg agctggcgaa agagctgttg 300
aagatctcca acgaacagaa ccagatgctg aaccacgtga atgcccaact gaacgccatc 360
aactcgaccc tgaacatcta cttgcccaag atcacctcga tgttgaacga ggtgatgaag 420
cagaaccatg tgctgagcct gcagattgag ttcctgagca agcagctgca ggaaatctcc 480
gacaagctgg atatcatcaa cctgaatgtg ctgatcaata gcaccttgac cgagatcacc 540
ccggcctatc agcgcatcaa gtacgtgaac gagaagttcg acgaactgac cagcaccgtc 600
gaaaagaatc ccaagtcgta ccaggacaat gtcaccaaag aagtgatcga gaacttgaac 660
gagctcaccg aactggccaa atcggtgacg aagaacgaca tggactcgtt cgaattctat 720
ctgcagacct tccacgacgt gatgaccggc aacaacctgt tcggccgcag cgccctgaaa 780
accgcctcgg aactgatcac gaaagagaat gtgaccaccc gtggctccga gatcggcaag 840
gtgtacaact tcctgattgt cctgacctcg ctgcaggcga aggcgtttct gaccttgacg 900
gcctgccgta agctgttggg cctgaccgac attgactaca cgcagatcat gaatcaccac 960
atcgacggtc agaagcggga attccgcatc aacatcctgc cgaccctgag caataacttc 1020
tccaacccca gctacagcaa gaaccggggc agcgatatcg acgacccgat cgtggtcctc 1080
gaagcggcgc ccggctacgc cctgatcggc ttcgagatcc tgaacgaccc gttgccgatc 1140
ctgaaagggt accaagcgcg cctgaagccg aattaccagg tggatcgcga gtcgatgagc 1200
gaaaccattt acggcgacat tcacaagctg ttctgcccca agcaactcga acagaagtac 1260
tatatcaagg acatcgaatt cccggaaggc tacgtgatta ccaagatcgt ctttgaaaag 1320
cgcctgaacc agctgggtta tgaggtcacc gccaacttct acgacccgag cacgggtagc 1380
atcgacctga ataaggtgaa ggtggaaagc tggaaagaga agtcgtgcga agaggattcg 1440
tgcgaggacg agtatagcat catcaaggcg gaaaccgatg gcatctacat gccgctcggc 1500
gtcgtcagcg aaacctttct caccccgatc tacgggtttg gcttgaccgt ggatgaaaag 1560
aaccagaaaa tcaccctgac cggtaagagc tatctccgcg agtccttgct ggaaacggac 1620
ctgttgaaca acgaaaccta cctgattgcc agcccggatg gttatatctc gtcgattgtg 1680
gagaactgga acatcaccag cgacaacacc ggctcgtggc gtgccaacaa taacaatgcc 1740
ttcgtcgaca aggccgatac gatcaagggt agctccagcc tgtacaccca caaagacggt 1800
gaatttagcc agtttattgg caataagttg aagcccaaga ccaattacgt catccaatac 1860
gtcattaagg gccgtccggc gatctatctg aaaaacaaca aggacaccct cttcgaggat 1920
accaagaaca acttcagcga cttccagacc gtgaccaaga aattcaattc cggtgtgaac 1980
ccgtccgaaa tctatttcct gttcaaaaac cagtcggaat acgaggcctg gggcaacaac 2040
tttatcatcc tggagatcaa gagcctggag tttctgccgc aaatgctgaa acccgaggat 2100
tggattccca gcgggaacgt gcagatgaag gatggtggcc gcctcgaaat cttgggcgac 2160
ggctacttca agcagttcat caagttggaa aacgacagca cctaccatct gcggctgtcc 2220
gtcaagggca ccggccgtgt gtccatcatc gacgagtcca agtatctgct cttcgtgaac 2280
gtgaaggatg aggatctgac ccgcgtgatc aagaacacca gcagcaaagg ggaatgtttc 2340
atcgccctgg aaggcacgta cgtcgagaac tccagcacca tcttcagcaa cgtgtccatc 2400
gtgaaagagt ga 2412
<210> 5
<211> 2412
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 5
atggtgcaga agtggatgca gaggatgata atcgttgaca ataataaatt aaacgtgagg 60
gcactgccga gcttcatcga ctacttcaac ggcatctacg gcttcgccac cggcatcaag 120
gacatcatgg gcatgatctt caagaccgac accggaggca gcaacctgac cctggacgag 180
atcctcaaga atcaaaacct cctgaacgac atcagcggga agctggacgg aattaatggc 240
gatcttggag acctgatcgc ccaaggcaac ctcaactccg agctggccaa ggagctgctg 300
aagatttcca acgagcagaa tcaaatgctg aaccacgtga acgctcagct gaacgcaatc 360
aactcaaccc tgaacatcta cctgcccaag atcacgtcca tgctgaacga ggtgatgaag 420
cagaaccatg tgctgtccct ccagatagag ttcctgagca agcagctgca agagatatcc 480
gacaagctgg acatcatcaa cctcaacgtg ctgatcaaca gcaccctcac cgagatcacc 540
cctgcgtacc agaggattaa atacgtgaac gagaagttcg acgaactgac ctccaccgtg 600
gagaagaacc ccaaatccta ccaagacaac gtcaccaagg aggtgatcga gaacctgaac 660
gagctgacgg agctggcgaa gagcgtcacc aagaacgaca tggactcctt cgagttctac 720
ctccagacct tccacgacgt gatgaccggg aataatctgt tcgggcgctc agcattaaag 780
accgcgagcg aattaatcac caaggagaac gtgaccacca gaggctccga gatcggcaag 840
gtgtacaact tcctgatcgt gctcacctcc ctgcaagcga aggcgttcct caccctgacc 900
gcgtgtagga agctgctggg cctgaccgac atcgattaca cccagatcat gaaccaccac 960
atcgacggcc agaagaggga gtttaggatt aatatcctgc cgaccctgtc caataatttc 1020
tccaacccga gctactccaa aaatagaggc tcggacatcg acgaccccat cgtggtgctg 1080
gaggcagccc ctggctacgc cctgatcggc ttcgagatcc tgaacgaccc gctgccgatc 1140
ctcaagggct accaagcgag gctgaaacca aactaccaag tggataggga gagcatgtca 1200
gagaccatct acggggacat ccataaactg ttctgcccaa agcagctgga gcaaaaatat 1260
tacattaaag atatcgagtt cccggagggc tacgtgatca ccaagatcgt gttcgagaag 1320
aggctgaatc aactgggcta cgaagtgacc gccaacttct acgacccgag caccggctcc 1380
atcgacctga acaaggtgaa ggtggagagc tggaaggaga agtcctgtga ggaggactcc 1440
tgcgaggacg agtactccat catcaaggcc gagaccgacg ggatctacat gccgctgggc 1500
gtggtgagcg agaccttcct gaccccgatc tacgggtttg gcctcaccgt ggacgagaaa 1560
aatcaaaaaa taaccctgac cggcaagtcc tacctgaggg agtccctgct ggagaccgac 1620
ctgctgaaca atgaaaccta cctcatcgcc agcccagacg gctacatcag cagcatcgtc 1680
gagaactgga atataacctc cgacaacacc gggagctgga gggccaataa taataatgcc 1740
ttcgtggaca aggcggatac tataaaaggc tcaagcagcc tgtacaccca taaagacggc 1800
gagttctcgc agttcatcgg caacaagctg aagcccaaga ccaactacgt catccagtac 1860
gttataaaag gaaggcctgc gatctacctg aaaaataata aagacaccct gttcgaggac 1920
accaaaaata acttcagcga cttccagacc gtgaccaaga agttcaactc tggcgtgaac 1980
ccgagcgaaa tctacttcct gttcaagaat caatccgagt acgaggcctg gggcaacaac 2040
ttcatcatcc tggaaattaa atccctggag ttcctgccgc agatgctgaa gccggaggac 2100
tggataccga gcggcaacgt gcagatgaaa gacggtggaa ggctggagat cctgggcgac 2160
ggctacttca agcagttcat taaactggag aacgacagca cctaccacct gaggctgagc 2220
gtgaagggaa ctgggagggt gtcgatcatc gacgagtcca agtacctgct gttcgtgaac 2280
gtgaaggacg aggacctgac gagggtgatt aaaaatacct cctccaaggg cgagtgcttc 2340
atcgcgctgg aaggcaccta cgtggagaac agctcaacca tcttctccaa cgtgagcatc 2400
gtgaaggagt ga 2412
<210> 6
<211> 1800
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 6
atgtcctacc aagacaacgt caccaaggag gtgatcgaga acctgaacga gctgacggag 60
ctggcgaaga gcgtcaccaa gaacgacatg gactccttcg agttctacct ccagaccttc 120
cacgacgtga tgaccgggaa taatctgttc gggcgctcag cattaaagac cgcgagcgaa 180
ttaatcacca aggagaacgt gaccaccaga ggctccgaga tcggcaaggt gtacaacttc 240
ctgatcgtgc tcacctccct gcaagcgaag gcgttcctca ccctgaccgc gtgtaggaag 300
ctgctgggcc tgaccgacat cgattacacc cagatcatga accaccacat cgacggccag 360
aagagggagt ttaggattaa tatcctgccg accctgtcca ataatttctc caacccgagc 420
tactccaaaa atagaggctc ggacatcgac gaccccatcg tggtgctgga ggcagcccct 480
ggctacgccc tgatcggctt cgagatcctg aacgacccgc tgccgatcct caagggctac 540
caagcgaggc tgaaaccaaa ctaccaagtg gatagggaga gcatgtcaga gaccatctac 600
ggggacatcc ataaactgtt ctgcccaaag cagctggagc aaaaatatta cattaaagat 660
atcgagttcc cggagggcta cgtgatcacc aagatcgtgt tcgagaagag gctgaatcaa 720
ctgggctacg aagtgaccgc caacttctac gacccgagca ccggctccat cgacctgaac 780
aaggtgaagg tggagagctg gaaggagaag tcctgtgagg aggactcctg cgaggacgag 840
tactccatca tcaaggccga gaccgacggg atctacatgc cgctgggcgt ggtgagcgag 900
accttcctga ccccgatcta cgggtttggc ctcaccgtgg acgagaaaaa tcaaaaaata 960
accctgaccg gcaagtccta cctgagggag tccctgctgg agaccgacct gctgaacaat 1020
gaaacctacc tcatcgccag cccagacggc tacatcagca gcatcgtcga gaactggaat 1080
ataacctccg acaacaccgg gagctggagg gccaataata ataatgcctt cgtggacaag 1140
gcggatacta taaaaggctc aagcagcctg tacacccata aagacggcga gttctcgcag 1200
ttcatcggca acaagctgaa gcccaagacc aactacgtca tccagtacgt tataaaagga 1260
aggcctgcga tctacctgaa aaataataaa gacaccctgt tcgaggacac caaaaataac 1320
ttcagcgact tccagaccgt gaccaagaag ttcaactctg gcgtgaaccc gagcgaaatc 1380
tacttcctgt tcaagaatca atccgagtac gaggcctggg gcaacaactt catcatcctg 1440
gaaattaaat ccctggagtt cctgccgcag atgctgaagc cggaggactg gataccgagc 1500
ggcaacgtgc agatgaaaga cggtggaagg ctggagatcc tgggcgacgg ctacttcaag 1560
cagttcatta aactggagaa cgacagcacc taccacctga ggctgagcgt gaagggaact 1620
gggagggtgt cgatcatcga cgagtccaag tacctgctgt tcgtgaacgt gaaggacgag 1680
gacctgacga gggtgattaa aaatacctcc tccaagggcg agtgcttcat cgcgctggaa 1740
ggcacctacg tggagaacag ctcaaccatc ttctccaacg tgagcatcgt gaaggagtga 1800
<210> 7
<211> 599
<212> PRT
<213> 人工序列
<220>
<223> 自合成的全长编码区翻译
<400> 7
Met Ser Tyr Gln Asp Asn Val Thr Lys Glu Val Ile Glu Asn Leu Asn
1 5 10 15
Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Met Asp Ser
20 25 30
Phe Glu Phe Tyr Leu Gln Thr Phe His Asp Val Met Thr Gly Asn Asn
35 40 45
Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile Thr Lys
50 55 60
Glu Asn Val Thr Thr Arg Gly Ser Glu Ile Gly Lys Val Tyr Asn Phe
65 70 75 80
Leu Ile Val Leu Thr Ser Leu Gln Ala Lys Ala Phe Leu Thr Leu Thr
85 90 95
Ala Cys Arg Lys Leu Leu Gly Leu Thr Asp Ile Asp Tyr Thr Gln Ile
100 105 110
Met Asn His His Ile Asp Gly Gln Lys Arg Glu Phe Arg Ile Asn Ile
115 120 125
Leu Pro Thr Leu Ser Asn Asn Phe Ser Asn Pro Ser Tyr Ser Lys Asn
130 135 140
Arg Gly Ser Asp Ile Asp Asp Pro Ile Val Val Leu Glu Ala Ala Pro
145 150 155 160
Gly Tyr Ala Leu Ile Gly Phe Glu Ile Leu Asn Asp Pro Leu Pro Ile
165 170 175
Leu Lys Gly Tyr Gln Ala Arg Leu Lys Pro Asn Tyr Gln Val Asp Arg
180 185 190
Glu Ser Met Ser Glu Thr Ile Tyr Gly Asp Ile His Lys Leu Phe Cys
195 200 205
Pro Lys Gln Leu Glu Gln Lys Tyr Tyr Ile Lys Asp Ile Glu Phe Pro
210 215 220
Glu Gly Tyr Val Ile Thr Lys Ile Val Phe Glu Lys Arg Leu Asn Gln
225 230 235 240
Leu Gly Tyr Glu Val Thr Ala Asn Phe Tyr Asp Pro Ser Thr Gly Ser
245 250 255
Ile Asp Leu Asn Lys Val Lys Val Glu Ser Trp Lys Glu Lys Ser Cys
260 265 270
Glu Glu Asp Ser Cys Glu Asp Glu Tyr Ser Ile Ile Lys Ala Glu Thr
275 280 285
Asp Gly Ile Tyr Met Pro Leu Gly Val Val Ser Glu Thr Phe Leu Thr
290 295 300
Pro Ile Tyr Gly Phe Gly Leu Thr Val Asp Glu Lys Asn Gln Lys Ile
305 310 315 320
Thr Leu Thr Gly Lys Ser Tyr Leu Arg Glu Ser Leu Leu Glu Thr Asp
325 330 335
Leu Leu Asn Asn Glu Thr Tyr Leu Ile Ala Ser Pro Asp Gly Tyr Ile
340 345 350
Ser Ser Ile Val Glu Asn Trp Asn Ile Thr Ser Asp Asn Thr Gly Ser
355 360 365
Trp Arg Ala Asn Asn Asn Asn Ala Phe Val Asp Lys Ala Asp Thr Ile
370 375 380
Lys Gly Ser Ser Ser Leu Tyr Thr His Lys Asp Gly Glu Phe Ser Gln
385 390 395 400
Phe Ile Gly Asn Lys Leu Lys Pro Lys Thr Asn Tyr Val Ile Gln Tyr
405 410 415
Val Ile Lys Gly Arg Pro Ala Ile Tyr Leu Lys Asn Asn Lys Asp Thr
420 425 430
Leu Phe Glu Asp Thr Lys Asn Asn Phe Ser Asp Phe Gln Thr Val Thr
435 440 445
Lys Lys Phe Asn Ser Gly Val Asn Pro Ser Glu Ile Tyr Phe Leu Phe
450 455 460
Lys Asn Gln Ser Glu Tyr Glu Ala Trp Gly Asn Asn Phe Ile Ile Leu
465 470 475 480
Glu Ile Lys Ser Leu Glu Phe Leu Pro Gln Met Leu Lys Pro Glu Asp
485 490 495
Trp Ile Pro Ser Gly Asn Val Gln Met Lys Asp Gly Gly Arg Leu Glu
500 505 510
Ile Leu Gly Asp Gly Tyr Phe Lys Gln Phe Ile Lys Leu Glu Asn Asp
515 520 525
Ser Thr Tyr His Leu Arg Leu Ser Val Lys Gly Thr Gly Arg Val Ser
530 535 540
Ile Ile Asp Glu Ser Lys Tyr Leu Leu Phe Val Asn Val Lys Asp Glu
545 550 555 560
Asp Leu Thr Arg Val Ile Lys Asn Thr Ser Ser Lys Gly Glu Cys Phe
565 570 575
Ile Ala Leu Glu Gly Thr Tyr Val Glu Asn Ser Ser Thr Ile Phe Ser
580 585 590
Asn Val Ser Ile Val Lys Glu
595
<210> 8
<211> 2412
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 8
atggttcaaa agtggatgca aagaatgata attgttgaca ataataaatt aaatgttaga 60
gcacttccat ctttcattga ttactttaac ggaatctatg gatttgctac tgggattaag 120
gatatcatgg gaatgatctt taagactgat actggtggtt ctaatcttac tcttgatgag 180
atattgaaga atcaaaattt gcttaatgac atttctggca agctggatgg aattaatgga 240
gaccttggtg atcttattgc tcaaggaaac cttaactctg aacttgctaa ggaacttctt 300
aagatttcaa atgagcaaaa tcaaatgttg aaccatgtta acgctcaact taacgcaatc 360
aactctacac ttaacatcta tcttccaaag attacttcaa tgcttaatga ggttatgaag 420
caaaaccacg ttctttctct tcaaattgaa tttctttcta agcaacttca agagatttct 480
gataagctgg atatcattaa ccttaacgtt ctcattaact ctactttgac tgagattact 540
cctgcttacc aaaggattaa atacgttaac gaaaagttcg atgaactgac ttctactgtt 600
gagaagaacc caaagtctta ccaagataac gttactaagg aagttattga aaaccttaat 660
gagcttactg agcttgctaa gtctgttaca aagaacgaca tggattcatt cgagttctat 720
cttcaaactt tccatgatgt tatgactggg aataatcttt tcgggaggtc tgcattaaag 780
actgcttctg aattaatcac taaggaaaac gtaactacta gaggttctga gattggaaag 840
gtgtataact ttcttattgt tcttacttca cttcaagcta aggctttcct tactcttact 900
gcttgtagaa agctccttgg tcttactgac attgattaca ctcagattat gaaccatcat 960
attgatggtc aaaagagaga gtttaggatt aatatccttc caactctttc taataatttc 1020
tcaaatccat cttactctaa aaatagaggt tctgatattg atgatcctat tgtagttctt 1080
gaagctgctc ctggatacgc tcttattgga tttgagatac ttaacgatcc acttcctatt 1140
cttaagggtt atcaagctag acttaaacca aactatcaag ttgatagaga gtctatgtca 1200
gagactatct acggagatat tcataaactc ttttgcccaa agcaacttga gcaaaaatat 1260
tacattaaag atattgaatt tcctgaggga tacgtcatta ctaagatagt ttttgaaaag 1320
agacttaatc aacttggtta cgaggttact gctaactttt acgatccatc taccggatct 1380
attgatctta acaaggttaa ggttgaatct tggaaggaaa agtcttgcga ggaggattct 1440
tgcgaagatg agtattcaat cattaaggct gaaacagatg gcatctatat gccacttgga 1500
gtggtttctg aaacttttct tactcctatc tatggtttcg gacttactgt tgatgagaag 1560
aatcaaaaaa taactttgac tgggaagtct tatcttagag aatctcttct tgaaactgat 1620
cttcttaaca atgaaactta ccttattgct tctccagacg gttacatttc ttctattgtt 1680
gagaactgga atataacttc tgacaatact ggatcttgga gagctaataa taataatgct 1740
tttgtggata aggctgatac tataaaaggt tcttcatctc tttacactca taaagatggt 1800
gagttttctc aattcattgg aaacaaactt aagcctaaga ccaattacgt tattcaatat 1860
gttataaaag ggagacctgc tatctatctt aaaaataata aagatactct tttcgaggat 1920
accaaaaata acttttctga ttttcaaact gttactaaga aattcaattc tggagttaat 1980
ccttctgaaa tctacttcct tttcaagaat caatctgaat acgaggcatg gggaaacaac 2040
tttatcattt tggaaattaa atcacttgag ttccttccac aaatgcttaa gcctgaggat 2100
tggattcctt ctggaaacgt tcagatgaaa gacggaggca gacttgagat tcttggagat 2160
ggttacttta agcaattcat taaattggag aatgattcaa cttaccatct gagactttct 2220
gttaagggaa ctggcagagt ttctatcatt gatgaatcta agtatcttct ttttgtaaat 2280
gttaaggatg aggatctgac tagagtcatt aaaaatactt cttctaaggg tgagtgtttc 2340
attgctcttg agggaactta cgttgaaaac tcttctacca ttttctctaa tgtttcaata 2400
gttaaggagt ga 2412
<210> 9
<211> 1800
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 9
atgtcttacc aagataacgt tactaaggaa gttattgaaa accttaatga gcttactgag 60
cttgctaagt ctgttacaaa gaacgacatg gattcattcg agttctatct tcaaactttc 120
catgatgtta tgactgggaa taatcttttc gggaggtctg cattaaagac tgcttctgaa 180
ttaatcacta aggaaaacgt aactactaga ggttctgaga ttggaaaggt gtataacttt 240
cttattgttc ttacttcact tcaagctaag gctttcctta ctcttactgc ttgtagaaag 300
ctccttggtc ttactgacat tgattacact cagattatga accatcatat tgatggtcaa 360
aagagagagt ttaggattaa tatccttcca actctttcta ataatttctc aaatccatct 420
tactctaaaa atagaggttc tgatattgat gatcctattg tagttcttga agctgctcct 480
ggatacgctc ttattggatt tgagatactt aacgatccac ttcctattct taagggttat 540
caagctagac ttaaaccaaa ctatcaagtt gatagagagt ctatgtcaga gactatctac 600
ggagatattc ataaactctt ttgcccaaag caacttgagc aaaaatatta cattaaagat 660
attgaatttc ctgagggata cgtcattact aagatagttt ttgaaaagag acttaatcaa 720
cttggttacg aggttactgc taacttttac gatccatcta ccggatctat tgatcttaac 780
aaggttaagg ttgaatcttg gaaggaaaag tcttgcgagg aggattcttg cgaagatgag 840
tattcaatca ttaaggctga aacagatggc atctatatgc cacttggagt ggtttctgaa 900
acttttctta ctcctatcta tggtttcgga cttactgttg atgagaagaa tcaaaaaata 960
actttgactg ggaagtctta tcttagagaa tctcttcttg aaactgatct tcttaacaat 1020
gaaacttacc ttattgcttc tccagacggt tacatttctt ctattgttga gaactggaat 1080
ataacttctg acaatactgg atcttggaga gctaataata ataatgcttt tgtggataag 1140
gctgatacta taaaaggttc ttcatctctt tacactcata aagatggtga gttttctcaa 1200
ttcattggaa acaaacttaa gcctaagacc aattacgtta ttcaatatgt tataaaaggg 1260
agacctgcta tctatcttaa aaataataaa gatactcttt tcgaggatac caaaaataac 1320
ttttctgatt ttcaaactgt tactaagaaa ttcaattctg gagttaatcc ttctgaaatc 1380
tacttccttt tcaagaatca atctgaatac gaggcatggg gaaacaactt tatcattttg 1440
gaaattaaat cacttgagtt ccttccacaa atgcttaagc ctgaggattg gattccttct 1500
ggaaacgttc agatgaaaga cggaggcaga cttgagattc ttggagatgg ttactttaag 1560
caattcatta aattggagaa tgattcaact taccatctga gactttctgt taagggaact 1620
ggcagagttt ctatcattga tgaatctaag tatcttcttt ttgtaaatgt taaggatgag 1680
gatctgacta gagtcattaa aaatacttct tctaagggtg agtgtttcat tgctcttgag 1740
ggaacttacg ttgaaaactc ttctaccatt ttctctaatg tttcaatagt taaggagtga 1800
<210> 10
<211> 2625
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 10
atgttagcta gacaaggtgg atctcttaga gcttctcaat gtaacgctgg acttgctaga 60
agagttgagg ttggtgctct tgttgttcct agaccaattt ctgttaacga cgttgttcca 120
cacgtctatt ctgctcctct ttctgttgct agacgttctt gctctaagtc ctctattaga 180
agtactagaa ggcttcaaac tactgtttgc tcaatggttc aaaagtggat gcaaagaatg 240
ataattgttg acaataataa attaaatgtt agagcacttc catctttcat tgattacttt 300
aacggaatct atggatttgc tactgggatt aaggatatca tgggaatgat ctttaagact 360
gatactggtg gttctaatct tactcttgat gagatattga agaatcaaaa tttgcttaat 420
gacatttctg gcaagctgga tggaattaat ggagaccttg gtgatcttat tgctcaagga 480
aaccttaact ctgaacttgc taaggaactt cttaagattt caaatgagca aaatcaaatg 540
ttgaaccatg ttaacgctca acttaacgca atcaactcta cacttaacat ctatcttcca 600
aagattactt caatgcttaa tgaggttatg aagcaaaacc acgttctttc tcttcaaatt 660
gaatttcttt ctaagcaact tcaagagatt tctgataagc tggatatcat taaccttaac 720
gttctcatta actctacttt gactgagatt actcctgctt accaaaggat taaatacgtt 780
aacgaaaagt tcgatgaact gacttctact gttgagaaga acccaaagtc ttaccaagat 840
aacgttacta aggaagttat tgaaaacctt aatgagctta ctgagcttgc taagtctgtt 900
acaaagaacg acatggattc attcgagttc tatcttcaaa ctttccatga tgttatgact 960
gggaataatc ttttcgggag gtctgcatta aagactgctt ctgaattaat cactaaggaa 1020
aacgtaacta ctagaggttc tgagattgga aaggtgtata actttcttat tgttcttact 1080
tcacttcaag ctaaggcttt ccttactctt actgcttgta gaaagctcct tggtcttact 1140
gacattgatt acactcagat tatgaaccat catattgatg gtcaaaagag agagtttagg 1200
attaatatcc ttccaactct ttctaataat ttctcaaatc catcttactc taaaaataga 1260
ggttctgata ttgatgatcc tattgtagtt cttgaagctg ctcctggata cgctcttatt 1320
ggatttgaga tacttaacga tccacttcct attcttaagg gttatcaagc tagacttaaa 1380
ccaaactatc aagttgatag agagtctatg tcagagacta tctacggaga tattcataaa 1440
ctcttttgcc caaagcaact tgagcaaaaa tattacatta aagatattga atttcctgag 1500
ggatacgtca ttactaagat agtttttgaa aagagactta atcaacttgg ttacgaggtt 1560
actgctaact tttacgatcc atctaccgga tctattgatc ttaacaaggt taaggttgaa 1620
tcttggaagg aaaagtcttg cgaggaggat tcttgcgaag atgagtattc aatcattaag 1680
gctgaaacag atggcatcta tatgccactt ggagtggttt ctgaaacttt tcttactcct 1740
atctatggtt tcggacttac tgttgatgag aagaatcaaa aaataacttt gactgggaag 1800
tcttatctta gagaatctct tcttgaaact gatcttctta acaatgaaac ttaccttatt 1860
gcttctccag acggttacat ttcttctatt gttgagaact ggaatataac ttctgacaat 1920
actggatctt ggagagctaa taataataat gcttttgtgg ataaggctga tactataaaa 1980
ggttcttcat ctctttacac tcataaagat ggtgagtttt ctcaattcat tggaaacaaa 2040
cttaagccta agaccaatta cgttattcaa tatgttataa aagggagacc tgctatctat 2100
cttaaaaata ataaagatac tcttttcgag gataccaaaa ataacttttc tgattttcaa 2160
actgttacta agaaattcaa ttctggagtt aatccttctg aaatctactt ccttttcaag 2220
aatcaatctg aatacgaggc atggggaaac aactttatca ttttggaaat taaatcactt 2280
gagttccttc cacaaatgct taagcctgag gattggattc cttctggaaa cgttcagatg 2340
aaagacggag gcagacttga gattcttgga gatggttact ttaagcaatt cattaaattg 2400
gagaatgatt caacttacca tctgagactt tctgttaagg gaactggcag agtttctatc 2460
attgatgaat ctaagtatct tctttttgta aatgttaagg atgaggatct gactagagtc 2520
attaaaaata cttcttctaa gggtgagtgt ttcattgctc ttgagggaac ttacgttgaa 2580
aactcttcta ccattttctc taatgtttca atagttaagg agtga 2625
<210> 11
<211> 874
<212> PRT
<213> 人工序列
<220>
<223> 自合成的全长编码区翻译
<400> 11
Met Leu Ala Arg Gln Gly Gly Ser Leu Arg Ala Ser Gln Cys Asn Ala
1 5 10 15
Gly Leu Ala Arg Arg Val Glu Val Gly Ala Leu Val Val Pro Arg Pro
20 25 30
Ile Ser Val Asn Asp Val Val Pro His Val Tyr Ser Ala Pro Leu Ser
35 40 45
Val Ala Arg Arg Ser Cys Ser Lys Ser Ser Ile Arg Ser Thr Arg Arg
50 55 60
Leu Gln Thr Thr Val Cys Ser Met Val Gln Lys Trp Met Gln Arg Met
65 70 75 80
Ile Ile Val Asp Asn Asn Lys Leu Asn Val Arg Ala Leu Pro Ser Phe
85 90 95
Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp
100 105 110
Ile Met Gly Met Ile Phe Lys Thr Asp Thr Gly Gly Ser Asn Leu Thr
115 120 125
Leu Asp Glu Ile Leu Lys Asn Gln Asn Leu Leu Asn Asp Ile Ser Gly
130 135 140
Lys Leu Asp Gly Ile Asn Gly Asp Leu Gly Asp Leu Ile Ala Gln Gly
145 150 155 160
Asn Leu Asn Ser Glu Leu Ala Lys Glu Leu Leu Lys Ile Ser Asn Glu
165 170 175
Gln Asn Gln Met Leu Asn His Val Asn Ala Gln Leu Asn Ala Ile Asn
180 185 190
Ser Thr Leu Asn Ile Tyr Leu Pro Lys Ile Thr Ser Met Leu Asn Glu
195 200 205
Val Met Lys Gln Asn His Val Leu Ser Leu Gln Ile Glu Phe Leu Ser
210 215 220
Lys Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Leu Asn
225 230 235 240
Val Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg
245 250 255
Ile Lys Tyr Val Asn Glu Lys Phe Asp Glu Leu Thr Ser Thr Val Glu
260 265 270
Lys Asn Pro Lys Ser Tyr Gln Asp Asn Val Thr Lys Glu Val Ile Glu
275 280 285
Asn Leu Asn Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp
290 295 300
Met Asp Ser Phe Glu Phe Tyr Leu Gln Thr Phe His Asp Val Met Thr
305 310 315 320
Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu
325 330 335
Ile Thr Lys Glu Asn Val Thr Thr Arg Gly Ser Glu Ile Gly Lys Val
340 345 350
Tyr Asn Phe Leu Ile Val Leu Thr Ser Leu Gln Ala Lys Ala Phe Leu
355 360 365
Thr Leu Thr Ala Cys Arg Lys Leu Leu Gly Leu Thr Asp Ile Asp Tyr
370 375 380
Thr Gln Ile Met Asn His His Ile Asp Gly Gln Lys Arg Glu Phe Arg
385 390 395 400
Ile Asn Ile Leu Pro Thr Leu Ser Asn Asn Phe Ser Asn Pro Ser Tyr
405 410 415
Ser Lys Asn Arg Gly Ser Asp Ile Asp Asp Pro Ile Val Val Leu Glu
420 425 430
Ala Ala Pro Gly Tyr Ala Leu Ile Gly Phe Glu Ile Leu Asn Asp Pro
435 440 445
Leu Pro Ile Leu Lys Gly Tyr Gln Ala Arg Leu Lys Pro Asn Tyr Gln
450 455 460
Val Asp Arg Glu Ser Met Ser Glu Thr Ile Tyr Gly Asp Ile His Lys
465 470 475 480
Leu Phe Cys Pro Lys Gln Leu Glu Gln Lys Tyr Tyr Ile Lys Asp Ile
485 490 495
Glu Phe Pro Glu Gly Tyr Val Ile Thr Lys Ile Val Phe Glu Lys Arg
500 505 510
Leu Asn Gln Leu Gly Tyr Glu Val Thr Ala Asn Phe Tyr Asp Pro Ser
515 520 525
Thr Gly Ser Ile Asp Leu Asn Lys Val Lys Val Glu Ser Trp Lys Glu
530 535 540
Lys Ser Cys Glu Glu Asp Ser Cys Glu Asp Glu Tyr Ser Ile Ile Lys
545 550 555 560
Ala Glu Thr Asp Gly Ile Tyr Met Pro Leu Gly Val Val Ser Glu Thr
565 570 575
Phe Leu Thr Pro Ile Tyr Gly Phe Gly Leu Thr Val Asp Glu Lys Asn
580 585 590
Gln Lys Ile Thr Leu Thr Gly Lys Ser Tyr Leu Arg Glu Ser Leu Leu
595 600 605
Glu Thr Asp Leu Leu Asn Asn Glu Thr Tyr Leu Ile Ala Ser Pro Asp
610 615 620
Gly Tyr Ile Ser Ser Ile Val Glu Asn Trp Asn Ile Thr Ser Asp Asn
625 630 635 640
Thr Gly Ser Trp Arg Ala Asn Asn Asn Asn Ala Phe Val Asp Lys Ala
645 650 655
Asp Thr Ile Lys Gly Ser Ser Ser Leu Tyr Thr His Lys Asp Gly Glu
660 665 670
Phe Ser Gln Phe Ile Gly Asn Lys Leu Lys Pro Lys Thr Asn Tyr Val
675 680 685
Ile Gln Tyr Val Ile Lys Gly Arg Pro Ala Ile Tyr Leu Lys Asn Asn
690 695 700
Lys Asp Thr Leu Phe Glu Asp Thr Lys Asn Asn Phe Ser Asp Phe Gln
705 710 715 720
Thr Val Thr Lys Lys Phe Asn Ser Gly Val Asn Pro Ser Glu Ile Tyr
725 730 735
Phe Leu Phe Lys Asn Gln Ser Glu Tyr Glu Ala Trp Gly Asn Asn Phe
740 745 750
Ile Ile Leu Glu Ile Lys Ser Leu Glu Phe Leu Pro Gln Met Leu Lys
755 760 765
Pro Glu Asp Trp Ile Pro Ser Gly Asn Val Gln Met Lys Asp Gly Gly
770 775 780
Arg Leu Glu Ile Leu Gly Asp Gly Tyr Phe Lys Gln Phe Ile Lys Leu
785 790 795 800
Glu Asn Asp Ser Thr Tyr His Leu Arg Leu Ser Val Lys Gly Thr Gly
805 810 815
Arg Val Ser Ile Ile Asp Glu Ser Lys Tyr Leu Leu Phe Val Asn Val
820 825 830
Lys Asp Glu Asp Leu Thr Arg Val Ile Lys Asn Thr Ser Ser Lys Gly
835 840 845
Glu Cys Phe Ile Ala Leu Glu Gly Thr Tyr Val Glu Asn Ser Ser Thr
850 855 860
Ile Phe Ser Asn Val Ser Ile Val Lys Glu
865 870
<210> 12
<211> 2013
<212> DNA
<213> 人工序列
<220>
<223> 合成的全长编码区
<400> 12
atgttagcta gacaaggtgg atctcttaga gcttctcaat gtaacgctgg acttgctaga 60
agagttgagg ttggtgctct tgttgttcct agaccaattt ctgttaacga cgttgttcca 120
cacgtctatt ctgctcctct ttctgttgct agacgttctt gctctaagtc ctctattaga 180
agtactagaa ggcttcaaac tactgtttgc tcaatgtctt accaagataa cgttactaag 240
gaagttattg aaaaccttaa tgagcttact gagcttgcta agtctgttac aaagaacgac 300
atggattcat tcgagttcta tcttcaaact ttccatgatg ttatgactgg gaataatctt 360
ttcgggaggt ctgcattaaa gactgcttct gaattaatca ctaaggaaaa cgtaactact 420
agaggttctg agattggaaa ggtgtataac tttcttattg ttcttacttc acttcaagct 480
aaggctttcc ttactcttac tgcttgtaga aagctccttg gtcttactga cattgattac 540
actcagatta tgaaccatca tattgatggt caaaagagag agtttaggat taatatcctt 600
ccaactcttt ctaataattt ctcaaatcca tcttactcta aaaatagagg ttctgatatt 660
gatgatccta ttgtagttct tgaagctgct cctggatacg ctcttattgg atttgagata 720
cttaacgatc cacttcctat tcttaagggt tatcaagcta gacttaaacc aaactatcaa 780
gttgatagag agtctatgtc agagactatc tacggagata ttcataaact cttttgccca 840
aagcaacttg agcaaaaata ttacattaaa gatattgaat ttcctgaggg atacgtcatt 900
actaagatag tttttgaaaa gagacttaat caacttggtt acgaggttac tgctaacttt 960
tacgatccat ctaccggatc tattgatctt aacaaggtta aggttgaatc ttggaaggaa 1020
aagtcttgcg aggaggattc ttgcgaagat gagtattcaa tcattaaggc tgaaacagat 1080
ggcatctata tgccacttgg agtggtttct gaaacttttc ttactcctat ctatggtttc 1140
ggacttactg ttgatgagaa gaatcaaaaa ataactttga ctgggaagtc ttatcttaga 1200
gaatctcttc ttgaaactga tcttcttaac aatgaaactt accttattgc ttctccagac 1260
ggttacattt cttctattgt tgagaactgg aatataactt ctgacaatac tggatcttgg 1320
agagctaata ataataatgc ttttgtggat aaggctgata ctataaaagg ttcttcatct 1380
ctttacactc ataaagatgg tgagttttct caattcattg gaaacaaact taagcctaag 1440
accaattacg ttattcaata tgttataaaa gggagacctg ctatctatct taaaaataat 1500
aaagatactc ttttcgagga taccaaaaat aacttttctg attttcaaac tgttactaag 1560
aaattcaatt ctggagttaa tccttctgaa atctacttcc ttttcaagaa tcaatctgaa 1620
tacgaggcat ggggaaacaa ctttatcatt ttggaaatta aatcacttga gttccttcca 1680
caaatgctta agcctgagga ttggattcct tctggaaacg ttcagatgaa agacggaggc 1740
agacttgaga ttcttggaga tggttacttt aagcaattca ttaaattgga gaatgattca 1800
acttaccatc tgagactttc tgttaaggga actggcagag tttctatcat tgatgaatct 1860
aagtatcttc tttttgtaaa tgttaaggat gaggatctga ctagagtcat taaaaatact 1920
tcttctaagg gtgagtgttt cattgctctt gagggaactt acgttgaaaa ctcttctacc 1980
attttctcta atgtttcaat agttaaggag tga 2013
<210> 13
<211> 670
<212> PRT
<213> 人工序列
<220>
<223> 自合成的全长编码区翻译
<400> 13
Met Leu Ala Arg Gln Gly Gly Ser Leu Arg Ala Ser Gln Cys Asn Ala
1 5 10 15
Gly Leu Ala Arg Arg Val Glu Val Gly Ala Leu Val Val Pro Arg Pro
20 25 30
Ile Ser Val Asn Asp Val Val Pro His Val Tyr Ser Ala Pro Leu Ser
35 40 45
Val Ala Arg Arg Ser Cys Ser Lys Ser Ser Ile Arg Ser Thr Arg Arg
50 55 60
Leu Gln Thr Thr Val Cys Ser Met Ser Tyr Gln Asp Asn Val Thr Lys
65 70 75 80
Glu Val Ile Glu Asn Leu Asn Glu Leu Thr Glu Leu Ala Lys Ser Val
85 90 95
Thr Lys Asn Asp Met Asp Ser Phe Glu Phe Tyr Leu Gln Thr Phe His
100 105 110
Asp Val Met Thr Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr
115 120 125
Ala Ser Glu Leu Ile Thr Lys Glu Asn Val Thr Thr Arg Gly Ser Glu
130 135 140
Ile Gly Lys Val Tyr Asn Phe Leu Ile Val Leu Thr Ser Leu Gln Ala
145 150 155 160
Lys Ala Phe Leu Thr Leu Thr Ala Cys Arg Lys Leu Leu Gly Leu Thr
165 170 175
Asp Ile Asp Tyr Thr Gln Ile Met Asn His His Ile Asp Gly Gln Lys
180 185 190
Arg Glu Phe Arg Ile Asn Ile Leu Pro Thr Leu Ser Asn Asn Phe Ser
195 200 205
Asn Pro Ser Tyr Ser Lys Asn Arg Gly Ser Asp Ile Asp Asp Pro Ile
210 215 220
Val Val Leu Glu Ala Ala Pro Gly Tyr Ala Leu Ile Gly Phe Glu Ile
225 230 235 240
Leu Asn Asp Pro Leu Pro Ile Leu Lys Gly Tyr Gln Ala Arg Leu Lys
245 250 255
Pro Asn Tyr Gln Val Asp Arg Glu Ser Met Ser Glu Thr Ile Tyr Gly
260 265 270
Asp Ile His Lys Leu Phe Cys Pro Lys Gln Leu Glu Gln Lys Tyr Tyr
275 280 285
Ile Lys Asp Ile Glu Phe Pro Glu Gly Tyr Val Ile Thr Lys Ile Val
290 295 300
Phe Glu Lys Arg Leu Asn Gln Leu Gly Tyr Glu Val Thr Ala Asn Phe
305 310 315 320
Tyr Asp Pro Ser Thr Gly Ser Ile Asp Leu Asn Lys Val Lys Val Glu
325 330 335
Ser Trp Lys Glu Lys Ser Cys Glu Glu Asp Ser Cys Glu Asp Glu Tyr
340 345 350
Ser Ile Ile Lys Ala Glu Thr Asp Gly Ile Tyr Met Pro Leu Gly Val
355 360 365
Val Ser Glu Thr Phe Leu Thr Pro Ile Tyr Gly Phe Gly Leu Thr Val
370 375 380
Asp Glu Lys Asn Gln Lys Ile Thr Leu Thr Gly Lys Ser Tyr Leu Arg
385 390 395 400
Glu Ser Leu Leu Glu Thr Asp Leu Leu Asn Asn Glu Thr Tyr Leu Ile
405 410 415
Ala Ser Pro Asp Gly Tyr Ile Ser Ser Ile Val Glu Asn Trp Asn Ile
420 425 430
Thr Ser Asp Asn Thr Gly Ser Trp Arg Ala Asn Asn Asn Asn Ala Phe
435 440 445
Val Asp Lys Ala Asp Thr Ile Lys Gly Ser Ser Ser Leu Tyr Thr His
450 455 460
Lys Asp Gly Glu Phe Ser Gln Phe Ile Gly Asn Lys Leu Lys Pro Lys
465 470 475 480
Thr Asn Tyr Val Ile Gln Tyr Val Ile Lys Gly Arg Pro Ala Ile Tyr
485 490 495
Leu Lys Asn Asn Lys Asp Thr Leu Phe Glu Asp Thr Lys Asn Asn Phe
500 505 510
Ser Asp Phe Gln Thr Val Thr Lys Lys Phe Asn Ser Gly Val Asn Pro
515 520 525
Ser Glu Ile Tyr Phe Leu Phe Lys Asn Gln Ser Glu Tyr Glu Ala Trp
530 535 540
Gly Asn Asn Phe Ile Ile Leu Glu Ile Lys Ser Leu Glu Phe Leu Pro
545 550 555 560
Gln Met Leu Lys Pro Glu Asp Trp Ile Pro Ser Gly Asn Val Gln Met
565 570 575
Lys Asp Gly Gly Arg Leu Glu Ile Leu Gly Asp Gly Tyr Phe Lys Gln
580 585 590
Phe Ile Lys Leu Glu Asn Asp Ser Thr Tyr His Leu Arg Leu Ser Val
595 600 605
Lys Gly Thr Gly Arg Val Ser Ile Ile Asp Glu Ser Lys Tyr Leu Leu
610 615 620
Phe Val Asn Val Lys Asp Glu Asp Leu Thr Arg Val Ile Lys Asn Thr
625 630 635 640
Ser Ser Lys Gly Glu Cys Phe Ile Ala Leu Glu Gly Thr Tyr Val Glu
645 650 655
Asn Ser Ser Thr Ile Phe Ser Asn Val Ser Ile Val Lys Glu
660 665 670
Claims (5)
1.一种核酸构建体,其包含选自下组的核酸:SEQ ID NO:1,SEQ ID NO:3,SEQ ID NO:4,SEQ ID NO:5,SEQ ID NO:6,SEQ ID NO:8,SEQ ID NO:9,SEQ ID NO:10和SEQ ID NO:12。
2.一种分离的杀虫蛋白,其选自下组:SEQ ID NO:2,SEQ ID NO:7,SEQ ID NO:11和SEQID NO:13。
3.一种控制易感性昆虫的方法,其包括使所述昆虫与有效量的权利要求2的蛋白质接触。
4.权利要求3的方法,其中所述易感性昆虫选自下组:欧洲玉米螟,大豆尺蠖,黧豆毛虫,烟草芽虫,棉铃虫,玉米穗虫和菱纹背蛾。
5.权利要求4的方法,其中所述易感性昆虫为Cry1F抗性欧洲玉米螟。
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US10316329B2 (en) | 2014-10-16 | 2019-06-11 | Monsanto Technology Llc | Proteins toxic or inhibitory to lepidopteran insects |
CA2996295A1 (en) | 2015-08-27 | 2017-03-02 | Monsanto Technology Llc | Novel insect inhibitory proteins |
MX2020007329A (es) * | 2017-09-26 | 2020-11-24 | Corteva Agriscience Llc | Proteínas insecticidas quiméricas. |
WO2022125639A1 (en) | 2020-12-08 | 2022-06-16 | Monsanto Technology Llc | Modified plant-associated bacteria and methods of their use |
MX2023007427A (es) | 2020-12-21 | 2023-07-03 | Monsanto Technology Llc | Proteinas inhibidoras de insectos novedosas. |
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