CN101492490A - 改良Cry3的方法、改良Cry3、质粒及其应用 - Google Patents
改良Cry3的方法、改良Cry3、质粒及其应用 Download PDFInfo
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Abstract
本发明提供了一种增强Cry3(crystalline protein 3)对玉米根虫的杀虫能力的方法,所述方法为:对Cry3的N端第1~50位氨基酸序列进行结构改良,所述结构改良为删除和/或插入至少一个多肽片段,所述多肽片段长度为1~50个氨基酸,所述Cry3为Cry3A或Cry3B。本发明的有益效果主要体现在:提供了一种增强Cry3(crystalline protein 3)对玉米根虫的杀虫能力的方法,利用这种方法获得的改良Cry3蛋白的转基因抗虫玉米,对玉米根虫有较好的杀虫能力。
Description
(一)技术领域
本发明涉及一种增强苏云金芽孢杆菌杀虫蛋白质Cry3对玉米根虫(Diabrotica virgifera virgifera)的杀虫能力的方法,以及利用这种方法获得的改良Cry3、所用质粒,及其在制备转基因抗虫玉米中的应用。
(二)背景技术
玉米根虫是一种最具有破坏性的玉米害虫。在美国,3种重要种分别为:西方玉米根虫(D.virgifera virgifera)、北方玉米根虫(D.undecimpunctatahowardi)和南方玉米根虫(D.undecimpunctata howardi)。西方玉米根虫和北方玉米根虫是美国玉米栽培中的首要害虫。玉米根虫通过取食植物根部引起植物严重损伤。这种损伤表现在增加玉米倒伏,减少玉米产量和生物量而改变植物的营养含量。玉米根虫的取食还间接影响植物病原生物的侵入,幼虫对根的损伤,使植物病原细菌、真菌等微生物可以更容易侵入到植物中。玉米根虫成虫在夏末玉米田中非常活跃,它们取食叶耳,叶丝和花粉,干扰了玉米的正常授粉。
苏云金芽孢杆菌(Bacillus thuringiensis)最早于1902年在日本国发现,1911年在德国再次被发现并得鉴定,是一种格兰氏阳性细菌。在它的孢子形成过程中,合成出大量的杀虫结晶包涵体(insecticidal crystallineinclusions),其中的晶体蛋白质(crystalline protein,Cry)是由叫做σ-内毒素(σ-endotoxins)的原毒素亚基(protoxin subunit)组成的。大多数的苏云金芽孢杆菌菌株都会产生一系列的σ-内毒素,它对许多昆虫都具有特异的毒性,故这种蛋白质又叫做杀虫晶体蛋白(insecticidal crystal protein,ICP),或苏云会芽孢杆菌毒蛋白(Bt toxin)。此外,苏云金芽孢杆菌在生长及芽孢形成期间,还能分泌一些外毒素,叫做苏云金毒素或β-外毒素(β-exotoxin),同样也具有抑制幼虫生长的能力
苏云金芽孢杆菌(Bacillus thuringiensis,Bt)δ-内毒素Cry3有杀甲虫的活性。Cry3A蛋白对科罗拉多马铃薯甲虫(Leptinotarsa decemlineata)有很好的杀虫活性,但对Diabrotica属的甲虫没有杀虫活性(Johnson et al.,1993,J.Econ.Entomol.86:330 333)。根据Slaney等所述(1992,InsectBiochem.Molec.Biol.22:918),Cry3A蛋白对科罗拉多马铃薯甲虫的杀虫活性是南方玉米根虫(D.undecimpunctata howardi)的2000倍。而且,Cry3A蛋白对西方玉米根虫有很小的或者没有杀虫活性。同样,Cry3B对西方玉米根虫有比较低的杀虫能力,但是杀虫活性仍然不够理想。
由于δ-内毒素杀虫机理的了解,改造δ-内毒素使其更有杀虫活性成为了可能。Li等人1991年确定Cry3A的三维结构时,改造δ-内毒素更成就为一种可能。Cry3A蛋白具有3个结构域:N-端的1-290个氨基酸是第一个结构域,由7个α螺旋组成;第二个结构域从291-500个氨基酸,包含3个β折叠;C-端的501-644个氨基酸是第三个结构域,是一个β-折叠。基于这种结构,提出了一个关于δ-内毒素结构和功能关系的一个假说。这个假说一般来说是结构域I是在昆虫肠膜形成穿孔的主要因素(Gazit and Shai,1993,Appl.Environ.Microbiol.57:28162820),结构域II是和肠膜受体结合的主要部分(Gazit and Shai,1993,Appl.Environ.Microbiol.57:28162820),而结构域III有调节离子通道活性的功能(Chen et al.,1993,PNAS 90:90419045),最有可能和蛋白的稳定性有关(Li et al.1991,supra)。
Van Rie等人1997年通过随机替换一个Cry3A蛋白质中在溶解性方面有重要作用的氨基酸(接近结构域II的丙胺酸)来改造Cry3A。这几个随机替换中有几个限制了结构域II对受体的结合,增加了西方玉米根虫的活性。然而,其他研究显示在Cry3A结构域II中有的丙胺酸的替换破坏了受体结合能力,引起结构的不稳定(Wu and Dean,1996,J.Mol.Biol.255:628640)。English等人1999年报道Cry3Bb中的氨基酸替代引起了其对南方玉米根虫和西方的杀虫活性的增加(Intl.Pat.Appl.Publ.No.WO99/31248)。然而,在35个Cry3Bb的突变中,只有3个主要在结构域II以及结构域II和I的交界处的突变可以对西方玉米根虫有杀虫活性。而且,在对西方玉米根虫相同生物测定中野生型的Cry3Bb毒性的区别远大于突变Cry3Bb和野生型Cry3Bb的区别。因此,Cry3Bb突变体毒性的提高仅限于南方玉米根虫。
Cry3A的另外一种改良是在Cry3A的氨基酸107~117插入一个Cathpsin-G的位点而增加了对西方玉米根虫的杀虫能力(美国专利:USPat.7030295;US Pat.7230167)。改造的Cry3A毒素可以形成一个蛋白酶G的识别位点,据说改造后位于107-115个氨基酸之间的蛋白酶Cathpsin-G的识别位点可以改变Cry3A毒素的杀虫能力。这样改造过的Cry3A对西方玉米根虫(D.virgifera virgifera)的杀虫能力比原Cry3A更高。
但是目前还没有一种对西方玉米根虫(D.virgifera virgifera)有理想杀虫活性的δ-内毒素(包括Cry3A,Cry3B,Cry3C,Cry7A,Cry8A,Cry8B,and Cry8C)可以用来发展优秀的转基因玉米来控制玉米根虫对作物的危害。
(三)发明内容
本发明针对控制玉米根虫的需要,提供了一种增强苏云金芽孢杆菌杀虫蛋白质Cry3对玉米根虫的杀虫能力的方法,以及利用这种方法获得的改良Cry3、所用质粒,及其在制备转基因抗虫玉米中的应用。
本发明采用的技术方案是:
一种增强苏云金芽孢杆菌杀虫蛋白质Cry3对玉米根虫的杀虫能力的方法,所述方法为:对Cry3的N端第1~40位氨基酸序列进行改变,所述结构改变为删除和/或插入至少一个多肽片段,所述多肽片段长度为1~40个氨基酸,所述Cry3为Cry3A或Cry3B。经过改变的Cry3可以通过昆虫生物测定来确定和选择增强了对玉米根虫的杀虫能力的改良Cry3。Cry3是一种苏云金芽孢杆菌晶体杀虫蛋白质,其在Bt中有二种不同的分子形式。一种是比较长的分子;另一种是同样有活性的比较短的分子,例如本发明涉及的599个氨基酸残基的Cry3A(SEQ ID No:1)。本发明要点在于通过将Cry3的N端第1~40位氨基酸序列改变,从而增强了其对玉米根虫的杀虫活性。
Bt的晶体杀虫蛋白质的N-端氨基酸序列对杀虫活性有重要影响。删除和插入一个多肽可能可以改变杀虫活性,特别是改变杀虫谱。特别是为了达到获得或者增强了对玉米根甲虫(D.virgifera virgifera)的杀虫能力,Cry3的N-端的1~32氨基酸序列可以被全部或者部分删除,也可以在氨基酸30~33之间插入一个氨基酸或者多肽。通过生物测定,可以选择获得或者增强了对玉米根甲虫(D.virgifera virgifera)杀虫能力的改良Cry3杀虫蛋白质。一个蛋白质在很多情况下增加或者减少N-端一个或者几个氨基酸通常对蛋白质的功能没有大的改变是已经知道的知识。因此,Cry3杀虫蛋白质在N-端删除的多肽序列的长度可能是可变的,它可以是32个氨基酸残基,更长或者更短。同样,Cry3A杀虫蛋白质在N-端的插入的位置和序列也可能是可变的。
Cry3基因目前有3个种类,分别是Cry3A(M22472:Herrnstadt et al.,1987;J02978:Sekar et al.,1987),Cry3B(X17123“Sick et al.,1990;M89794:Donovan et al.,1992),和Cry3C(X59797:Lambert et al.,1992)。它们的氨基酸序列的相同性相当高,例如Cry3Aa1和Cry3Bb1的氨基酸序列相同性是68%,并且它们的杀虫能力也相似。Cry3B同样可以通过N-端删除一个多肽而获得或者增强对玉米根甲虫(D.virgifera virgifera)的杀虫能力。例如在Cry3B的N-端删除32个氨基酸多肽(SEQ ID NO:4)。Cry3B同样可以通过N-端插入的氨基酸而获得的改良了的Cry3B(SEQID NO:5).
具体的,所述方法可为:在Cry3的N端第30~33位氨基酸中插入至少一个多肽片段。为了获得改良的Cry3杀虫蛋白质而需要在N-端插入氨基酸的序列和长度可能是可变的。这些氨基酸的序列的插入有助其N-端氨基酸多肽在目标昆虫中肠中被昆虫中肠蛋白酶切除。
所述方法为:删除Cry3的N端第1~32位氨基酸中至少一个多肽片段。在N端删除的多肽序列的长度是可变的,可以是34个氨基酸残基,也可以更短,也可删除不连续的多个多肽片段。
优选的所述方法为:将Cry3的N端第32~33位氨基酸Val-Val替换为Gly-Pro-Gly-Lys。
本发明还涉及利用本发明方法获得的一种改良Cry3。
优选的,所述改良Cry3为Cry3A,具有SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列,这种改良Cry3A对西方玉米根虫和北方玉米根虫的杀虫能力要远远高于天然Cry3A。
优选的,所述改良Cry3为Cry3B,具有SEQ ID NO.4或SEQ ID NO.5所示的氨基酸序列,这种改良Cry3B对南方玉米根虫的杀虫能力要远远高于天然Cry3B。
上述改良的Cry3蛋白质,可用于控制害虫。
本发明还涉及含有编码SEQ ID NO.2或SEQ ID NO.3所示的Cry3A蛋白质的核苷酸序列的质粒。以及含有编码SEQ ID NO.4或SEQ ID NO.5所示的Cry3B蛋白质的核苷酸序列的质粒。
本发明方法可应用于制备转基因抗虫玉米。具体的,所述应用为:将前述含有编码改良Cry3的核苷酸的质粒转化玉米细胞,获得转基因抗虫玉米。
具体的,所述转基因抗虫玉米的获得包括下列步骤:1)获得编码改良Cry3蛋白的核苷酸序列(优选为SEQ ID NO:7或SEQ ID NO:8的序列);2)将这个核苷酸序列和一个启动子和一个终止子功能性地连接而形成一个能够在植物中表达的人工基因;启动子可以选择玉米泛素(Ubiqutin-1)启动子(Christensen and Quail,Transgenic Res.,1996,5:213-8),水稻actin启动子,或者是玉米根特异性表达启动子等;3)这个基因被克隆到转化载体,转化玉米细胞并且进一步获得转基因玉米。转基因的方法为基因枪方法、农杆菌介导方法,花粉管导入方法,或者其他方法。这些方法是已有的技术(Hiei et al.(1994)The Plant Journal 6:271-282;Ishidaet al.(1996)Nature Biotechnology 14:745-750;Ayres and Park(1994)Critical Reviews in Plant Science 13:219-239;Bommineni and Jauhar(1997)Maydica 42:107-120)。
本发明的有益效果主要体现在:提供了一种通过对Cry3的N端第1~40位氨基酸序列改变,增强Cry3(crystalline protein 3)对玉米根虫的杀虫能力的方法,利用这种方法获得的改良Cry3蛋白导入玉米细胞获得的转基因抗虫玉米,对玉米根虫有较好的杀虫能力。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
本发明实施例所使用分子生物学的方法均为已知的技术。在Ausubel编写的John Wiley and Sons公司出版的Current Protocols in MolecularBiology,和J.Sambrook等编写Cold Spring Harbor Laboratory Press(2001)出版的Molecular Cloning:A Labortory Manual,3rd ED.等文献均有详细的说明。
实施例1:构建改造的cry3A基因
经过玉米密码子优化的Cry3A改良基因cry3A-32GPGK(SEQ ID NO:3)是设计后由上海生工(上海)合成。天然Cry3A蛋白质的氨基酸序列和Cry3A-32GPGK的差别在氨基酸32-36之间,天然Cry3A是Val Val(32-33)而Cry3A-32GPGK是Gly Pro Gly Lys(32-36)。改良基因cry3A-32GPGK是克隆在pUC57载体中BamH1和Sac1位点之间,质粒命名为pUC57-cry3A-32GPGK。玉米密码子优化的Cry3A天然基因和N-端删除的基因是利用cry3A-32GPGK作为模板通过PCR而获得的。
pET28a载体的改造:pET28a中表达蛋白质时N-端的His和T7多肽标记的删除。
从上海生工(上海)合成以下引物:
Del-His-T7-F:5’CTAGAAATAATTTTGTTTAACTTTAAGAAGGAG
Del-His-T7-R:5’GATCCTCCTTCTTAAAGTTAAACAAAATTATTT
将这2个引物配制成10uM的浓度并等量混合,72℃反应10分钟,作为Linker,命名为Del-His-T7-Linker。使用Xba1和BamH1对pET28a载体进行酶切回收,然后和Del-His-T7-Linker做连接,并转化入克隆菌株TG1中,酶切和测序鉴定,获得了删除了表达蛋白质时N-端的His和T7多肽标记的pET28a改造载体,命名为pET28a-Del-His-T7。
1、Cry3A改良基因(cry3A-32GPGK)表达载体的构建
使用BamH1和Sac1这2种酶分别酶切pUC57-cry3A-32GPGK和pET28a-Del-His-T7载体,凝胶回收,获得1.8kb的cry3A-32GPGK和5.3kb的pET28a-Del-His-T7载体,并将2个片段连接,转入克隆菌株TG1中,酶切和测序鉴定获得pET28a-del-His-T7-cry3A-32GPGK,并将其转入表达菌株BL21star(InVitrogen)。
2.天然Cry3A基因(cry3A-syn)基因的构建
天然Cry3A基因cry3A-syn基因是从cry3A-32GPGK通过PCR获得的。从上海生工(上海)合成以下引物:
cry3-VVG-F:
5’-CCTCGGCGTGGTGGGCTTCCCGTTC GGCGGCGCCCTCGT.cry3-VVG-R:
5’-GGAAGCCCACCACGCCGAGGAGGTCGCCCACCACGG
以pUC57-cry3A-32GPGK为模板分别用cry3-VVG-F和通用引物M13(-20)5’GTAAAACGACGGCCAGT,以及cry3-VVG-R和通用引物,M13(-26)5’CAGGAAACAGCTATGAC,进行PCR扩增反应。然后混合以上述2个PCR反应的产物为模板,利用通用引物M13(-20)和M13(-26)为引物进行PCR扩增反应,得到一条1.8kb的PCR产物条带。将这个1.8kb的PCR产物条带进行克隆,获得含有质粒pMD20-cry3A-syn的TG1菌株。酶切和测序鉴定说明获得的质粒pMD20-cry3A-syn是正确的。用BamH1和Sac1这2种酶分别酶切pMD20-cry3A-syn和pET28a-Del-His-T7载体,凝胶回收,获得1.8kb的cry3A-syn和5.3kb的pET28a-Del-His-T7载体,并将2个片段连接,转入克隆菌株TG1中,经过酶切和测序鉴定获得pET28a-Del-His-T7-cry3A-syn,并将此质粒转入表达菌株BL21star中。
3.Cry3A改良基因cry3A-del34基因的构建
从上海生工(上海)合成以下引物:
cry3-del34:
5’TGAGGATCCATGGGCTTCCCGTTCGGCGGCGCCCTCGT.
以pUC57-cry3A-32GPGK为模板使用cry3-del34和通用引物M13(-20)进行PCR扩增反应,得到一条1.7kb的PCR产物条带。将这个1.7kb的PCR产物条带进行克隆。经过酶切和测序鉴定后,获得含有质粒pMD20-cry3A-del34的TG1菌株。使用BamH1和Sac1同时酶切pMD20-cry3A-del34和pET28a-Del-His-T7载体,凝胶回收1.7kb的cry3A-del34和5.3kb的pET28a-Del-His-T7载体,并将这2个片段连接,转入克隆菌株TG1中,经过酶切和测序鉴定获得pET28a-Del-His-T7-cry3A-del34,并将质粒转入表达菌株BL21star中。
实施例2:cry3A及其改良基因在E.coli中的表达
用含有pET28a-Del-His-T7-cry3A-32GPGK,pET28a-Del-His-T7-cry3A-syn以及pET28a-Del-His-T7-cry3A-del34质粒的E.coli表达菌株BL21star分别在LB培养基平版(含20ng/mL卡那霉素)上以划单菌落的方式涂板,并进行以下步骤诱导表达:
(1)使用无菌牙签在平板上挑取2~3mm直径的单菌落,接种至1000ml锥形瓶中的200ml液体LB培养基中(含20ng/mL卡那霉素);
(2)将锥形瓶于37℃恒温摇床中,以250转/分的速度培养5-8小时,直至菌液的OD600值达到0.6;
(3)在锥形瓶中加入终浓度为1mM的IPTG,诱导表达3~5小时;
(4)诱导完毕后,将菌液从锥形瓶移至3个100ml的无菌离心管中,以4℃,10000rmp的程序离心3分钟,去除上清;
(5)在每个离心管中加入70ml的10mM PBS,重悬细胞,并以4℃,10,000rmp的程序离心3分钟,去除上清,清洗残留LB;
(6)在每个离心管中加入10ml的10mM PBS,重悬细胞,以250W的功率,破碎2秒,间隔时间8秒,超声破碎时间5分钟的程序超声波破碎细胞。
(7)将离心管以4℃,10000rmp的程序离心10分钟,去除上清,收集包涵体;
在每个离心管中各加入2ml的10mM PBS,重悬包涵体,于-80℃保存,供杀虫活性测定用。
实施例3:cry3A及其改良基因的生物测定
cry3A改良毒素对西方玉米根虫和北方玉米根虫的杀虫活性进行了昆虫生物测定。在生物测定中在人工饲料添加本发明在大肠杆菌表达的cry3A改良毒素,然后对昆虫饲喂。500ml的表达培样品和500ml的熔化饲料相混合(Marrone et al.(1985)J.of Economic Entomology 78:290293)。熔化饲料被分装到培养板小孔中,其凝固后在培养板每个小孔的饲料上放入20头玉米根虫幼虫。培养板置于30℃环境中,6天后记录昆虫的死亡率。cry3A改良毒素(Cry3A-32GPGK和Cry3A-del34)可以引起西方玉米根虫和北方玉米根虫70%~100%的死亡率,而野生型Cry3A(cry3A-syn)只有0%-20%的死亡率。
实施例4:T-DNA转化质粒的构建
1、Cry3A改良基因cry3A-32GPGK的T-DNA转化质粒构建
利用PCR的方法从玉米中扩增获得玉米泛素(ZmUbi-1)启动子。引物分别是ZmUbiF(5’-GCGAAGCTTGCATGCCTACAGTGCAGCGTGACCCGGTCGTGC(在5’设计有Hind3酶切位点)ZmUbiR(5’-GTGGGATCCTCTAGAGTCGACCTGCAGAAGTAACACCAAACAACAG,5’设计有BamH1酶切位点)。
利用BamH1和Sac1酶切pUC57-cry3A-32GPGK、Hind3和Sac1酶切pCAMBIAL1300载体,以及Hind3和BamH1酶切ZmUbi-1启动子,凝胶回收,获得1.8kb的cry3A-32GPGK、8.9kb的pCAMBIAL1300载体以及1.9kb的ZmUbi-1启动子,并将3个片段连接,转入克隆菌株TG1中,酶切和测序鉴定获得含有质粒pCAMBIAL1300-Ubi-cry3A-32GPGK的菌株。
2、Cry3A改良基因cry3A-del34的T-DNA转化质粒构建
用BamH1和Sac1酶切pET28a-Del-His-T7-cry3A-del34、Hind3和Sac1酶切pCAMBIAL1300载体以及使用Hind3和BamH1酶切上述ZmUbi-1启动子,凝胶回收1.7kb的cry3A-del34、8.9kb的pCAMBIAL1300载体以及1.9kb的ZmUbi-1启动子,并将3个片段连接,转入克隆菌株TG1中,酶切和测序鉴定获得含有质粒pCAMBIAL1300-Ubi-cry3A-del34的菌株。
实施例5:转基因抗虫玉米的获得
将pCAMBIAL1300-Ubi-cry3A-32GPGK 和pCAMBIAL 1300-Ubi-cry3A-del34用电击方法导入Agrobacteriumtumefaciens(LAB4404)。
取授粉后8~10天的Hi-2玉米穗。收集所有的未成熟胚(大小为1.0~1.5mm)。将含有T-DNA载体的农杆菌与未成熟胚共培育共培养2~3天(22℃)。转移未成熟胚到愈伤诱导培养基上(含200mg/L的Timentin杀农杆菌),28℃暗培养10-14天。将所有的愈伤转到带有50ng/mL潮霉素的筛选培养基上,28℃暗培养2~3周。
转移所有的组织到新鲜潮霉素的筛选培养基上,28℃暗培养2~3周。然后,转移所有筛选后成活的胚性组织到再生培养基上,28℃暗培养10~14天,每皿一个株系。转移胚性组织到新鲜的再生培养基上,26℃光照培养10~14天。转移所有发育完全的植株到生根培养基上,26℃光照培养直到根发育完全。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
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Met Thr Ala Asp Asn Asn Thr Glu Ala Leu Asp Ser Ser Thr Thr Lys
1 5 10 15
Asp Val Ile Gln Lys Gly Ile Ser Val Val Gly Asp Leu Leu Gly Gly
20 25 30
Pro Gly Lys Gly Phe Pro Phe Gly Gly Ala Leu Val Ser Phe Tyr Thr
35 40 45
Asn Phe Leu Asn Thr Ile Trp Pro Ser Glu Asp Pro Trp Lys Ala Phe
50 55 60
Met Glu Gln Val Glu Ala Leu Met Asp Gln Lys Ile Ala Asp Tyr Ala
65 70 75 80
Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Val Glu
85 90 95
Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys Asn Pro Val Ser Ser
100 105 110
Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala
115 120 125
Glu Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Ile Ser Gly Tyr
130 135 140
Glu Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala Ala Asn Thr His Leu
145 150 155 160
Phe Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu Glu Trp Gly Tyr Glu
165 170 175
Lys Glu Asp Ile Ala Glu Phe Tyr Lys Arg Gln Leu Lys Leu Thr Gln
180 185 190
Glu Tyr Thr Asp His Cys Val Lys Trp Tyr Asn Val Gly Leu Asp Lys
195 200 205
Leu Arg Gly Ser Ser Tyr Glu Ser Trp Val Asn Phe Asn Arg Tyr Arg
210 215 220
Arg Glu Met Thr Leu Thr Val Leu Asp Leu Ile Ala Leu Phe Pro Leu
225 230 235 240
Tyr Asp Val Arg Leu Tyr Pro Lys Glu Val Lys Thr Glu Leu Thr Arg
245 250 255
Asp Val Leu Thr Asp Pro Ile Val Gly Val Asn Asn Leu Arg Gly Tyr
260 265 270
Gly Thr Thr Phe Ser Asn Ile Glu Asn Tyr Ile Arg Lys Pro His Leu
275 280 285
Phe Asp Tyr Leu His Arg Ile Gln Phe His Thr Arg Phe Gln Pro Gly
290 295 300
Tyr Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Ser
305 310 315 320
Thr Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile Thr Ser Pro Phe Tyr
325 330 335
Gly Asn Lys Ser Ser Glu Pro Val Gln Asn Leu Glu Phe Asn Gly Glu
340 345 350
Lys Val Tyr Arg Ala Val Ala Asn Thr Asn Leu Ala Val Trp Pro Ser
355 360 365
Ala Val Tyr Ser Gly Val Thr Lys Val Glu Phe Ser Gln Tyr Asn Asp
370 375 380
Gln Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Val
385 390 395 400
Gly Ala Val Ser Trp Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr
405 410 415
Asp Glu Pro Leu Glu Lys Gly Tyr Ser His Gln Leu Asn Tyr Val Met
420 425 430
Cys Phe Leu Met Gln Gly Ser Arg Gly Thr Ile Pro Val Leu Thr Trp
435 440 445
Thr His Lys Ser Val Asp Phe Phe Asn Met Ile Asp Ser Lys Lys Ile
450 455 460
Thr Gln Leu Pro Leu Val Lys Ala Tyr Lys Leu Gln Ser Gly Ala Ser
465 470 475 480
Val Val Ala Gly Pro Arg Phe Thr Gly Gly Asp Ile Ile Gln Cys Thr
485 490 495
Glu Asn Gly Ser Ala Ala Thr Ile Tyr Val Thr Pro Asp Val Ser Tyr
500 505 510
Ser Gln Lys Tyr Arg Ala Arg Ile His Tyr Ala Ser Thr Ser Gln Ile
515 520 525
Thr Phe Thr Leu Ser Leu Asp Gly Ala Pro Phe Asn Gln Tyr Tyr Phe
530 535 540
Asp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr Tyr Asn Ser Phe Asn
545 550 555 560
Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser Gly Asn Asn Leu Gln
565 570 575
Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys Val Tyr Ile Asp Lys
580 585 590
Ile Glu Phe Ile Pro Val Asn
595
<210>4
<211>569
<212>PRT
<213>Unknown
<220>
<223>人工合成
<400>4
Met Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu Asn Ala
1 5 10 15
Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala Gln Val
20 25 30
Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys Ala
35 40 45
Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val Asn
50 55 60
Ala Leu Asp Ser Trp Lys Lys Ala Pro Val Asn Leu Arg Ser Arg Arg
65 70 75 80
Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe
85 90 95
Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val Leu Phe
100 105 110
Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu Lys
115 120 125
Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp Ile
130 135 140
Ala Glu Phe Tyr Gln Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr Asp
145 150 155 160
His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Ser Leu Arg Gly Ser
165 170 175
Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met Thr
180 185 190
Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp Val Arg
195 200 205
Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile Phe Thr
210 215 220
Asp Pro Ile Phe Thr Leu Asn Ala Leu Gln Glu Tyr Gly Pro Thr Phe
225 230 235 240
Ser Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp Tyr Leu
245 250 255
Arg Gly Ile Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Ser Gly Lys
260 265 270
Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro Ser
275 280 285
Ile Gly Ser Asn Asp Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys Ser
290 295 300
Ile Glu Pro Ile Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr Arg
305 310 315 320
Thr Ile Ala Asn Thr Asp Ile Ala Ala Phe Pro Asp Gly Lys Ile Tyr
325 330 335
Phe Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys Asn
340 345 350
Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Tyr Asn Gly Tyr Leu
355 360 365
Gly Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu
370 375 380
Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys Phe
385 390 395 400
Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr His
405 410 415
Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr Gln
420 425 430
Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile Ile
435 440 445
Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu Ser
450 455 460
Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala Ala Leu
465 470 475 480
Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu
485 490 495
Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr Ile
500 505 510
Asn Lys Thr Met Asn Ile Asp Gly Asp Leu Thr Tyr Gln Thr Phe Asp
515 520 525
Phe Ala Thr Ser Asn Ser Asn Met Gly Phe Ser Gly Asp Thr Asn Asp
530 535 540
Phe Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr Ile
545 550 555 560
Asp Lys Ile Glu Phe Ile Pro Val Gln
565
<210>5
<211>602
<212>PRT
<213>Unknown
<220>
<223>人工合成
<400>5
Met Thr Ala Asp Asn Ser Thr Glu Val Leu Asp Ser Ser Thr Val Lys
1 5 l0 15
Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Gly
20 25 30
Lys Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu Asn
35 40 45
Ala Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala Gln
50 55 60
Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys
65 70 75 80
Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val
85 90 95
Asn Ala Leu Asp Ser Trp Lys Lys Ala Pro Val Asn Leu Arg Ser Arg
100 105 110
Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser His
115 120 125
Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val Leu
130 135 140
Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu Leu
145 150 155 160
Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp
165 170 175
Ile Ala Glu Phe Tyr Gln Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr
180 185 190
Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Ser Leu Arg Gly
195 200 205
Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg Glu Met
210 215 220
Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp Val
225 230 235 240
Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile Phe
245 250 255
Thr Asp Pro Ile Phe Thr Leu Asn Ala Leu Gln Glu Tyr Gly Pro Thr
260 265 270
Phe Ser Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp Tyr
275 280 285
Leu Arg Gly Ile Glu Phe His Thr Arg Leu Arg Pro Gly Tyr Ser Gly
290 295 300
Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro
305 310 315 320
Ser Ile Gly Ser Asn Asp Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys
325 330 335
Ser Ile Glu Pro Ile Gln Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr
340 345 350
Arg Thr Ile Ala Asn Thr Asp Ile Ala Ala Phe Pro Asp Gly Lys Ile
355 360 365
Tyr Phe Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys
370 375 380
Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Tyr Asn Gly Tyr
385 390 395 400
Leu Gly Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp
405 410 415
Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys
420 425 430
Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr
435 440 445
His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr
450 455 460
Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile
465 470 475 480
Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu
485 490 495
Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala Ala
500 505 510
Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn
515 520 525
Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr
530 535 540
Ile Asn Lys Thr Met Asn Ile Asp Gly Asp Leu Thr Tyr Gln Thr Phe
545 550 555 560
Asp Phe Ala Thr Ser Asn Ser Asn Met Gly Phe Ser Gly Asp Thr Asn
565 570 575
Asp Phe Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile Tyr
580 585 590
Ile Asp Lys Ile Glu Phe Ile Pro Val Gln
595 600
<210>6
<211>1800
<212>DNA
<213>Bacillus thuringiensis
<400>6
atgaccgccg acaacaacac cgaggccctc gactcctcca ccaccaagga cgtgatccag 60
aagggcatct ccgtggtggg cgacctcctc ggcgtggtgg gcttcccgtt cggcggcgcc 120
ctcgtgtcct tctacaccaa cttcctcaac accatctggc cgtccgagga cccgtggaag 180
gccttcatgg agcaggtgga ggccctcatg gaccagaaga tcgccgacta cgccaagaac 240
aaggccctcg ccgagcttca gggcctccag aacaacgtgg aggactacgt gtccgccctc 300
tcctcctggc agaagaaccc ggtgtcctcc cgcaacccgc actcccaggg ccgcatccgc 360
gagctgttct cccaggccga gtcccacttc cgcaactcca tgccgtcctt cgccatctcc 420
ggctacgagg tgctcttcct caccacctac gcccaggccg ccaacaccca cctcttcctc 480
ctcaaggacg cccaaatcta cggcgaggag tggggctacg agaaggagga catcgccgag 540
ttctacaagc gccagctcaa gctcacccag gagtacaccg accactgcgt gaagtggtac 600
aacgtgggcc tcgacaagct ccgcggctcc tcctacgagt cctgggtgaa cttcaaccgc 660
taccgccgcg agatgaccct caccgtgctc gacctcatcg ccctcttccc gctctacgac 720
gtgcgcctct acccgaagga ggtgaagacc gaactcaccc gcgacgtgct caccgacccg 780
atcgtgggcg tgaacaacct ccgcggctac ggcaccacct tctccaacat cgagaactac 840
atccgcaagc cgcacctctt cgactacctc caccgcatcc agttccacac ccgcttccag 900
ccgggctact acggcaacga ctccttcaac tactggtccg gcaactacgt gtccacccgc 960
ccgtccatcg gctccaacga catcatcacc tccccgttct acggcaacaa gtcctccgag 1020
ccggtgcaga acctcgagtt caacggcgag aaggtgtacc gcgccgtggc caacaccaac 1080
ctcgccgtgt ggccgtccgc cgtgtactcc ggcgtgacca aggtggagtt ctcccagtac 1140
aacgaccaga ccgacgaggc ctccacccag acctacgact ccaagcgcaa cgtgggcgcc 1200
gtgtcctggg actccatcga ccagctcccg ccggagacca ccgacgagcc gcttgagaag 1260
ggctactccc accagctcaa ctacgtgatg tgcttcctca tgcagggctc ccgcggcacc 1320
atcccggtgc tcacctggac ccacaagtcc gtggacttct tcaacatgat cgactccaag 1380
aagatcaccc agctcccgct cgtgaaggcc tacaagctcc agtccggcgc ctccgtggtg 1440
gccggcccgc gcttcaccgg cggcgacatc atccagtgca ccgagaacgg ctccgccgcc 1500
accatctacg tgaccccgga cgtgtcctac tcccagaagt accgcgcccg catccactac 1560
gcctccacct cccagatcac cttcaccctc tccctcgacg gcgccccgtt caaccagtac 1620
tacttcgaca agaccatcaa caagggcgac accctcacct acaactcctt caacctcgcc 1680
tccttctcca ccccgttcga gctatccggc aacaacctcc agatcggcgt gaccggcctc 1740
tccgccggcg acaaggtgta catcgacaag atcgagttca tcccggtgaa ctaagagctc 1800
<210>7
<211>1704
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>7
atgggcttcc cgttcggcgg cgccctcgtg tccttctaca ccaacttcct caacaccatc 60
tggccgtccg aggacccgtg gaaggccttc atggagcagg tggaggccct catggaccag 120
aagatcgccg actacgccaa gaacaaggcc ctcgccgagc ttcagggcct ccagaacaac 180
gtggaggact acgtgtccgc cctctcctcc tggcagaaga acccggtgtc ctcccgcaac 240
ccgcactccc agggccgcat ccgcgagctg ttctcccagg ccgagtccca cttccgcaac 300
tccatgccgt ccttcgccat ctccggctac gaggtgctct tcctcaccac ctacgcccag 360
gccgccaaca cccacctctt cctcctcaag gacgcccaaa tctacggcga ggagtggggc 420
tacgagaagg aggacatcgc cgagttctac aagcgccagc tcaagctcac ccaggagtac 480
accgaccact gcgtgaagtg gtacaacgtg ggcctcgaca agctccgcgg ctcctcctac 540
gagtcctggg tgaacttcaa ccgctaccgc cgcgagatga ccctcaccgt gctcgacctc 600
atcgccctct tcccgctcta cgacgtgcgc ctctacccga aggaggtgaa gaccgaactc 660
acccgcgacg tgctcaccga cccgatcgtg ggcgtgaaca acctccgcgg ctacggcacc 720
accttctcca acatcgagaa ctacatccgc aagccgcacc tcttcgacta cctccaccgc 780
atccagttcc acacccgctt ccagccgggc tactacggca acgactcctt caactactgg 840
tccggcaact acgtgtccac ccgcccgtcc atcggctcca acgacatcat cacctccccg 900
ttctacggca acaagtcctc cgagccggtg cagaacctcg agttcaacgg cgagaaggtg 960
taccgcgccg tggccaacac caacctcgcc gtgtggccgt ccgccgtgta ctccggcgtg 1020
accaaggtgg agttctccca gtacaacgac cagaccgacg aggcctccac ccagacctac 1080
gactccaagc gcaacgtggg cgccgtgtcc tgggactcca tcgaccagct cccgccggag 1140
accaccgacg agccgcttga gaagggctac tcccaccagc tcaactacgt gatgtgcttc 1200
ctcatgcagg gctcccgcgg caccatcccg gtgctcacct ggacccacaa gtccgtggac 1260
ttcttcaaca tgatcgactc caagaagatc acccagctcc cgctcgtgaa ggcctacaag 1320
ctccagtccg gcgcctccgt ggtggccggc ccgcgcttca ccggcggcga catcatccag 1380
tgcaccgaga acggctccgc cgccaccatc tacgtgaccc cggacgtgtc ctactcccag 1440
aagtaccgcg cccgcatcca ctacgcctcc acctcccaga tcaccttcac cctctccctc 1500
gacggcgccc cgttcaacca gtactacttc gacaagacca tcaacaaggg cgacaccctc 1560
acctacaact ccttcaacct cgcctccttc tccaccccgt tcgagctatc cggcaacaac 1620
ctccagatcg gcgtgaccgg cctctccgcc ggcgacaagg tgtacatcga caagatcgag 1680
ttcatcccgg tgaactaaga gctc 1704
<210>8
<211>1806
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>8
atgaccgccg acaacaacac cgaggccctc gactcctcca ccaccaagga cgtgatccag 60
aagggcatct ccgtggtggg cgacctcctc ggcggacccg ggaagggctt cccgttcggc 120
ggcgccctcg tgtccttcta caccaacttc ctcaacacca tctggccgtc cgaggacccg 180
tggaaggcct tcatggagca ggtggaggcc ctcatggacc agaagatcgc cgactacgcc 240
aagaacaagg ccctcgccga gcttcagggc ctccagaaca acgtggagga ctacgtgtcc 300
gccctctcct cctggcagaa gaacccggtg tcctcccgca acccgcactc ccagggccgc 360
atccgcgagc tgttctccca ggccgagtcc cacttccgca actccatgcc gtccttcgcc 420
atctccggct acgaggtgct cttcctcacc acctacgccc aggccgccaa cacccacctc 480
ttcctcctca aggacgccca aatctacggc gaggagtggg gctacgagaa ggaggacatc 540
gccgagttct acaagcgcca gctcaagctc acccaggagt acaccgacca ctgcgtgaag 600
tggtacaacg tgggcctcga caagctccgc ggctcctcct acgagtcctg ggtgaacttc 660
aaccgctacc gccgcgagat gaccctcacc gtgctcgacc tcatcgccct cttcccgctc 720
tacgacgtgc gcctctaccc gaaggaggtg aagaccgaac tcacccgcga cgtgctcacc 780
gacccgatcg tgggcgtgaa caacctccgc ggctacggca ccaccttctc caacatcgag 840
aactacatcc gcaagccgca cctcttcgac tacctccacc gcatccagtt ccacacccgc 900
ttccagccgg gctactacgg caacgactcc ttcaactact ggtccggcaa ctacgtgtcc 960
acccgcccgt ccatcggctc caacgacatc atcacctccc cgttctacgg caacaagtcc 1020
tccgagccgg tgcagaacct cgagttcaac ggcgagaagg tgtaccgcgc cgtggccaac 1080
accaacctcg ccgtgtggcc gtccgccgtg tactccggcg tgaccaaggt ggagttctcc 1140
cagtacaacg accagaccga cgaggcctcc acccagacct acgactccaa gcgcaacgtg 1200
ggcgccgtgt cctgggactc catcgaccag ctcccgccgg agaccaccga cgagccgctt 1260
gagaagggct actcccacca gctcaactac gtgatgtgct tcctcatgca gggctcccgc 1320
ggcaccatcc cggtgctcac ctggacccac aagtccgtgg acttcttcaa catgatcgac 1380
tccaagaaga tcacccagct cccgctcgtg aaggcctaca agctccagtc cggcgcctcc 1440
gtggtggccg gcccgcgctt caccggcggc gacatcatcc agtgcaccga gaacggctcc 1500
gccgccacca tctacgtgac cccggacgtg tcctactccc agaagtaccg cgcccgcatc 1560
cactacgcct ccacctccca gatcaccttc accctctccc tcgacggcgc cccgttcaac 1620
cagtactact tcgacaagac catcaacaag ggcgacaccc tcacctacaa ctccttcaac 1680
ctcgcctcct tctccacccc gttcgagcta tccggcaaca acctccagat cggcgtgacc 1740
ggcctctccg ccggcgacaa ggtgtacatc gacaagatcg agttcatccc ggtgaactaa 1800
gagctc 1806
<210>9
<211>33
<212>DNA
<213>Unknown
<220>
<223>人工合成
<400>9
ctagaaataa ttttgtttaa ctttaagaag gag 33
<210>10
<211>33
<212>DNA
<213>Unknown
<220>
<223>人工合成
<400>10
gatcctcctt cttaaagtta aacaaaatta ttt 33
<210>11
<211>39
<212>DNA
<213>Unknown
<220>
<223>人工合成
<400>11
cctcggcgtg gtgggcttcc cgttcggcgg cgccctcgt 39
<210>12
<211>38
<212>DNA
<213>Unknown
<220>
<223>人工合成
<400>12
tgaggatcca tgggcttccc gttcggcggc gccctcgt 38
<210>13
<211>36
<212>DNA
<213>Unknown
<220>
<223>人工合成
<400>13
ggaagcccac cacgccgagg aggtcgceca ccacgg 36
<210>14
<211>42
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>14
gcgaagcttg catgcctaca gtgcagcgtg acccggtcgt gc 42
<210>15
<211>46
<212>DNA
<213>Unknown
<220>
<223>人工序列
<400>15
gtgggatcct ctagagtcgacc tgcagaag taacaccaaa caacag 46
Claims (10)
1.一种增强苏云金芽孢杆菌杀虫蛋白质Cry3对玉米根虫(Diabroticavirgifera virgifera)的杀虫能力的方法,所述方法为:对Cry3的N端第1~40位氨基酸序列进行删除和/或插入至少一个多肽片段,所述多肽片段长度为1~40个氨基酸,所述Cry3为Cry3A或Cry3B。
2.如权利要求1所述的方法,其特征在于所述方法为:在Cry3的N端第30~33位氨基酸中插入至少一个多肽片段。
3.如权利要求1所述的方法,其特征在于所述方法为:删除Cry3的N端第1~32位氨基酸多肽片段。
4.如权利要求1所述的方法,其特征在于所述方法为:将Cry3的N端第32~33位氨基酸替换为Gly-Pro-Gly-Lys。
5.一种按照如权利要求1所述方法获得的改良Cry3。
6.如权利要求5所述的改良Cry3,所述Cry3为Cry3A,具有SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列。
7.如权利要求5所述的改良Cry3,所述Cry3为Cry3B,具有SEQ ID NO.4或SEQ ID NO.5所示的氨基酸序列。
8.含有编码如权利要求5所述Cry3蛋白质的核苷酸序列的质粒。
9.如权利要8所述的质粒在制备转基因抗虫玉米中的应用。
10.如权利要求9所述的应用,其特征在于所述应用为:将所述质粒转化玉米细胞,获得转基因抗虫玉米。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103215290A (zh) * | 2013-04-01 | 2013-07-24 | 浙江大学 | 抗虫融合基因、融合蛋白及其应用 |
CN103748228A (zh) * | 2011-03-30 | 2014-04-23 | 墨西哥国立大学 | 突变型苏云金芽孢杆菌cry基因及其使用方法 |
CN104388421A (zh) * | 2014-08-28 | 2015-03-04 | 上海市农业科学院 | 转基因水稻品系134Bt的外源插入片段旁侧序列、其扩增引物及其应用 |
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CN103194460B (zh) * | 2013-04-17 | 2015-04-08 | 中国农业大学 | 一种苏云金芽孢杆菌晶体融合蛋白Bt Cry3Bb的制备方法 |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN103748228A (zh) * | 2011-03-30 | 2014-04-23 | 墨西哥国立大学 | 突变型苏云金芽孢杆菌cry基因及其使用方法 |
CN103748228B (zh) * | 2011-03-30 | 2017-02-15 | 墨西哥国立大学 | 突变型苏云金芽孢杆菌cry基因及其使用方法 |
CN103215290A (zh) * | 2013-04-01 | 2013-07-24 | 浙江大学 | 抗虫融合基因、融合蛋白及其应用 |
CN104388421A (zh) * | 2014-08-28 | 2015-03-04 | 上海市农业科学院 | 转基因水稻品系134Bt的外源插入片段旁侧序列、其扩增引物及其应用 |
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