CN102031266A - 抗虫融合基因、融合蛋白质及其应用 - Google Patents
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Abstract
本发明公开了一种抗虫融合基因,该基因包括从5’-3’依次含有编码BT晶体毒素Cry1的核苷酸序列和编码Cry9Aa毒素的核苷酸序列;且上述2个核苷酸序列位于同一个开放阅读框内。该抗虫融合基因包括Cry1Aa、Cry1Ab、Cry1Ac、Cry1Aa的修饰基因、Cry1Ab的修饰基因或Cry1Ac的修饰基因;还包括Cry9Aa或者Cry9Aa经过修饰的基因。本发明还同时公开了上述抗虫融合基因所编码的融合蛋白,该融合蛋白从N端至C端依次为Cry1晶体毒素和Cry9Aa毒素。本发明的融合蛋白能用于制备转基因抗虫农作物。
Description
技术领域
本发明涉及一种获得高杀虫能力的抗虫融合基因的方法和一种高杀虫能力的抗虫融合基因和融合蛋白、以及该融合蛋白的具体应用。
背景技术
害虫给全球农业生产带来每年约80亿美元的损失。害虫的防治目前主要依靠使用化学农药,但是农药残留会对人体健康带来危害。因此,人们成功的选择了利用杀虫毒素转基因作物进行抗虫,目前转基因抗虫玉米和棉花已经大量种植。
转基因作物抗虫的关键技术是获得性能优良的杀虫蛋白质,杀虫蛋白质有多种。比较常用的是Bt晶体蛋白质,例如Cry1Ab,Cry1C等,它们已被大量运用于转基因抗虫作物。但是单个杀虫毒素往往杀虫谱都比较窄,杀虫活性比较低。同时长期大量使用单一的杀虫蛋白质还可能引起害虫抗性的发展。因此,获得高杀虫活性的新型蛋白质杀虫毒素,对提高杀虫能力和减缓害虫抗性的发生具有重要应用价值。
参考文献如下:
(1)、Crickmore,N.,Zeigler,D.R.,Feitelson,J.,Schnepf,E.,Van Rie,J.,Lereclus,D.,Baum,J.&Dean,D.H.(1998)Microbiol.Mol.Biol.Rev.62,807-813.(苏云金芽孢杆菌及杀虫晶体蛋白)
(2)、Ferre,J.&Van Rie,J.(2002)Annu.Rev.Entomol.47,501-533.(抗苏云金芽孢杆菌昆虫的生物化学和遗传学)
(3)、Estruch,J.J.,Warren,G.W.,Mullins,M.A.,Nye,G.J.,Craig,J.A.&Koziel,M.G.(1996)Proc.Natl..Acad.Sci.USA 93,5389-5394.(一种对鳞翅目昆虫具有光谱杀虫活性的苏云金芽孢杆菌营养期杀虫蛋白——VIP3A)
发明内容
本发明要解决的技术问题是提供一种获得高杀虫能力的抗虫融合基因的方法;同时提供高杀虫能力的融合抗虫基因,相应的融合蛋白及其用途。
为了解决上述技术问题,本发明提供一种抗虫融合基因,该基因包括从5’-3’依次为含有编码BT晶体毒素Cry1的核苷酸序列和编码Cry9Aa毒素的核苷酸序列;且上述2个核苷酸序列位于同一个开放阅读框内;Cry1晶体毒素为C端切除的活性毒素。
作为本发明的抗虫融合基因的一步改进:Cry1晶体毒素为Cry1Ab、Cry1Ac,Cry1Aa。
作为本发明的抗虫融合基因的进一步改进:当Cry1晶体毒素为Cry1Ab时,融合基因的核苷酸序列为SEQ ID NO:1;当Cry1晶体毒素为Cry1Ac时,融合基因的核苷酸序列为SEQID NO:2。
本发明还提供了上述抗虫融合基因编码的融合蛋白,该融合蛋白从N端至C端依次为Cry1晶体毒素和Cry9Aa毒素。
作为本发明的融合蛋白的一种改进:当Cry1晶体毒素为Cry1Ab时,融合蛋白的氨基酸序列为SEQ ID NO:3;当Cry1晶体毒素为Cry1Ac时,融合蛋白的氨基酸序列为SEQ ID NO:4。
本发明还提供了上述融合蛋白在转基因抗虫作物和转基因杀虫微生物方面的应用。
本行业的技术人员均知道以下理论:
1、二个Cry杀虫基因的融合并不是都能够增强杀虫能力的。例如Cry1Ab和Cry1Ca的融合蛋白质的杀虫能力比相同重量的单独Cry1Ab和Cry1Ca的混合物的杀虫能力还要低。
2、即使两个单独蛋白质组合使用具有增效作用,但也并不能得出它们的融合蛋白具有高效杀虫能力;这是因为:在蛋白质生物化学上,两个单独蛋白质的物理混和与融合是不同的,不能根据物理混和所具有的增效作用来肯定融合后所获得的增效作用;且在不同位置融合所得的融合蛋白的活性是大相径庭的。
本发明所设计的用一个Cry1晶体毒素与Cry9Aa毒素融合成的人工蛋白质分子,与原先的Cry1和Cry9Aa晶体毒素物理混合相比,具有如下优点:杀虫活性提高了10倍以上,杀虫谱更广。本发明的杀虫蛋白质可以杀灭粘虫、甜菜夜蛾、玉米螟、二化螟等水稻、玉米和棉花的主要鳞翅目害虫。此外,本发明发现的融合蛋白质还可以有效的减缓害虫抗性的发生。
具体实施方式
本发明的以下实施例所使用的分子生物学和生物化学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 Molecular Biology,和J.Sambrook等编写Cold Spring Harbor Laboratory Press(2001)出版的Molecular Cloning:ALabortory Manual,3rd ED.等文献均有详细的说明。
实施例1、Cry1Ab-Cry9A和Cry1Ac-Cry9A融合基因的构建:
编码Cry9Aa、Cry1Ab、Cry1Ac、和Cry1Ca的杀虫蛋白的基因均由上海生工合成,其DNA序列分别为SEQ ID NO:5,SEQ ID NO:6,SEQ ID NO:7和SEQ ID NO:8。这些基因分别被克隆到载体pET28a表达载体中限制性内切酶BamHI和SacI的位点之间,得到的载体分别命名为pET-9Aa、pET-1Ab、pET-1Ac和pET-1Ca。
Cry1Ab-Cry9Aa构建过程:Cry1Ab片段由BamHI和XmaI酶切pET-1Ab获得,Cry9Aa片段由XmaI和SacI酶切pET-9Aa获得。载体pET28a经过BamHI和SacI酶切以后和Cry1Ab以及Cry9Aa连接,得到载体pET-Cry1Ab-Cry9Aa。这个载体包含一个融合基因,Cry1Ab-Cry9Aa(如SEQ ID NO:1),其编码一个氨基酸序列为SEQ ID No:3的融合蛋白质。
Cry1Ac-Cry9Aa构建过程:Cry1Ac片段由BamHI和XmaI酶切pET-1Ac获得,Cry9Aa片段由XmaI和SacI酶切pET-9Aa获得。载体pET28a经过BamHI和SacI酶切以后和Cry1Ac以及Cry9Aa连接,得到载体pET-CryAc-Cry9Aa。这个载体包含一个融合基因,CryAc-Cry9Aa(如SEQ ID NO:2),其编码一个氨基酸序列为SEQ ID No:4的融合蛋白质。
说明:SEQ ID NO:1和SEQ ID NO:2的核酸序列的表达框从第一个起始密码子(ATG)开始,到最后终止密码子(TAA)前结束,长度均为3933bp,与SEQ ID NO:3和SEQ ID NO:4对应。第一个ATG前的核酸序列GGATCC为BamH1酶切位点,其后的ACC为核糖体结合位点,最后GAGCTC为Sac1酶切位点。
Cry1Ab-Cry1Ca的构建过程:Cry1Ab片段由BamHI和XmaI酶切pET-1Ab获得,Cry1Ca片段由XmaI和SacI酶切pET-1Ca获得。载体pET28a经过BamHI和SacI酶切以后和Cry1Ab以及Cry1Ca连接,得到载体pET-Cry1Ab-Cry1Ca。这个载体包含一个融合基因,CryAb-Cry1Ca,其核酸序列为SEQ ID No:9。
实施例2、杀虫蛋白质的制备:
包含杀虫基因的载体pET-9Aa、pET-1Ab、pET-1Ac、pET-1Ca、Cry1Ab-Cry9Aa、CryAc-Cry9Aa和Cry1Ab-Cry1Ca分别导入BL21Star细胞系(E.coli),在包含50mg/L卡那霉素的LB固体培养基上选取单克隆。单个菌落接种到100毫升LB细菌培养液,在37℃下震动培养至OD600=0.6,然后加IPTG(Isopropyl-β-D-thiogalactoside)到0.5mM,并继续在同样的条件下培养4小时。培养液经过5000g离心10分钟沉淀大肠杆菌细胞,然后弃上清收集沉淀。沉淀中加30毫升20mM Tris-HCl缓冲液,超声粉碎。这样获得的重组蛋白质用来进行杀虫活性的测定。
实施例3、在大肠杆菌中表达的杀虫蛋白的杀虫活性测定:
实施例2所获得的杀虫蛋白各100微升单独或者混合涂在0.5平方厘米昆虫人工饲料的表面,饲养新生一龄幼虫用来进行杀虫活性测定。阴性对照的准备方法与实施例2相同,但质粒为不含任何插入DNA的pET28a载体本身。饲养7天以后统计杀虫率,结果如表1和2所示:
表1、Cry1Ab-Cry9Aa的杀虫率
杀虫蛋白质 | 棉铃虫 | 玉米螟 | 甜菜夜蛾 |
Cry1Ab(100微升) | 0% | 100% | 60% |
Cry9Aa(100微升) | 0% | 55% | 0% |
Cry1Ab+Cry9Aa(各100微升) | 0% | 100% | 60% |
本发明的Cry1Ab-cry9Aa(100微升) | 100% | 100% | 100% |
阴性对照(100微升) | 0% | 0% | 0% |
表2、Cry1Ab-Cry1Ca的杀虫率
杀虫蛋白质 | 棉铃虫 | 玉米螟 | 甜菜夜蛾 |
Cry1Ab(100微升) | 0% | 100% | 60% |
Cry1Ca(100微升) | 35% | 20% | 60% |
Cry1Ab+Cry1Ca(各100微升) | 60% | 100% | 90% |
Cry1Ab-Cry1Ca(100微升) | 50% | 90% | 65% |
阴性对照(100微升) | 0% | 0% | 0% |
结果表明,融合蛋白质Cry1Ab-Cry9Aa的杀虫活性比单独的Cry1Ab或者Cry9Aa明显提高,同时也比Cry1Ab和Cry9Aa混合的活性明显提高。进一步的测定表明Cry1Ab-Cry9Aa对棉铃虫的活性的致死中量低于Cry1Ab和Cry9Aa混合物10倍左右。但是融合蛋白质Cry1Ab-Cry1Ca的杀虫活性比单独的Cry1Ab或者Cry1Ca没有明显提高,比Cry1Ab和Cry9Aa混合的活性反而有所下降。因此并不是二个Cry杀虫基因都可以通过融合而提高杀虫活性。
实施例4、农杆菌转化T-DNA载体的构建:
农杆菌转化T-DNA载体是基于pCambia 1300载体而构建的。编码杀虫蛋白质的合成核苷酸序列Cry1Ab-Cry9Aa(SEQ ID No:1,5’端被设计上BamHI位点,3’端被设计上SacI位点,BamHI-SacI酶切片段)与玉米的pepc终止子(SEQ ID No:10,5’端被设计上SacI位点,3’端被设计上KpnI位点,SacI-KpnI片段)连接,获得包括基因和终止子的BamHI-KpnI片段。
玉米ubiquitin-1启动子从玉米的基因组中通过PCR获得,使用的引物分别是:
ZmUbiF (5’GCGAAGCTTGCATGCCTACAGTGC AGCGTGACCCGGTCGTGC,添加了HindIII位点)。
ZmUbiR(5’GTGGGATCCTCTAGAGTCGACCTGCAGAAGTAACACCAAACAACAG,添加了BamHI位点)。
玉米ubiquitin-1启动子经过HindIII和BamHI酶切后和基因-终止子片段(BamHI-KpnI片段)共同连接到经过HindIII和KpnI酶切的pCambia 1300载体中,获得T-DNA载体pCAM-Cry1Ab-Cry9Aa。由于玉米ubiquitin-1启动子在禾本科植物中均有启动子活性,pCAM-Cry1Ab-Cry9Aa可以作为多种禾本科植物的抗虫转化T-DNA载体。
实施例5、转基因水稻的获得:
转基因植物的获得方法是采用现有技术(卢雄斌龚祖埙,1998生命科学10:125-131;刘凡等,2003分子植物育种1:108-115)。选取成熟饱满种子去壳,诱导产生愈伤组织作为转化材料。取含目的基因的农杆菌(pCAM-Cry1Ab-Cry9Aa)划板,挑单菌落接种准备转化用农杆菌。将待转化的愈伤组织放入适当浓度的农杆菌液中(含乙酰丁香酮),让农杆菌结合到愈伤组织表面,然后把愈伤组织转移到共培养基中,共培养2-3天。用无菌水冲洗转化后的愈伤,转移到含抗生素的筛选培养基上,筛选培养(50ng/mL潮霉素)两个月(中间继代一次)。把筛选后,生长活力良好的愈伤转移到预分化培养基上培养20天左右,然后将预分化好的愈伤组织移到分化培养基,14小时光照分化发芽。2-3周后,把抗性再生植株转移到生根培养基上壮苗生根,最后将再生植株洗去琼脂移植于温室,作为鉴定材料。
实施例6、转基因玉米的获得:
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pCAM-Cry1Ab-Cry9Aa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。
实施例7、转基因农作物抗虫能力的测定:
利用二化螟和稻纵卷叶螟测定了通过实施例5获得的10个转基因水稻系的杀虫活性。新生二化螟和稻纵卷叶螟的一龄幼虫取食转基因水稻后,有8个系的致死率是100%。
利用玉米螟和棉铃虫测定了通过实施例6获得的10个转基因玉米系的杀虫活性。新生玉米螟一龄幼虫取食10个转基因玉米系后,全部100%死亡,新生棉铃虫一龄幼虫取食10个转基因玉米系后,有7个系100%杀灭棉铃虫,其他3个系对棉铃虫杀灭活性在30%到70%之间。
实施例8、Cry1Ac-Cry9Aa在大肠杆菌中表达的杀虫蛋白的杀虫活性测定:
实施例2所获得的杀虫蛋白各100微升单独或者混合涂在0.5平方厘米昆虫人工饲料的表面,饲养新生一龄幼虫用来进行杀虫活性测定。阴性对照的准备方法与实施例2相同,但质粒为不含任何插入DNA的pET28a载体本身。饲养7天以后统计杀虫率,结果如表3所示:
表3、Cry1Ac-Cry9Aa的杀虫率
杀虫蛋白质 | 棉铃虫 | 玉米螟 | 甜菜夜蛾 |
Cry1Ac(100微升) | 100% | 80% | 80% |
Cry9Aa(100微升) | 0% | 55% | 0% |
Cry1Ac+Cry9Aa(各100微升) | 100% | 80% | 80% |
本发明的Cry1Ac-cry9Aa(100微升) | 100% | 100% | 100% |
阴性对照(100微升) | 0% | 0% | 0% |
结果表明,融合蛋白质Cry1Ac-Cry9Aa的杀虫活性比单独的Cry1Ac或者Cry9Aa明显提高,同时也比Cry1Ac和Cry9Aa混合的活性明显提高。进一步的测定表明Cry1Ac-Cry9Aa对棉铃虫的活性的致死中量低于Cry1Ac和Cry9Aa混合物15倍左右。
实施例9、农杆菌转化T-DNA载体的构建:
农杆菌转化T-DNA载体是基于pCambia 1300载体而构建的。编码杀虫蛋白质的合成核苷酸序列Cry1Ac-Cry9Aa(SEQ ID No:2,5’端被设计上BamHI位点,3’端被设计上SacI位点,BamHI-SacI酶切片段)与玉米的pepc终止子(SEQ ID No:10,5’端被设计上SacI位点,3’端被设计上KpnI位点,SacI-KpnI片段)连接,获得包括基因和终止子的BamHI-KpnI片段。
玉米ubiquitin-1启动子从玉米的基因组中通过PCR获得,使用的引物分别是:
ZmUbiF(5’GCGAAGCTTGCATGCCTACAGTGCAGCGTGACCCGGTCGTGC,添加了HindIII位点)。
ZmUbiR(5’GTGGGATCCTCTAGAGTCGACCTGCAGAAGTAACACCAAACAACAG,添加了BamHI位点)。
玉米ubiquitin-1启动子经过HindIII和BamHI酶切后和基因-终止子片段(BamHI-KpnI片段)共同连接到经过HindIII和KpnI酶切的pCambia 1300载体中,获得T-DNA载体pCAM-Cry1Ac-Cry9Aa。由于玉米ubiquitin-1启动子在禾本科植物中均有启动子活性,pCAM-Cry1Ac-Cry9Aa可以作为多种禾本科植物的抗虫转化T-DNA载体。
实施例10、转Cry1Ac-Cry9Aa基因水稻的获得:
转基因植物的获得方法是采用现有技术(卢雄斌龚祖埙,1998生命科学10:125-131;刘凡等,2003分子植物育种1:108-115)。选取成熟饱满种子去壳,诱导产生愈伤组织作为转化材料。取含目的基因的农杆菌(pCAM-Cry1Ac-Cry9Aa)划板,挑单菌落接种准备转化用农杆菌。将待转化的愈伤组织放入适当浓度的农杆菌液中(含乙酰丁香酮),让农杆菌结合到愈伤组织表面,然后把愈伤组织转移到共培养基中,共培养2-3天。用无菌水冲洗转化后的愈伤,转移到含抗生素的筛选培养基上,筛选培养(50ng/mL潮霉素)两个月(中间继代一次)。把筛选后,生长活力良好的愈伤转移到预分化培养基上培养20天左右,然后将预分化好的愈伤组织移到分化培养基,14小时光照分化发芽。2-3周后,把抗性再生植株转移到生根培养基上壮苗生根,最后将再生植株洗去琼脂移植于温室,作为鉴定材料。
实施例11、转Cry1Ac-Cry9Aa基因玉米的获得:
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pCAM-Cry1Ac-Cry9Aa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。
实施例12、转Cry1Ac-Cry9Aa基因农作物抗虫能力的测定:
利用二化螟和稻纵卷叶螟测定了通过实施例10获得的10个转基因水稻系的杀虫活性。新生二化螟和稻纵卷叶螟的一龄幼虫取食转基因水稻后,有6个转化系的致死率都是100%。
利用玉米螟和棉铃虫测定了通过实施例11获得的10个转基因玉米系的杀虫活性。新生棉铃虫一龄幼虫取食7个转基因玉米系后,全部100%死亡,新生玉米螟一龄幼虫取食7个转基因玉米系后,全部100%死亡。
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。
序列表
SEQ ID NO:1(Cryl Ab-Cry9Aa)
1 GGATCCACCA TGGACAACAA CCCCAACATC AACGAGTGCA TCCCCTACAA CTGCCTGAGC
61 AACCCCGAGG TGGAGGTGCT GGGCGGCGAG CGCATCGAGA CCGGCTACAC CCCCATCGAC
121 ATCAGCCTGA GCCTGACCCA GTTCCTGCTG AGCGAGTTCG TGCCCGGCGC CGGCTTCGTG
181 CTGGGCCTGG TGGACATCAT CTGGGGCATC TTCGGCCCCA GCCAGTGGGA CGCCTTCCTG
241 GTGCAGATCG AGCAGCTGAT CAACCAGCGC ATCGAGGAGT TCGCCCGCAA CCAGGCCATC
301 AGCCGCCTGG AGGGCCTGAG CAACCTGTAC CAAATCTACG CCGAGAGCTT CCGCGAGTGG
361 GAGGCCGACC CCACCAACCC CGCCCTGCGC GAGGAGATGC GCATCCAGTT CAACGACATG
421 AACAGCGCCC TGACCACCGC CATCCCCCTG TTCGCCGTGC AGAACTACCA GGTGCCCCTG
481 CTGAGCGTGT ACGTGCAGGC CGCCAACCTG CACCTGAGCG TGCTGCGCGA CGTCAGCGTG
541 TTCGGCCAGC GCTGGGGCTT CGACGCCGCC ACCATCAACA GCCGCTACAA CGACCTGACC
601 CGCCTGATCG GCAACTACAC CGACCACGCC GTGCGCTGGT ACAACACCGG CCTGGAGCGC
661 GTGTGGGGTC CCGACAGCCG CGACTGGATC AGGTACAACC AGTTCCGCCG CGAGCTGACC
721 CTGACCGTGC TGGACATCGT GAGCCTGTTC CCCAACTACG ACAGCCGCAC CTACCCCATC
781 CGCACCGTGA GCCAGCTGAC CCGCGAGATT TACACCAACC CCGTGCTGGA GAACTTCGAC
841 GGCAGCTTCC GCGGCAGCGC CCAGGGCATC GAGGGCAGCA TCCGCAGCCC CCACCTGATG
901 GACATCCTGA ACAGCATCAC CATCTACACC GACGCCCACC GCGGCGAGTA CTACTGGAGC
961 GGCCACCAGA TCATGGCCAG CCCCGTCGGC TTCAGCGGCC CCGAGTTCAC CTTCCCCCTG
1021 TACGGCACCA TGGGCAACGC TGCACCTCAG CAGCGCATAG TGGCACAGCT GGGCCAGGGA
1081 GTGTACCGCA CCCTGAGCAG CACCCTGTAC CGTCGACCTT TCAACATCGG CATCAACAAC
1141 CAGCAGCTGA GCGTGCTGGA CGGCACCGAG TTCGCCTACG GCACCAGCAG CAACCTGCCC
1201 AGCGCCGTGT ACCGCAAGAG CGGCACCGTG GACAGCCTGG ACGAGATCCC CCCTCAGAAC
1261 AACAACGTGC CACCTCGACA GGGCTTCAGC CACCGTCTGA GCCACGTGAG CATGTTCCGC
1321 AGTGGCTTCA GCAACAGCAG CGTGAGCATC ATCCGTGCAC CTATGTTCAG CTGGATTCAC
1381 CGCAGTGCCG AGTTCAACAA CATCATCCCC AGCAGCCAGA TCACCCAGAT CCCCCTGACC
1441 AAGAGCACCA ACCTGGGCAG CGGCACCAGC GTGGTGAAGG GCCCCGGCTT CACCGGCGGC
1501 GACATCCTGC GCCGCACCAG CCCCGGCCAG ATCAGCACCC TGCGCGTGAA CATCACCGCC
1561 CCCCTGAGCC AGCGCTACCG CGTCCGCATC CGCTACGCCA GCACCACCAA CCTGCAGTTC
1621 CACACCAGCA TCGACGGCCG CCCCATCAAC CAGGGCAACT TCAGCGCCAC CATGAGCAGC
1681 GGCAGCAACC TGCAGAGCGG CAGCTTCCGC ACCGTGGGCT TCACCACCCC CTTCAACTTC
1741 AGCAACGGCA GCAGCGTGTT CACCCTGAGC GCCCACGTGT TCAACAGCGG CAACGAGGTG
1801 TACATCGACC GCATCGAGTT CGTGCCCGCC GAGGTGACCT TCGAGGCCGA GTACGACCTG
1861 GAGAGGGCTC AGAAGGCCGT GAACGAGCTG TTCACCAGCA GCAACCAGAT CGGCCTGAAG
1921 ACCGACGTGA CCGACTACCA CATCGATCAG GTGCGACCCG GGAAGGGTAA AGGGGGTAAC
1981 CAGAACAAGC ACGGCATCAT CGGCGCCTCC AACTGCGGCT GCGCCTCCGA CGACGTGGCC
2041 AAGTACCCGC TCGCCAACAA CCCGTACTCC TCCGCCCTCA ACCTCAACTC CTGCCAGAAC
2101 TCCTCCATCC TCAACTGGAT CAACATCATC GGCGACGCCG CCAAGGAGGC CGTGTCCATC
2161 GGCACCACCA TCGTGTCCCT CATCACCGCC CCGTCCCTCA CCGGCCTCAT CTCCATCGTG
2221 TACGACCTCA TCGGCAAGGT GCTCGGCGGC TCCTCCGGCC AGTCCATCTC CGACCTCTCC
2281 ATCTGCGACC TCCTCTCCAT CATCGACCTC CGCGTGTCCC AGTCCGTGCT CAACGACGGC
2341 ATCGCCGACT TCAACGGCTC CGTGCTCCTC TACCGCAACT ACCTTGAGGC CCTCGACTCC
2401 TGGAACAAGA ACCCGAACTC CGCCTCCGCC GAGGAGCTTC GCACCCGCTT CCGCATCGCC
2461 GACTCCGAGT TCGACCGCAT CCTCACCCGC GGCTCCCTCA CCAACGGCGG CTCCCTCGCC
2521 CGCCAGAACG CCCAGATCCT CCTCCTCCCG TCCTTCGCCT CCGCCGCCTT CTTCCACCTC
2581 CTCCTCCTCC GCGACGCCAC CCGCTACGGC ACCAACTGGG GCCTCTACAA CGCCACCCCG
2641 TTCATCAACT ACCAGTCCAA GCTCGTGGAA CTCATCGAAC TCTACACCGA CTACTGCGTG
2701 CACTGGTACA ACCGCGGCTT CAACGAGCTT CGCCAGCGCG GCACCTCCGC CACCGCCTGG
2761 CTGGAGTTCC ACCGCTACCG CCGCGAGATG ACCCTCATGG TGCTCGACAT CGTGGCCTCC
2821 TTCTCCTCCC TCGACATCAC CAACTACCCG ATCGAGACCG ACTTCCAGCT CTCCCGCGTG
2881 ATCTACACCG ACCCGATCGG CTTCGTGCAC CGCTCCTCCC TCCGCGGCGA GTCCTGGTTC
2941 TCCTTCGTGA ACCGCGCCAA CTTCTCCGAC CTGGAGAACG CCATCCCGAA CCCGCGCCCG
3001 TCCTGGTTCC TCAACAACAT GATCATCTCC ACCGGCTCCC TCACCCTCCC GGTGTCCCCG
3061 TCCACCGACC GCGCCCGCGT GTGGTACGGC TCCCGCGACC GCATCTCCCC GGCCAACTCC
3121 CAGTTCATCA CCGAGCTTAT CTCCGGCCAG CACACCACCG CCACCCAGAC CATCCTCGGC
3181 CGCAACATCT TCCGCGTGGA CTCCCAGGCC TGCAACCTCA ACGACACCAC CTACGGCGTG
3241 AACCGCGCCG TGTTCTACCA CGACGCCTCC GAGGGCTCCC AGCGCTCCGT GTACGAGGGC
3301 TACATCCGCA CCACCGGCAT CGACAACCCG CGCGTGCAGA ACATCAACAC CTACCTTCCG
3361 GGCGAGAACT CCGACATCCC GACCCCGGAG GACTACACCC ACATCCTCTC CACCACCATC
3421 AACCTCACCG GCGGCCTCCG CCAGGTGGCC TCCAACCGCC GCTCCTCCCT CGTGATGTAC
3481 GGCTGGACCC ACAAGTCCCT CGCCCGCAAC AACACCATCA ACCCGGACCG CATCACCCAG
3541 ATCCCGCTCA CCAAGGTGGA CACCCGCGGC ACCGGCGTGT CCTACGTGAA CGACCCCGGC
3601 TTCATCGGCG GCGCCCTCCT CCAGCGCACC GACCACGGCT CCCTCGGCGT GCTCCGCGTG
3661 CAGTTCCCGC TCCACCTCCG CCAGCAGTAC CGCATCCGCG TGCGCTACGC CTCCACCACC
3721 AACATCCGCC TCTCCGTGAA CGGCTCCTTC GGCACCATCT CCCAGAACCT CCCGTCCACC
3781 ATGCGCCTCG GCGAGGACCT CCGCTACGGC TCCTTCGCCA TCCGCGAGTT CAACACCTCC
3841 ATCCGCCCGA CCGCCTCCCC GGACCAGATC CGCCTCACCA TCGAGCCGTC CTTCATCCGC
3901 CAGGAGGTGT ACGTGGACCG CATCGAGTTC ATCCCGGTGA ACTAAGAGCT C
SEQ ID NO:2(Cryl Ac-Cry9Aa)
1 GGATCCACCA TGGACAACAA CCCCAACATC AACGAGTGCA TCCCCTACAA CTGCCTGAGC
61 AACCCCGAGG TGGAGGTGCT GGGCGGCGAG CGCATCGAGA CCGGCTACAC CCCCATCGAC
121 ATCAGCCTGA GCCTGACCCA GTTCCTGCTG AGCGAGTTCG TGCCCGGCGC CGGCTTCGTG
181 CTGGGCCTGG TGGACATCAT CTGGGGCATC TTCGGCCCCA GCCAGTGGGA CGCCTTCCTG
241 GTGCAGATCG AGCAGCTGAT CAACCAGCGC ATCGAGGAGT TCGCCCGCAA CCAGGCCATC
301 AGCCGCCTGG AGGGCCTGAG CAACCTGTAC CAAATCTACG CCGAGAGCTT CCGCGAGTGG
361 GAGGCCGACC CCACCAACCC CGCCCTGCGC GAGGAGATGC GCATCCAGTT CAACGACATG
421 AACAGCGCCC TGACCACCGC CATCCCCCTG TTCGCCGTGC AGAACTACCA GGTGCCCCTG
481 CTGAGCGTGT ACGTGCAGGC CGCCAACCTG CACCTGAGCG TGCTGCGCGA CGTCAGCGTG
541 TTCGGCCAGC GCTGGGGCTT CGACGCCGCC ACCATCAACA GCCGCTACAA CGACCTGACC
601 CGCCTGATCG GCAACTACAC CGACCACGCC GTGCGCTGGT ACAACACCGG CCTGGAGCGC
661 GTGTGGGGTC CCGACAGCCG CGACTGGATC AGGTACAACC AGTTCCGCCG CGAGCTGACC
721 CTGACCGTGC TGGACATCGT GAGCCTGTTC CCCAACTACG ACAGCCGCAC CTACCCCATC
781 CGCACCGTGA GCCAGCTGAC CCGCGAGATT TACACCAACC CCGTGCTGGA GAACTTCGAC
841 GGCAGCTTCC GCGGCAGCGC CCAGGGCATC GAGGGCAGCA TCCGCAGCCC CCACCTGATG
901 GACATCCTGA ACAGCATCAC CATCTACACC GACGCCCACC GCGGCGAGTA CTACTGGAGC
961 GGCCACCAGA TCATGGCCAG CCCCGTCGGC TTCAGCGGCC CCGAGTTCAC CTTCCCCCTG
1021 TACGGCACCA TGGGCAACGC TGCACCTCAG CAGCGCATAG TGGCACAGCT GGGCCAGGGA
1081 GTGTACCGCA CCCTGAGCAG CACCCTGTAC CGTCGACCTT TCAACATCGG CATCAACAAC
1141 CAGCAGCTGA GCGTGCTGGA CGGCACCGAG TTCGCCTACG GCACCAGCAG CAACCTGCCC
1201 AGCGCCGTGT ACCGCAAGAG CGGCACCGTG GACAGCCTGG ACGAGATCCC CCCTCAGAAC
1261 AACAACGTGC CACCTCGACA GGGCTTCAGC CACCGTCTGA GCCACGTGAG CATGTTCCGC
1321 AGTGGCTTCA GCAACAGCAG CGTGAGCATC ATCCGTGCAC CTATGTTCAG CTGGATTCAC
1381 CGCAGTGCCG AGTTCAACAA CATCATCGCC TCCGACTCCA TCACCCAGAT CCCGGCTGTG
1441 AAGGGCAACT TCCTCTTCAA CGGCTCCGTG ATCTCCGGTC CAGGCTTCAC CGGTGGCGAC
1501 CTCGTGCGCC TCAACTCCTC CGGCAACAAC ATCCAGAACC GCGGTTACAT CGAGGTGCCG
1561 ATCCACTTCC CGTCCACCTC CACCCGCTAC CGCGTGCGCG TGCGCTACGC CTCCGTGACC
1621 CCGATCCACC TCAACGTGAA CTGGGGCAAC TCCTCCATCT TCTCCAACAC CGTGCCAGCC
1681 ACCGCCACCT CCCTCGACAA CCTCCAGTCC TCCGACTTCG GCTACTTCGA GTCCGCCAAC
1741 GCCTTCACCT CCTCCCTCGG CAACATCGTG GGCGTGCGCA ACTTCTCCGG CACCGCTGGC
1801 GTGATCATCG ACCGCTTCGA GTTCATCCCG GTGACCGCCA CCCTCGAAGC CGAGTACAAC
1861 CTTGAGCGCG CTCAGAAGGC CGTGAACGCC CTCTTCACCT CCACCAACCA GCTCGGCCTC
1921 AAGACCAACG TGACCGACTA CCACATCGAC CAGGTGCCCG GGAAGGGTAA AGGGGGTAAC
1981 CAGAACAAGC ACGGCATCAT CGGCGCCTCC AACTGCGGCT GCGCCTCCGA CGACGTGGCC
2041 AAGTACCCGC TCGCCAACAA CCCGTACTCC TCCGCCCTCA ACCTCAACTC CTGCCAGAAC
2101 TCCTCCATCC TCAACTGGAT CAACATCATC GGCGACGCCG CCAAGGAGGC CGTGTCCATC
2161 GGCACCACCA TCGTGTCCCT CATCACCGCC CCGTCCCTCA CCGGCCTCAT CTCCATCGTG
2221 TACGACCTCA TCGGCAAGGT GCTCGGCGGC TCCTCCGGCC AGTCCATCTC CGACCTCTCC
2281 ATCTGCGACC TCCTCTCCAT CATCGACCTC CGCGTGTCCC AGTCCGTGCT CAACGACGGC
2341 ATCGCCGACT TCAACGGCTC CGTGCTCCTC TACCGCAACT ACCTTGAGGC CCTCGACTCC
2401 TGGAACAAGA ACCCGAACTC CGCCTCCGCC GAGGAGCTTC GCACCCGCTT CCGCATCGCC
2461 GACTCCGAGT TCGACCGCAT CCTCACCCGC GGCTCCCTCA CCAACGGCGG CTCCCTCGCC
2521 CGCCAGAACG CCCAGATCCT CCTCCTCCCG TCCTTCGCCT CCGCCGCCTT CTTCCACCTC
2581 CTCCTCCTCC GCGACGCCAC CCGCTACGGC ACCAACTGGG GCCTCTACAA CGCCACCCCG
2641 TTCATCAACT ACCAGTCCAA GCTCGTGGAA CTCATCGAAC TCTACACCGA CTACTGCGTG
2701 CACTGGTACA ACCGCGGCTT CAACGAGCTT CGCCAGCGCG GCACCTCCGC CACCGCCTGG
2761 CTGGAGTTCC ACCGCTACCG CCGCGAGATG ACCCTCATGG TGCTCGACAT CGTGGCCTCC
2821 TTCTCCTCCC TCGACATCAC CAACTACCCG ATCGAGACCG ACTTCCAGCT CTCCCGCGTG
2881 ATCTACACCG ACCCGATCGG CTTCGTGCAC CGCTCCTCCC TCCGCGGCGA GTCCTGGTTC
2941 TCCTTCGTGA ACCGCGCCAA CTTCTCCGAC CTGGAGAACG CCATCCCGAA CCCGCGCCCG
3001 TCCTGGTTCC TCAACAACAT GATCATCTCC ACCGGCTCCC TCACCCTCCC GGTGTCCCCG
3061 TCCACCGACC GCGCCCGCGT GTGGTACGGC TCCCGCGACC GCATCTCCCC GGCCAACTCC
3121 CAGTTCATCA CCGAGCTTAT CTCCGGCCAG CACACCACCG CCACCCAGAC CATCCTCGGC
3181 CGCAACATCT TCCGCGTGGA CTCCCAGGCC TGCAACCTCA ACGACACCAC CTACGGCGTG
3241 AACCGCGCCG TGTTCTACCA CGACGCCTCC GAGGGCTCCC AGCGCTCCGT GTACGAGGGC
3301 TACATCCGCA CCACCGGCAT CGACAACCCG CGCGTGCAGA ACATCAACAC CTACCTTCCG
3361 GGCGAGAACT CCGACATCCC GACCCCGGAG GACTACACCC ACATCCTCTC CACCACCATC
3421 AACCTCACCG GCGGCCTCCG CCAGGTGGCC TCCAACCGCC GCTCCTCCCT CGTGATGTAC
3481 GGCTGGACCC ACAAGTCCCT CGCCCGCAAC AACACCATCA ACCCGGACCG CATCACCCAG
3541 ATCCCGCTCA CCAAGGTGGA CACCCGCGGC ACCGGCGTGT CCTACGTGAA CGACCCCGGC
3601 TTCATCGGCG GCGCCCTCCT CCAGCGCACC GACCACGGCT CCCTCGGCGT GCTCCGCGTG
3661 CAGTTCCCGC TCCACCTCCG CCAGCAGTAC CGCATCCGCG TGCGCTACGC CTCCACCACC
3721 AACATCCGCC TCTCCGTGAA CGGCTCCTTC GGCACCATCT CCCAGAACCT CCCGTCCACC
3781 ATGCGCCTCG GCGAGGACCT CCGCTACGGC TCCTTCGCCA TCCGCGAGTT CAACACCTCC
3841 ATCCGCCCGA CCGCCTCCCC GGACCAGATC CGCCTCACCA TCGAGCCGTC CTTCATCCGC
3901 CAGGAGGTGT ACGTGGACCG CATCGAGTTC ATCCCGGTGA ACTAAGAGCT C
SEQ ID NO:3(Cryl Ab-Cry9Aa)
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu
Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu
Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu
Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu
Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp
Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala
Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile
Gly Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly
Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val
Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe
Arg Gly Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu
Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg
Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu
Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val
Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe
Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala
Glu Phe Asn Asn Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr Lys Ser Thr
Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu
Arg Arg Thr Ser Pro Gly Gln Ile Ser Thr Leu Arg Val Asn Ile Thr Ala Pro Leu Ser
Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Thr Ser
Ile Asp Gly Arg Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Met Ser Ser Gly Ser Asn
Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe Ser Asn Gly
Ser Ser Val Phe Thr Leu Ser Ala His Val Phe Asn Ser Gly Asn Glu Val Tyr Ile Asp
Arg Ile Glu Phe Val Pro Ala Glu Val Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala
Gln Lys Ala Val Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val
Thr Asp Tyr His Ile Asp Gln Val Arg Pro Gly Lys Gly Lys Gly Gly Asn Gln Asn Lys
His Gly Ile Ile Gly Ala Ser Asn Cys Gly Cys Ala Ser Asp Asp Val Ala Lys Tyr Pro
Leu Ala Asn Asn Pro Tyr Ser Ser Ala Leu Asn Leu Asn Ser Cys Gln Asn Ser Ser Ile
Leu Asn Trp Ile Asn Ile Ile Gly Asp Ala Ala Lys Glu Ala Val Ser Ile Gly Thr Thr
Ile Val Ser Leu Ile Thr Ala Pro Ser Leu Thr Gly Leu Ile Ser Ile Val Tyr Asp Leu
Ile Gly Lys Val Leu Gly Gly Ser Ser Gly Gln Ser Ile Ser Asp Leu Ser Ile Cys Asp
Leu Leu Ser Ile Ile Asp Leu Arg Val Ser Gln Ser Val Leu Asn Asp Gly Ile Ala Asp
Phe Asn Gly Sar Val Leu Leu Tyr Arg Asn Tyr Leu Glu Ala Leu Asp Ser Trp Asn Lys
Asn Pro Asn Ser Ala Ser Ala Glu Glu Leu Arg Thr Arg Phe Arg Ile Ala Asp Ser Glu
Phe Asp Arg Ile Leu Thr Arg Gly Ser Leu Thr Asn Gly Gly Ser Leu Ala Arg Gln Asn
Ala Gln Ile Leu Leu Leu Pro Ser Phe Ala Ser Ala Ala Phe Phe His Leu Leu Leu Leu
Arg Asp Ala Thr Arg Tyr Gly Thr Asn Trp Gly Leu Tyr Asn Ala Thr Pro Phe Ile Asn
Tyr Gln Ser Lys Leu Val Glu Leu Ile Glu Leu Tyr Thr Asp Tyr Cys Val His Trp Tyr
Asn Arg Gly Phe Asn Glu Leu Arg Gln Arg Gly Thr Ser Ala Thr Ala Trp Leu Glu Phe
His Arg Tyr Arg Arg Glu Met Thr Leu Met Val Leu Asp Ile Val Ala Ser Phe Ser Ser
Leu Asp Ile Thr Asn Tyr Pro Ile Glu Thr Asp Phe Gln Leu Ser Arg Val Ile Tyr Thr
Asp Pro Ile Gly Phe Val His Arg Ser Ser Leu Arg Gly Glu Ser Trp Phe Ser Phe Val
Asn Arg Ala Asn Phe Ser Asp Leu Glu Asn Ala Ile Pro Asn Pro Arg Pro Ser Trp Phe
Leu Asn Asn Met Ile Ile Ser Thr Gly Ser Leu Thr Leu Pro Val Ser Pro Ser Thr Asp
Arg Ala Arg Val Trp Tyr Gly Ser Arg Asp Arg Ile Ser Pro Ala Asn Ser Gln Phe Ile
Thr Glu Leu Ile Ser Gly Gln His Thr Thr Ala Thr Gln Thr Ile Leu Gly Arg Asn Ile
Phe Arg Val Asp Ser Gln Ala Cys Asn Leu Asn Asp Thr Thr Tyr Gly Val Asn Arg Ala
Val Phe Tyr His Asp Ala Ser Glu Gly Ser Gln Arg Ser Val Tyr Glu Gly Tyr Ile Arg
Thr Thr Gly Ile Asp Asn Pro Arg Val Gln Asn Ile Asn Thr Tyr Leu Pro Gly Glu Asn
Ser Asp Ile Pro Thr Pro Glu Asp Tyr Thr His Ile Leu Ser Thr Thr Ile Asn Leu Thr
Gly Gly Leu Arg Gln Val Ala Ser Asn Arg Arg Ser Ser Leu Val Met Tyr Gly Trp Thr
His Lys Ser Leu Ala Arg Asn Asn Thr Ile Asn Pro Asp Arg Ile Thr Gln Ile Pro Leu
Thr Lys Val Asp Thr Arg Gly Thr Gly Val Ser Tyr Val Asn Asp Pro Gly Phe Ile Gly
Gly Ala Leu Leu Gln Arg Thr Asp His Gly Ser Leu Gly Val Leu Arg Val Gln Phe Pro
Leu His Leu Arg Gln Gln Tyr Arg Ile Arg Val Arg Tyr Ala Ser Thr Thr Asn Ile Arg
Leu Ser Val Asn Gly Ser Phe Gly Thr Ile Ser Gln Asn Leu Pro Ser Thr Met Arg Leu
Gly Glu Asp Leu Arg Tyr Gly Ser Phe Ala Ile Arg Glu Phe Asn Thr Ser Ile Arg Pro
Thr Ala Ser Pro Asp Gln Ile Arg Leu Thr Ile Glu Pro Ser Phe Ile Arg Gln Glu Val
Tyr Val Asp Arg Ile Glu Phe Ile Pro Val Asn
SEQ ID NO:4(CrylAc-Cry9Aa)
Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu Ser Asn Pro Glu
Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly Tyr Thr Pro Ile Asp Ile Ser Leu
Ser Leu Thr Gln Phe Leu Leu Ser Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu
Val Asp Ile Ile Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile
Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala Ile Ser Arg Leu
Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu Ser Phe Arg Glu Trp Glu Ala Asp
Pro Thr Asn Pro Ala Leu Arg Glu Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala
Leu Thr Thr Ala Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser Val Phe Gly Gln
Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg Tyr Asn Asp Leu Thr Arg Leu Ile
Gly Asn Tyr Thr Asp His Ala Val Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly
Pro Asp Ser Arg Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val
Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro Ile Arg Thr Val
Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val Leu Glu Asn Phe Asp Gly Ser Phe
Arg Gly Ser Ala Gln Gly Ile Glu Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu
Asn Ser Ile Thr Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln
Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro Leu Tyr Gly Thr
Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala Gln Leu Gly Gln Gly Val Tyr Arg
Thr Leu Ser Ser Thr Leu Tyr Arg Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu
Ser Val Leu Asp Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val
Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln Asn Asn Asn Val
Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His Val Ser Met Phe Arg Ser Gly Phe
Ser Asn Ser Ser Val Ser Ile Ile Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala
Glu Phe Asn Asn Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys Gly Asn
Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly Asp Leu Val Arg
Leu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly Tyr Ile Glu Val Pro Ile His Phe
Pro Ser Thr Ser Thr Arg Tyr Arg Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His
Leu Asn Val Asn Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr
Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala Asn Ala Phe Thr
Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe Ser Gly Thr Ala Gly Val Ile Ile
Asp Arg Phe Glu Phe Ile Pro Val Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg
Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn
Val Thr Asp Tyr His Ile Asp Gln Val Pro Gly Lys Gly Lys Gly Gly Asn Gln Asn Lys
His Gly Ile Ile Gly Ala Ser Asn Cys Gly Cys Ala Ser Asp Asp Val Ala Lys Tyr Pro
Leu Ala Asn Asn Pro Tyr Ser Ser Ala Leu Asn Leu Asn Ser Cys Gln Asn Ser Ser Ile
Leu Asn Trp Ile Asn Ile Ile Gly Asp Ala Ala Lys Glu Ala Val Ser Ile Gly Thr Thr
Ile Val Ser Leu Ile Thr Ala Pro Ser Leu Thr Gly Leu Ile Ser Ile Val Tyr Asp Leu
Ile Gly Lys Val Leu Gly Gly Ser Ser Gly Gln Ser Ile Ser Asp Leu Ser Ile Cys Asp
Leu Leu Ser Ile Ile Asp Leu Arg Val Ser Gln Ser Val Leu Asn Asp Gly Ile Ala Asp
Phe Asn Gly Ser Val Leu Leu Tyr Arg Asn Tyr Leu Glu Ala Leu Asp Ser Trp Asn Lys
Asn Pro Asn Ser Ala Ser Ala Glu Glu Leu Arg Thr Arg Phe Arg Ile Ala Asp Ser Glu
Phe Asp Arg Ile Leu Thr Arg Gly Ser Leu Thr Asn Gly Gly Ser Leu Ala Arg Gln Asn
Ala Gln Ile Leu Leu Leu Pro Ser Phe Ala Ser Ala Ala Phe Phe His Leu Leu Leu Leu
Arg Asp Ala Thr Arg Tyr Gly Thr Asn Trp Gly Leu Tyr Asn Ala Thr Pro Phe Ile Asn
Tyr Gln Ser Lys Leu Val Glu Leu Ile Glu Leu Tyr Thr Asp Tyr Cys Val His Trp Tyr
Asn Arg Gly Phe Asn Glu Leu Arg Gln Arg Gly Thr Ser Ala Thr Ala Trp Leu Glu Phe
His Arg Tyr Arg Arg Glu Met Thr Leu Met Val Leu Asp Ile Val Ala Ser Phe Ser Ser
Leu Asp Ile Thr Asn Tyr Pro Ile Glu Thr Asp Phe Gln Leu Ser Arg Val Ile Tyr Thr
Asp Pro Ile Gly Phe Val His Arg Ser Ser Leu Arg Gly Glu Ser Trp Phe Ser Phe Val
Asn Arg Ala Asn Phe Ser Asp Leu Glu Asn Ala Ile Pro Asn Pro Arg Pro Ser Trp Phe
Leu Asn Asn Met Ile Ile Ser Thr Gly Ser Leu Thr Lau Pro Val Ser Pro Ser Thr Asp
Arg Ala Arg Val Trp Tyr Gly Ser Arg Asp Arg Ile Ser Pro Ala Asn Ser Gln Phe Ile
Thr Glu Leu Ile Ser Gly Gln His Thr Thr Ala Thr Gln Thr Ile Leu Gly Arg Asn Ile
Phe Arg Val Asp Ser Gln Ala Cys Asn Leu Asn Asp Thr Thr Tyr Gly Val Asn Arg Ala
Val Phe Tyr His Asp Ala Ser Glu Gly Ser Gln Arg Ser Val Tyr Glu Gly Tyr Ile Arg
Thr Thr Gly Ile Asp Asn Pro Arg Val Gln Asn Ile Asn Thr Tyr Leu Pro Gly Glu Asn
Ser Asp Ile Pro Thr Pro Glu Asp Tyr Thr His Ile Leu Ser Thr Thr Ile Asn Leu Thr
Gly Gly Leu Arg Gln Val Ala Ser Asn Arg Arg Ser Ser Leu Val Met Tyr Gly Trp Thr
His Lys Ser Leu Ala Arg Asn Asn Thr Ile Asn Pro Asp Arg Ile Thr Gln Ile Pro Leu
Thr Lys Val Asp Thr Arg Gly Thr Gly Val Ser Tyr Val Asn Asp Pro Gly Phe Ile Gly
Gly Ala Leu Leu Gln Arg Thr Asp His Gly Ser Leu Gly Val Leu Arg Val Gln Phe Pro
Leu His Leu Arg Gln Gln Tyr Arg Ile Arg Val Arg Tyr Ala Ser Thr Thr Asn Ile Arg
Leu Ser Val Asn Gly Ser Phe Gly Thr Ile Ser Gln Asn Leu Pro Ser Thr Met Arg Leu
Gly Glu Asp Leu Arg Tyr Gly Ser Phe Ala Ile Arg Glu Phe Asn Thr Ser Ile Arg Pro
Thr Ala Ser Pro Asp Gln Ile Arg Leu Thr Ile Glu Pro Ser Phe Ile Arg Gln Glu Val
Tyr Val Asp Arg Ile Glu Phe Ile Pro Val Asn
SEQ ID NO:5(Cry9Aa)
1 GGATCCAACC CCGGGAAGGG TAAAGGGGGT AACCAGAACA AGCACGGCAT CATCGGCGCC
61 TCCAACTGCG GCTGCGCCTC CGACGACGTG GCCAAGTACC CGCTCGCCAA CAACCCGTAC
121 TCCTCCGCCC TCAACCTCAA CTCCTGCCAG AACTCCTCCA TCCTCAACTG GATCAACATC
181 ATCGGCGACG CCGCCAAGGA GGCCGTGTCC ATCGGCACCA CCATCGTGTC CCTCATCACC
241 GCCCCGTCCC TCACCGGCCT CATCTCCATC GTGTACGACC TCATCGGCAA GGTGCTCGGC
301 GGCTCCTCCG GCCAGTCCAT CTCCGACCTC TCCATCTGCG ACCTCCTCTC CATCATCGAC
361 CTCCGCGTGT CCCAGTCCGT GCTCAACGAC GGCATCGCCG ACTTCAACGG CTCCGTGCTC
421 CTCTACCGCA ACTACCTTGA GGCCCTCGAC TCCTGGAACA AGAACCCGAA CTCCGCCTCC
481 GCCGAGGAGC TTCGCACCCG CTTCCGCATC GCCGACTCCG AGTTCGACCG CATCCTCACC
541 CGCGGCTCCC TCACCAACGG CGGCTCCCTC GCCCGCCAGA ACGCCCAGAT CCTCCTCCTC
601 CCGTCCTTCG CCTCCGCCGC CTTCTTCCAC CTCCTCCTCC TCCGCGACGC CACCCGCTAC
661 GGCACCAACT GGGGCCTCTA CAACGCCACC CCGTTCATCA ACTACCAGTC CAAGCTCGTG
721 GAACTCATCG AACTCTACAC CGACTACTGC GTGCACTGGT ACAACCGCGG CTTCAACGAG
781 CTTCGCCAGC GCGGCACCTC CGCCACCGCC TGGCTGGAGT TCCACCGCTA CCGCCGCGAG
841 ATGACCCTCA TGGTGCTCGA CATCGTGGCC TCCTTCTCCT CCCTCGACAT CACCAACTAC
901 CCGATCGAGA CCGACTTCCA GCTCTCCCGC GTGATCTACA CCGACCCGAT CGGCTTCGTG
961 CACCGCTCCT CCCTCCGCGG CGAGTCCTGG TTCTCCTTCG TGAACCGCGC CAACTTCTCC
1021 GACCTGGAGA ACGCCATCCC GAACCCGCGC CCGTCCTGGT TCCTCAACAA CATGATCATC
1081 TCCACCGGCT CCCTCACCCT CCCGGTGTCC CCGTCCACCG ACCGCGCCCG CGTGTGGTAC
1141 GGCTCCCGCG ACCGCATCTC CCCGGCCAAC TCCCAGTTCA TCACCGAGCT TATCTCCGGC
1201 CAGCACACCA CCGCCACCCA GACCATCCTC GGCCGCAACA TCTTCCGCGT GGACTCCCAG
1261 GCCTGCAACC TCAACGACAC CACCTACGGC GTGAACCGCG CCGTGTTCTA CCACGACGCC
1321 TCCGAGGGCT CCCAGCGCTC CGTGTACGAG GGCTACATCC GCACCACCGG CATCGACAAC
1381 CCGCGCGTGC AGAACATCAA CACCTACCTT CCGGGCGAGA ACTCCGACAT CCCGACCCCG
1441 GAGGACTACA CCCACATCCT CTCCACCACC ATCAACCTCA CCGGCGGCCT CCGCCAGGTG
1501 GCCTCCAACC GCCGCTCCTC CCTCGTGATG TACGGCTGGA CCCACAAGTC CCTCGCCCGC
1561 AACAACACCA TCAACCCGGA CCGCATCACC CAGATCCCGC TCACCAAGGT GGACACCCGC
1621 GGCACCGGCG TGTCCTACGT GAACGACCCC GGCTTCATCG GCGGCGCCCT CCTCCAGCGC
1681 ACCGACCACG GCTCCCTCGG CGTGCTCCGC GTGCAGTTCC CGCTCCACCT CCGCCAGCAG
1741 TACCGCATCC GCGTGCGCTA CGCCTCCACC ACCAACATCC GCCTCTCCGT GAACGGCTCC
1801 TTCGGCACCA TCTCCCAGAA CCTCCCGTCC ACCATGCGCC TCGGCGAGGA CCTCCGCTAC
1861 GGCTCCTTCG CCATCCGCGA GTTCAACACC TCCATCCGCC CGACCGCCTC CCCGGACCAG
1921 ATCCGCCTCA CCATCGAGCC GTCCTTCATC CGCCAGGAGG TGTACGTGGA CCGCATCGAG
1981 TTCATCCCGG TGAACTAAGA GCTC
SEQ ID NO:6(CrylAb)
1 GGATCCACCA TGGACAACAA CCCCAACATC AACGAGTGCA TCCCCTACAA CTGCCTGAGC
61 AACCCCGAGG TGGAGGTGCT GGGCGGCGAG CGCATCGAGA CCGGCTACAC CCCCATCGAC
121 ATCAGCCTGA GCCTGACCCA GTTCCTGCTG AGCGAGTTCG TGCCCGGCGC CGGCTTCGTG
181 CTGGGCCTGG TGGACATCAT CTGGGGCATC TTCGGCCCCA GCCAGTGGGA CGCCTTCCTG
241 GTGCAGATCG AGCAGCTGAT CAACCAGCGC ATCGAGGAGT TCGCCCGCAA CCAGGCCATC
301 AGCCGCCTGG AGGGCCTGAG CAACCTGTAC CAAATCTACG CCGAGAGCTT CCGCGAGTGG
361 GAGGCCGACC CCACCAACCC CGCCCTGCGC GAGGAGATGC GCATCCAGTT CAACGACATG
421 AACAGCGCCC TGACCACCGC CATCCCCCTG TTCGCCGTGC AGAACTACCA GGTGCCCCTG
481 CTGAGCGTGT ACGTGCAGGC CGCCAACCTG CACCTGAGCG TGCTGCGCGA CGTCAGCGTG
541 TTCGGCCAGC GCTGGGGCTT CGACGCCGCC ACCATCAACA GCCGCTACAA CGACCTGACC
601 CGCCTGATCG GCAACTACAC CGACCACGCC GTGCGCTGGT ACAACACCGG CCTGGAGCGC
661 GTGTGGGGTC CCGACAGCCG CGACTGGATC AGGTACAACC AGTTCCGCCG CGAGCTGACC
721 CTGACCGTGC TGGACATCGT GAGCCTGTTC CCCAACTACG ACAGCCGCAC CTACCCCATC
781 CGCACCGTGA GCCAGCTGAC CCGCGAGATT TACACCAACC CCGTGCTGGA GAACTTCGAC
841 GGCAGCTTCC GCGGCAGCGC CCAGGGCATC GAGGGCAGCA TCCGCAGCCC CCACCTGATG
901 GACATCCTGA ACAGCATCAC CATCTACACC GACGCCCACC GCGGCGAGTA CTACTGGAGC
961 GGCCACCAGA TCATGGCCAG CCCCGTCGGC TTCAGCGGCC CCGAGTTCAC CTTCCCCCTG
1021 TACGGCACCA TGGGCAACGC TGCACCTCAG CAGCGCATAG TGGCACAGCT GGGCCAGGGA
1081 GTGTACCGCA CCCTGAGCAG CACCCTGTAC CGTCGACCTT TCAACATCGG CATCAACAAC
1141 CAGCAGCTGA GCGTGCTGGA CGGCACCGAG TTCGCCTACG GCACCAGCAG CAACCTGCCC
1201 AGCGCCGTGT ACCGCAAGAG CGGCACCGTG GACAGCCTGG ACGAGATCCC CCCTCAGAAC
1261 AACAACGTGC CACCTCGACA GGGCTTCAGC CACCGTCTGA GCCACGTGAG CATGTTCCGC
1321 AGTGGCTTCA GCAACAGCAG CGTGAGCATC ATCCGTGCAC CTATGTTCAG CTGGATTCAC
1381 CGCAGTGCCG AGTTCAACAA CATCATCCCC AGCAGCCAGA TCACCCAGAT CCCCCTGACC
1441 AAGAGCACCA ACCTGGGCAG CGGCACCAGC GTGGTGAAGG GCCCCGGCTT CACCGGCGGC
1501 GACATCCTGC GCCGCACCAG CCCCGGCCAG ATCAGCACCC TGCGCGTGAA CATCACCGCC
1561 CCCCTGAGCC AGCGCTACCG CGTCCGCATC CGCTACGCCA GCACCACCAA CCTGCAGTTC
1621 CACACCAGCA TCGACGGCCG CCCCATCAAC CAGGGCAACT TCAGCGCCAC CATGAGCAGC
1681 GGCAGCAACC TGCAGAGCGG CAGCTTCCGC ACCGTGGGCT TCACCACCCC CTTCAACTTC
1741 AGCAACGGCA GCAGCGTGTT CACCCTGAGC GCCCACGTGT TCAACAGCGG CAACGAGGTG
1801 TACATCGACC GCATCGAGTT CGTGCCCGCC GAGGTGACCT TCGAGGCCGA GTACGACCTG
1861 GAGAGGGCTC AGAAGGCCGT GAACGAGCTG TTCACCAGCA GCAACCAGAT CGGCCTGAAG
1921 ACCGACGTGA CCGACTACCA CATCGATCAG GTGCGACCCG GGAAAGAGCT C
SEQ ID NO:7(CrylAc)
1 GGATCCACCA TGGACAACAA CCCCAACATC AACGAGTGCA TCCCCTACAA CTGCCTGAGC
61 AACCCCGAGG TGGAGGTGCT GGGCGGCGAG CGCATCGAGA CCGGCTACAC CCCCATCGAC
121 ATCAGCCTGA GCCTGACCCA GTTCCTGCTG AGCGAGTTCG TGCCCGGCGC CGGCTTCGTG
181 CTGGGCCTGG TGGACATCAT CTGGGGCATC TTCGGCCCCA GCCAGTGGGA CGCCTTCCTG
241 GTGCAGATCG AGCAGCTGAT CAACCAGCGC ATCGAGGAGT TCGCCCGCAA CCAGGCCATC
301 AGCCGCCTGG AGGGCCTGAG CAACCTGTAC CAAATCTACG CCGAGAGCTT CCGCGAGTGG
361 GAGGCCGACC CCACCAACCC CGCCCTGCGC GAGGAGATGC GCATCCAGTT CAACGACATG
421 AACAGCGCCC TGACCACCGC CATCCCCCTG TTCGCCGTGC AGAACTACCA GGTGCCCCTG
481 CTGAGCGTGT ACGTGCAGGC CGCCAACCTG CACCTGAGCG TGCTGCGCGA CGTCAGCGTG
541 TTCGGCCAGC GCTGGGGCTT CGACGCCGCC ACCATCAACA GCCGCTACAA CGACCTGACC
601 CGCCTGATCG GCAACTACAC CGACCACGCC GTGCGCTGGT ACAACACCGG CCTGGAGCGC
661 GTGTGGGGTC CCGACAGCCG CGACTGGATC AGGTACAACC AGTTCCGCCG CGAGCTGACC
721 CTGACCGTGC TGGACATCGT GAGCCTGTTC CCCAACTACG ACAGCCGCAC CTACCCCATC
781 CGCACCGTGA GCCAGCTGAC CCGCGAGATT TACACCAACC CCGTGCTGGA GAACTTCGAC
841 GGCAGCTTCC GCGGCAGCGC CCAGGGCATC GAGGGCAGCA TCCGCAGCCC CCACCTGATG
901 GACATCCTGA ACAGCATCAC CATCTACACC GACGCCCACC GCGGCGAGTA CTACTGGAGC
961 GGCCACCAGA TCATGGCCAG CCCCGTCGGC TTCAGCGGCC CCGAGTTCAC CTTCCCCCTG
1021 TACGGCACCA TGGGCAACGC TGCACCTCAG CAGCGCATAG TGGCACAGCT GGGCCAGGGA
1081 GTGTACCGCA CCCTGAGCAG CACCCTGTAC CGTCGACCTT TCAACATCGG CATCAACAAC
1141 CAGCAGCTGA GCGTGCTGGA CGGCACCGAG TTCGCCTACG GCACCAGCAG CAACCTGCCC
1201 AGCGCCGTGT ACCGCAAGAG CGGCACCGTG GACAGCCTGG ACGAGATCCC CCCTCAGAAC
1261 AACAACGTGC CACCTCGACA GGGCTTCAGC CACCGTCTGA GCCACGTGAG CATGTTCCGC
1321 AGTGGCTTCA GCAACAGCAG CGTGAGCATC ATCCGTGCAC CTATGTTCAG CTGGATTCAC
1381 CGCAGTGCCG AGTTCAACAA CATCATCGCC TCCGACTCCA TCACCCAGAT CCCGGCTGTG
1441 AAGGGCAACT TCCTCTTCAA CGGCTCCGTG ATCTCCGGTC CAGGCTTCAC CGGTGGCGAC
1501 CTCGTGCGCC TCAACTCCTC CGGCAACAAC ATCCAGAACC GCGGTTACAT CGAGGTGCCG
1561 ATCCACTTCC CGTCCACCTC CACCCGCTAC CGCGTGCGCG TGCGCTACGC CTCCGTGACC
1621 CCGATCCACC TCAACGTGAA CTGGGGCAAC TCCTCCATCT TCTCCAACAC CGTGCCAGCC
1681 ACCGCCACCT CCCTCGACAA CCTCCAGTCC TCCGACTTCG GCTACTTCGA GTCCGCCAAC
1741 GCCTTCACCT CCTCCCTCGG CAACATCGTG GGCGTGCGCA ACTTCTCCGG CACCGCTGGC
1801 GTGATCATCG ACCGCTTCGA GTTCATCCCG GTGACCGCCA CCCTCGAAGC CGAGTACAAC
1861 CTTGAGCGCG CTCAGAAGGC CGTGAACGCC CTCTTCACCT CCACCAACCA GCTCGGCCTC
1921 AAGACCAACG TGACCGACTA CCACATCGAC CAGGTGCCCG GG AAAGAGCT C
SEQ ID NO:8(CrylCa)
1 GGATCCACCC CCGGGAAAGG TAAGAAGGGC AAGGGAGGCC AGTGCATCCC GTACAACTGC
61 CTCTCCAACC CGGAGGAGGT GCTCCTCGAC GGCGAGCGCA TCTCCACCGG CAACTCCTCC
121 ATCGACATCT CCCTCTCCCT CGTGCAGTTC CTCGTGTCCA ACTTCGTGCC GGGCGGCGGC
181 TTCCTCGTGG GCCTCATCGA CTTCGTGTGG GGCATCGTGG GCCCGTCCCA GTGGGACGCC
241 TTCCTCGTGC AGATCGAGCA GCTCATCAAC GAGCGCATCG CCGAGTTCGC CCGCAACGCC
301 GCCATCGCCA ACCTCGAAGG CCTCGGCAAC AACTTCAACA TCTACGTGGA GGCCTTCAAG
361 GAGTGGGAGG AGGACCCGAA CAACCCGGAG ACCCGCACCC GCGTGATCGA CCGCTTCCGC
421 ATCCTCGACG GCCTCCTTGA GCGCGACATC CCGTCCTTCC GCATCTCCGG CTTCGAGGTG
481 CCGCTCCTCT CCGTGTACGC CCAGGCCGCC AACCTCCACC TCGCCATCCT CCGCGACTCC
541 GTGATCTTCG GCGAGCGCTG GGGCCTCACC ACCATCAACG TGAACGAGAA CTACAACCGC
601 CTCATCCGCC ACATCGACGA GTACGCCGAC CACTGCGCCA ACACCTACAA CCGCGGCCTC
661 AACAACCTCC CGAAGTCCAC CTACCAGGAC TGGATCACCT ACAACCGCCT CCGCCGCGAC
721 CTCACCCTCA CCGTGCTCGA CATCGCCGCC TTCTTCCCGA ACTACGACAA CCGCCGCTAC
781 CCGATCCAGC CGGTGGGCCA GCTCACCCGC GAGGTGTACA CCGACCCGCT CATCAACTTC
841 AACCCGCAGC TCCAGTCCGT GGCCCAGCTC CCGACCTTCA ACGTGATGGA GTCCTCCCGC
901 ATCCGCAACC CGCACCTCTT CGACATCCTC AACAACCTCA CCATCTTCAC CGACTGGTTC
961 TCCGTGGGCC GCAACTTCTA CTGGGGCGGC CACCGCGTGA TCTCCTCCCT CATCGGCGGC
1021 GGCAACATCA CCTCCCCGAT CTACGGCCGC GAGGCCAACC AGGAGCCGCC GCGCTCCTTC
1081 ACCTTCAACG GCCCGGTGTT CCGCACCCTC TCCAACCCGA CCCTCCGCCT CCTCCAGCAG
1141 CCGTGGCCGG CCCCGCCGTT CAACCTCCGC GGCGTGGAGG GCGTGGAGTT CTCCACCCCG
1201 ACCAACTCCT TCACCTACCG CGGCCGCGGC ACCGTGGACT CCCTCACCGA GCTTCCGCCG
1261 GAGGACAACT CCGTGCCGCC GCGCGAGGGC TACTCCCACC GCCTCTGCCA CGCCACCTTC
1321 GTGCAGCGCT CCGGCACCCC GTTCCTCACC ACCGGCGTGG TGTTCTCCTG GACCGACCGC
1381 TCCGCCACCC TCACCAACAC CATCGACCCG GAGCGCATCA ACCAGATCCC GCTCGTGAAG
1441 GGCTTCCGCG TGTGGGGCGG CACCTCCGTG ATCACCGGTC CGGGCTTCAC CGGCGGCGAC
1501 ATCCTCCGCC GCAACACCTT CGGCGACTTC GTGTCCCTCC AGGTGAACAT CAACTCCCCG
1561 ATCACCCAGC GCTACCGCCT CCGCTTCCGC TACGCCTCCT CCCGCGACGC CCGCGTGATC
1621 GTGCTCACCG GCGCCGCCTC CACCGGCGTG GGCGGCCAGG TGTCCGTGAA CATGCCGCTC
1681 CAGAAGACCA TGGAGATCGG CGAGAACCTC ACCTCCCGCA CCTTCCGCTA CACCGACTTC
1741 TCCAACCCGT TCTCCTTCCG CGCCAACCCG GACATCATCG GCATCTCCGA GCAGCCGCTC
1801 TTCGGCGCCG GCTCCATCTC CTCCGGCGAA CTCTACATCG ACAAGATCGA GATCATCCTC
1861 GCCGACTAAG AGCTC
SEQ ID NO:9(crylAb-crylCa)
1 GGATCCACCA TGGACAACAA CCCCAACATC AACGAGTGCA TCCCCTACAA CTGCCTGAGC
61 AACCCCGAGG TGGAGGTGCT GGGCGGCGAG CGCATCGAGA CCGGCTACAC CCCCATCGAC
121 ATCAGCCTGA GCCTGACCCA GTTCCTGCTG AGCGAGTTCG TGCCCGGCGC CGGCTTCGTG
181 CTGGGCCTGG TGGACATCAT CTGGGGCATC TTCGGCCCCA GCCAGTGGGA CGCCTTCCTG
241 GTGCAGATCG AGCAGCTGAT CAACCAGCGC ATCGAGGAGT TCGCCCGCAA CCAGGCCATC
301 AGCCGCCTGG AGGGCCTGAG CAACCTGTAC CAAATCTACG CCGAGAGCTT CCGCGAGTGG
361 GAGGCCGACC CCACCAACCC CGCCCTGCGC GAGGAGATGC GCATCCAGTT CAACGACATG
421 AACAGCGCCC TGACCACCGC CATCCCCCTG TTCGCCGTGC AGAACTACCA GGTGCCCCTG
481 CTGAGCGTGT ACGTGCAGGC CGCCAACCTG CACCTGAGCG TGCTGCGCGA CGTCAGCGTG
541 TTCGGCCAGC GCTGGGGCTT CGACGCCGCC ACCATCAACA GCCGCTACAA CGACCTGACC
601 CGCCTGATCG GCAACTACAC CGACCACGCC GTGCGCTGGT ACAACACCGG CCTGGAGCGC
661 GTGTGGGGTC CCGACAGCCG CGACTGGATC AGGTACAACC AGTTCCGCCG CGAGCTGACC
721 CTGACCGTGC TGGACATCGT GAGCCTGTTC CCCAACTACG ACAGCCGCAC CTACCCCATC
781 CGCACCGTGA GCCAGCTGAC CCGCGAGATT TACACCAACC CCGTGCTGGA GAACTTCGAC
841 GGCAGCTTCC GCGGCAGCGC CCAGGGCATC GAGGGCAGCA TCCGCAGCCC CCACCTGATG
901 GACATCCTGA ACAGCATCAC CATCTACACC GACGCCCACC GCGGCGAGTA CTACTGGAGC
961 GGCCACCAGA TCATGGCCAG CCCCGTCGGC TTCAGCGGCC CCGAGTTCAC CTTCCCCCTG
1021 TACGGCACCA TGGGCAACGC TGCACCTCAG CAGCGCATAG TGGCACAGCT GGGCCAGGGA
1081 GTGTACCGCA CCCTGAGCAG CACCCTGTAC CGTCGACCTT TCAACATCGG CATCAACAAC
1141 CAGCAGCTGA GCGTGCTGGA CGGCACCGAG TTCGCCTACG GCACCAGCAG CAACCTGCCC
1201 AGCGCCGTGT ACCGCAAGAG CGGCACCGTG GACAGCCTGG ACGAGATCCC CCCTCAGAAC
1261 AACAACGTGC CACCTCGACA GGGCTTCAGC CACCGTCTGA GCCACGTGAG CATGTTCCGC
1321 AGTGGCTTCA GCAACAGCAG CGTGAGCATC ATCCGTGCAC CTATGTTCAG CTGGATTCAC
1381 CGCAGTGCCG AGTTCAACAA CATCATCCCC AGCAGCCAGA TCACCCAGAT CCCCCTGACC
1441 AAGAGCACCA ACCTGGGCAG CGGCACCAGC GTGGTGAAGG GCCCCGGCTT CACCGGCGGC
1501 GACATCCTGC GCCGCACCAG CCCCGGCCAG ATCAGCACCC TGCGCGTGAA CATCACCGCC
1561 CCCCTGAGCC AGCGCTACCG CGTCCGCATC CGCTACGCCA GCACCACCAA CCTGCAGTTC
1621 CACACCAGCA TCGACGGCCG CCCCATCAAC CAGGGCAACT TCAGCGCCAC CATGAGCAGC
1681 GGCAGCAACC TGCAGAGCGG CAGCTTCCGC ACCGTGGGCT TCACCACCCC CTTCAACTTC
1741 AGCAACGGCA GCAGCGTGTT CACCCTGAGC GCCCACGTGT TCAACAGCGG CAACGAGGTG
1801 TACATCGACC GCATCGAGTT CGTGCCCGCC GAGGTGACCT TCGAGGCCGA GTACGACCTG
1861 GAGAGGGCTC AGAAGGCCGT GAACGAGCTG TTCACCAGCA GCAACCAGAT CGGCCTGAAG
1921 ACCGACGTGA CCGACTACCA CATCGATCAG GTGCGAGGCC CCGGGAAAGG TAAGAAGGGC
1981 AAGGGAGGCC AGTGCATCCC GTACAACTGC CTCTCCAACC CGGAGGAGGT GCTCCTCGAC
2041 GGCGAGCGCA TCTCCACCGG CAACTCCTCC ATCGACATCT CCCTCTCCCT CGTGCAGTTC
2101 CTCGTGTCCA ACTTCGTGCC GGGCGGCGGC TTCCTCGTGG GCCTCATCGA CTTCGTGTGG
2161 GGCATCGTGG GCCCGTCCCA GTGGGACGCC TTCCTCGTGC AGATCGAGCA GCTCATCAAC
2221 GAGCGCATCG CCGAGTTCGC CCGCAACGCC GCCATCGCCA ACCTCGAAGG CCTCGGCAAC
2281 AACTTCAACA TCTACGTGGA GGCCTTCAAG GAGTGGGAGG AGGACCCGAA CAACCCGGAG
2341 ACCCGCACCC GCGTGATCGA CCGCTTCCGC ATCCTCGACG GCCTCCTTGA GCGCGACATC
2401 CCGTCCTTCC GCATCTCCGG CTTCGAGGTG CCGCTCCTCT CCGTGTACGC CCAGGCCGCC
2461 AACCTCCACC TCGCCATCCT CCGCGACTCC GTGATCTTCG GCGAGCGCTG GGGCCTCACC
2521 ACCATCAACG TGAACGAGAA CTACAACCGC CTCATCCGCC ACATCGACGA GTACGCCGAC
2581 CACTGCGCCA ACACCTACAA CCGCGGCCTC AACAACCTCC CGAAGTCCAC CTACCAGGAC
2641 TGGATCACCT ACAACCGCCT CCGCCGCGAC CTCACCCTCA CCGTGCTCGA CATCGCCGCC
2701 TTCTTCCCGA ACTACGACAA CCGCCGCTAC CCGATCCAGC CGGTGGGCCA GCTCACCCGC
2761 GAGGTGTACA CCGACCCGCT CATCAACTTC AACCCGCAGC TCCAGTCCGT GGCCCAGCTC
2821 CCGACCTTCA ACGTGATGGA GTCCTCCCGC ATCCGCAACC CGCACCTCTT CGACATCCTC
2881 AACAACCTCA CCATCTTCAC CGACTGGTTC TCCGTGGGCC GCAACTTCTA CTGGGGCGGC
2941 CACCGCGTGA TCTCCTCCCT CATCGGCGGC GGCAACATCA CCTCCCCGAT CTACGGCCGC
3001 GAGGCCAACC AGGAGCCGCC GCGCTCCTTC ACCTTCAACG GCCCGGTGTT CCGCACCCTC
3061 TCCAACCCGA CCCTCCGCCT CCTCCAGCAG CCGTGGCCGG CCCCGCCGTT CAACCTCCGC
3121 GGCGTGGAGG GCGTGGAGTT CTCCACCCCG ACCAACTCCT TCACCTACCG CGGCCGCGGC
3181 ACCGTGGACT CCCTCACCGA GCTTCCGCCG GAGGACAACT CCGTGCCGCC GCGCGAGGGC
3241 TACTCCCACC GCCTCTGCCA CGCCACCTTC GTGCAGCGCT CCGGCACCCC GTTCCTCACC
3301 ACCGGCGTGG TGTTCTCCTG GACCGACCGC TCCGCCACCC TCACCAACAC CATCGACCCG
3361 GAGCGCATCA ACCAGATCCC GCTCGTGAAG GGCTTCCGCG TGTGGGGCGG CACCTCCGTG
3421 ATCACCGGTC CGGGCTTCAC CGGCGGCGAC ATCCTCCGCC GCAACACCTT CGGCGACTTC
3481 GTGTCCCTCC AGGTGAACAT CAACTCCCCG ATCACCCAGC GCTACCGCCT CCGCTTCCGC
3541 TACGCCTCCT CCCGCGACGC CCGCGTGATC GTGCTCACCG GCGCCGCCTC CACCGGCGTG
3601 GGCGGCCAGG TGTCCGTGAA CATGCCGCTC CAGAAGACCA TGGAGATCGG CGAGAACCTC
3661 ACCTCCCGCA CCTTCCGCTA CACCGACTTC TCCAACCCGT TCTCCTTCCG CGCCAACCCG
3721 GACATCATCG GCATCTCCGA GCAGCCGCTC TTCGGCGCCG GCTCCATCTC CTCCGGCGAA
3781 CTCTACATCG ACAAGATCGA GATCATCCTC GCCGACTAAG AGCTC
SEQ ID NO:10
1 AAGAGCTCAC TGGCTAGGCG GCTTCTCTTC ACTCACCTGC AGAGTGCACC GCAATAATCA
61 GCTTCCGGAT GGTGGCCGTT TTGTCAGTTT TGGATGGAAA TGCCGAACTG GCCAGCGTCT
121 GTTTTCCCTA TGCATATGTA ATTTCCTGCC TCTTTATATT CACTCTTGTT GTCAAGTCCA
181 AGTGGAAAAT CTTGGCATAT TATACATATT GTAATAATAA ACATCGTACA ATCTGCATGC
241 TGTTTTGTAA TAATTAATTA ATATCCCAGC CCATTGGATG GACTTGTTTA AGGTACC
Claims (6)
1.一种抗虫融合基因,其特征是:所述基因包括从5’-3’依次含有编码BT晶体毒素Cry1的核苷酸序列和编码Cry9Aa毒素的核苷酸序列;且上述2个核苷酸序列位于同一个开放阅读框内。
2.根据权利要求1所述的抗虫融合基因,其特征是:所述抗虫融合基因包括Cry1Aa、Cry1Ab、Cry1Ac、Cry1Aa的修饰基因、Cry1Ab的修饰基因或Cry1Ac的修饰基因;所述抗虫融合基因还包括Cry9Aa或者Cry9Aa经过修饰的基因。
3.根据权利要求1或2的抗虫融合基因,其特征是:所述抗虫融合基因的核苷酸序列为以下任意一种:
SEQ ID NO:3、SEQ ID NO:4、与SEQ ID NO:3相比具有≥90%的相同性、与SEQ ID NO:4相比具有≥90%的相同性。
4.根据权利要求1~3中任意一种抗虫融合基因所编码的融合蛋白,其特征是:所述融合蛋白从N端至C端依次为Cry1晶体毒素和Cry9Aa毒素。
5.根据权利要求4所述的融合蛋白,其特征是:所述融合蛋白的氨基酸序列为以下任意一种:
SEQ ID NO:1、SEQ ID NO:2、与SEQ ID NO:1相比具有≥90%的相同性、与SEQ ID NO:2相比具有≥90%的相同性。
6.如权利要求4或5所述的融合蛋白在转基因抗虫农作物中的应用。
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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 |
CN103525834A (zh) * | 2013-04-25 | 2014-01-22 | 湖南农业大学 | CrylAc基因及其应用 |
CN104861074A (zh) * | 2015-04-14 | 2015-08-26 | 中国农业科学院作物科学研究所 | 融合杀虫蛋白Cry1Am、其编码基因及应用 |
CN105017391A (zh) * | 2015-06-30 | 2015-11-04 | 杭州瑞丰生物科技有限公司 | 抗虫蛋白、抗虫融合蛋白、编码基因、载体及应用 |
CN105753951A (zh) * | 2016-05-18 | 2016-07-13 | 中国农业科学院生物技术研究所 | 一种Bt抗虫基因及其编码的蛋白质和应用 |
WO2016188332A1 (zh) * | 2015-05-22 | 2016-12-01 | 杭州瑞丰生物科技有限公司 | 一种玉米转化事件及其特异性鉴定方法和应用 |
WO2017012577A1 (zh) * | 2015-07-23 | 2017-01-26 | 中国种子集团有限公司 | 编码苏云金芽孢杆菌晶体蛋白质的基因及其的应用 |
CN106832001A (zh) * | 2017-01-21 | 2017-06-13 | 浙江大学 | 一种杀虫融合蛋白、编码基因及其应用 |
CN114853858A (zh) * | 2022-03-29 | 2022-08-05 | 河南大学 | 一种抗虫性的苏铁毒蛋白基因、表达载体及应用 |
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CN103525834A (zh) * | 2013-04-25 | 2014-01-22 | 湖南农业大学 | CrylAc基因及其应用 |
CN104861074A (zh) * | 2015-04-14 | 2015-08-26 | 中国农业科学院作物科学研究所 | 融合杀虫蛋白Cry1Am、其编码基因及应用 |
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WO2016188332A1 (zh) * | 2015-05-22 | 2016-12-01 | 杭州瑞丰生物科技有限公司 | 一种玉米转化事件及其特异性鉴定方法和应用 |
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WO2017012577A1 (zh) * | 2015-07-23 | 2017-01-26 | 中国种子集团有限公司 | 编码苏云金芽孢杆菌晶体蛋白质的基因及其的应用 |
US10626411B2 (en) | 2015-07-23 | 2020-04-21 | China National Seed Group Corporation, Ltd. | Gene for encoding Bacillus thuringiensis crystal proteins, and use thereof |
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CN114853858A (zh) * | 2022-03-29 | 2022-08-05 | 河南大学 | 一种抗虫性的苏铁毒蛋白基因、表达载体及应用 |
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