CN114134171A - 一种抑制或杀灭东方黏虫的方法及其应用 - Google Patents

一种抑制或杀灭东方黏虫的方法及其应用 Download PDF

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CN114134171A
CN114134171A CN202111274192.1A CN202111274192A CN114134171A CN 114134171 A CN114134171 A CN 114134171A CN 202111274192 A CN202111274192 A CN 202111274192A CN 114134171 A CN114134171 A CN 114134171A
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贾志伟
马小伟
邸萌亮
李晓娇
王强
赵丽媛
李胜兵
孙宇
易金麒
韩雨颖
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Longping Biotechnology Hainan Co ltd
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Abstract

本发明涉及一种抑制或杀灭东方黏虫的方法及其应用,具体涉及一种抑制或杀灭东方黏虫的方法和检测植物基因组是否插入Cry1B.868基因的方法。其中,所述抑制或杀灭东方黏虫的方法包括:(1)提供基因组插入编码SEQ ID NO:2所示Cry1B.868蛋白的基因的植物,所述植物表达Cry1B.868蛋白,产生东方黏虫抗性;和(2)所述植物与东方黏虫接触,抑制或杀灭所述东方黏虫。本发明发现插入该基因后,植物显示出优良的东方黏虫抗性,东方黏虫的杀虫率可达68%~76%。

Description

一种抑制或杀灭东方黏虫的方法及其应用
技术领域
本发明涉及抗虫基因领域,具体而言,涉及一种抑制或杀灭东方黏虫的方法及其应用。
背景技术
目前,农业生产上面临的生物胁迫(如病害和虫害等)和非生物胁迫(如旱害、寒害和盐害等)造成农作物生长势减弱,产量降低,给全球粮食安全造成巨大威胁。其中,虫害是影响农林生产力的主要生物胁迫因素之一。例如,东方黏虫(Mythinmaseparata)又名行军虫、剃枝虫,是中国重大农作物病虫之一,主要危害小麦、水稻、玉米、高粱、糜子等禾谷类作物,有报道称东方黏虫已经成为我国危害最为严重的玉米害虫。因此,开发有效针对东方黏虫的防控方法具有重要意义。
随着使用化学农药进行害虫防治造成的环境问题愈发严重,生物杀虫剂的使用逐渐进入人们的视野。苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)是一种革兰氏阳性菌,能够产生杀虫晶体蛋白(Insecticidal crystal proteins,ICPs)和营养期杀虫蛋白(Vegetative insecticidal proteins,Vips)等不同类型的杀虫蛋白。其中,Cry蛋白是在芽胞形成期于芽胞内形成的一类杀虫晶体蛋白,对大多数鳞翅目害虫具有良好抗性。
在中国专利CN201980049875.1中证明Cry1B.868和Cry1Da_7基因共表达可表现出对鳞翅目害虫秋夜蛾、玉米穗蛾、西南玉米蛀虫和甘蔗蛀虫的抗性。然而,目前还未见通过Cry1B.868蛋白控制东方黏虫的相关报道。
有鉴于此,特提供本发明。
发明内容
本发明提供一种抑制或杀灭东方黏虫的方法和检测植物基因组DNA是否插入Cry1B.868基因的方法。
在第一方面,本发明提供一种抑制或杀灭东方黏虫的方法,所述方法包括:(1)提供基因组插入编码SEQ ID NO:2所示Cry1B.868蛋白的基因的植物,所述植物表达Cry1B.868蛋白,产生东方黏虫抗性;和(2)所述植物与东方黏虫接触,抑制或杀灭所述东方黏虫。
在一些具体的实施方式中,所述表达Cry1B.868蛋白的基因具有如SEQ ID NO:1所示核苷酸序列。
在一些具体的实施方式中,所述植物为玉米、小麦、水稻或高粱。
在一些具体的实施方式中,所述表达Cry1B.868蛋白的基因以单拷贝插入所述植物的基因组。
在一些具体的实施方式中,所述植物的Cry1B.868蛋白表达量为3~4μg/g叶片。
在第二方面,本发明还提供一种检测植物基因组是否插入Cry1B.868基因的方法,取植物样本的基因组DNA进行PCR反应,根据PCR结果判断所基因组DNA是否插入Cry1B.868基因;其中PCR反应使用具有如SEQ ID NO:7和SEQ ID NO:8所示序列的引物对,或使用SEQID NO:9和SEQ ID NO:10所示序列的引物对和SEQ ID NO:11所示的探针。
在一些具体的实施方式中,所述Cry1B.868基因表达如SEQ ID NO:2所示的氨基酸序列。
在一些具体的实施方式中,所述Cry1B.868基因具有如SEQ ID NO:1所示核苷酸序列。
术语定义
如本文所用,术语“重组”是指通常不会在自然界中发现并由人为干预产生的非天然DNA、蛋白质或生物体。“重组DNA分子”是这样的DNA分子,其包含不会天然地一起出现并且是人为干预的结果的DNA分子的组合。例如,由至少两个彼此异源的DNA分子的组合构成的DNA分子是重组DNA分子,所述至少两个DNA分子是诸如包含转基因和与该转基因相邻的植物基因组DNA的DNA分子。
本文所指的术语“DNA”是指脱氧核糖核酸(DNA)分子。DNA分子可以是基因组或合成来源的,并且按照惯例是从5'(上游)端到3'(下游)端。如本文所用,术语“DNA序列”是指DNA分子的核苷酸序列。按照惯例,本发明的DNA序列及其片段根据两条互补DNA序列链中的仅一条链来公开。通过暗示和意图,此处提供的序列的互补序列(互补链的序列)在本领域中也称为反向互补序列,它们在本发明的范围内,并且明确地意在要求保护的主题的范围内。
本文所述的术语“PCR”,是一种需要2个位于待合成靶序列两侧的引物的体外DNA扩增方法。引物是一段寡核苷酸序列,其能够以序列特异性的方式杂交到靶序列上并在PCR过程中延伸。扩增子(Amplicons)或PCR产物或PCR片段是包括引物和新合成的靶序列拷贝的延伸产物。多重PCR系统含有多套引物,其导致同时产生多个扩增子。引物可与靶序列完全匹配或者它们可含有内部错配的碱基,该碱基可导致在特异的靶序列中导入限制性核酸酶识别/切割位点。引物也可含有额外的序列和/或修饰或标记的核苷酸以便于获取或检测扩增子。DNA的热变性、引物与其互补序列的退火和退火引物以DNA聚合酶的延伸的重复循环导致靶序列的指数扩增。术语靶或靶序列指待扩增的核酸序列。术语模板指待扩增的原始核酸。
有益效果
本发明发现将Cry1B.868基因插入植物(玉米)基因组DNA后,Cry1B.868转基因植物(玉米)显示出了东方黏虫抗性,并且在转基因植物(玉米)单拷贝插入Cry1B.868基因,Cry1B.868蛋白表达量在3μg/g样品时,具有优良的抗东方黏虫特性,东方黏虫的杀伤率在68%~76%左右。
附图说明
图1为本发明中含有Cry1B.868核苷酸序列的重组克隆载体LP19-T构建流程图;
图2为本发明中含有Cry1B.868核苷酸序列的重组表达载体LP-PT19构建流程图;
图3为本发明中转基因玉米植株接种东方黏虫的叶片损伤图,其中WT为野生型植株,NGM为经PCR检测为非转基因的玉米植株,Cry1B.868为转基因玉米植株。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1 Cry1B.868基因的获得和合成
由南京金斯瑞生物科技公司合成Cry1B.868基因(SEQ ID NO:1),合成的所述Cry1B.868基因的5’端连接有NcoI酶切位点,3’端连接有EcoRI酶切位点。所述Cry1B.868基因编码SEQ ID NO:2所示的Cry1B.868杀虫蛋白。
实施例2载体构建
1、构建克隆载体
将实施例1合成的Cry1B.868基因核苷酸序列连入克隆载体pEASY-T5(Transgen,Beijing,China,CAT:CT501-01)上,操作步骤按Transgen公司产品pEASY-T5载体说明书进行,得到重组克隆载体LP19-T,其构建流程如图1所示(其中Kan+表示卡那霉素抗性基因;Amp+表示氨苄青霉素抗性基因;pUC origin表示质粒pUC的复制区序列,可引导双链DNA复制过程;LacZ为LacZ起始密码子;Cry1B.868为实施例1合成Cry1B.868基因的核苷酸序列)。
将重组克隆载体LP19-T用热激法转化大肠杆菌T1感受态细胞(Transgen,Beijing,China;Cat.No:CD501)。其转化过程为:50μl大肠杆菌T1感受态细胞和10μl质粒DNA(重组克隆载体LP19-T)混合后42℃水浴30s,37℃水浴45min,转化后在200rpm摇床上摇动1h,随后涂布在含有的氨苄青霉素(100mg/L)的LB平板(胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L,琼脂15g/L,用NaOH调pH至7.5)上生长过夜。挑取白色菌落,在LB液体培养基(胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L,氨苄青霉素100mg/L,用NaOH调pH至7.5)中于37℃条件的摇床上培养过夜。利用碱法提取质粒,具体步骤为:将菌液12000rpm离心1min,弃上清液,沉淀菌体用100μl提前用冰预冷的溶液I(25mMTris-HCl,10mM EDTA(乙二胺四乙酸),50mM葡萄糖,pH调至8.0)悬浮;加入150μl新配制的溶液II(0.2M NaOH,1%SDS(十二烷基硫酸钠)),将离心管上下颠倒4次,混合,置冰上3-5min;加入150μl冰冷的溶液III(4M醋酸钾,2M醋酸),立即充分混匀,冰上放置5-10min;于温度4℃、转速12000rpm条件下离心5min,在上清液中加入2倍体积无水乙醇,混匀后室温放置5min;于温度4℃、转速12000rpm条件下离心5min,弃上清液,沉淀用质量浓度为70%的乙醇洗涤后晾干;加入30μl含Rnase(20μg/ml)的TE(10mMTris-HCl,1mM EDTA,PH调至8.0)溶解沉淀;37℃下水浴30min,消化RNA;最后保存在-20℃冰箱中备用。
提取的质粒经NcoI和EcoRI酶切鉴定后,对阳性克隆进行测序验证,结果表明重组克隆载体LP19-T中插入的所述Cry1B.868核苷酸序列为序列表中SEQ ID NO:1所示的核苷酸序列,即Cry1B.868核苷酸序列正确插入。
2、构建含有Cry1B.868基因的重组表达载体
用限制性内切酶NcoI和EcoRI分别酶切重组克隆载体LP19-T和表达载体LP-BB1(载体骨架:pCAMBIA3301(CAMBIA机构提供)),将切下的Cry1B.868核苷酸序列片段插入到表达载体LP-BB1的NcoI和EcoRI位点之间,构建成重组表达载体LP-PT19,其构建流程如图2所示(KanR:卡那霉素抗性基因;RB:右边界;Cry1B.868:Cry1B.868核苷酸序列(SEQ ID NO:1);Nos:胭脂碱合成酶的终止子(SEQ ID NO:3);PAT:编码磷丝菌素乙酰转移酶基因(SEQID NO:4);pZmUbi1:玉米Ubiquitin(泛素)基因启动子(SEQ ID NO:5);35S:指来自花椰菜花叶病毒(CaMV)的终止子(SEQ ID NO:6);LB:左边界)。
将重组表达载体LP-PT19用热激方法转化大肠杆菌T1感受态细胞。其转化过程为:50μl大肠杆菌T1感受态细胞和10μl质粒DNA(重组表达载体LP-PT19)混合后42℃水浴30s,37℃水浴45min,转化后在200rpm摇床上摇动1h,随后涂布在含有的氨苄青霉素(100mg/L)的LB平板(胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L,琼脂15g/L,用NaOH调pH至7.5)上生长过夜。挑取白色菌落,在LB液体培养基(胰蛋白胨10g/L,酵母提取物5g/L,NaCl 10g/L,卡那霉素50mg/L,用NaOH调pH至7.5)中于37℃摇床上培养过夜。碱法提取其质粒,提取方法同上。将提取的质粒用限制性内切酶NcoI和EcoRI进行酶切后鉴定,并将阳性克隆进行测序,结果表明重组表达载体LP-PT19在NcoI和EcoRI位点间的核苷酸序列为序列表中SEQ IDNO:1所示核苷酸序列,即Cry1B.868核苷酸序列。
实施例3重组表达载体转化农杆菌及检测
(一)重组表达载体转化农杆菌
对己构建正确的重组表达载体LP-PT19用液氮法转化到农杆菌LBA4404(Invitrgen,Chicago,USA;Cat.No:18313-015)中,其转化条件为:100μL农杆菌LBA4404和3μL质粒DNA(重组表达载体)置于液氮中冷冻10min,37℃水浴10min;将转化后的农杆菌LBA4404接种于装有LB液体培养基的离心管中于置于28℃、200rpm条件的摇床上培养2h,涂布于含50mg/L利福平(Rifampicin)和50mg/L卡那霉素(Kanamycin)的LB固体培养基上直至长出阳性单克隆,挑取单克隆培养并提取其质粒,用限制性内切酶NotI和SalI对重组表达载体LP-PT19酶切后进行酶切验证,结果表明重组表达载体LP-PT19结构完全正确。
(二)农杆菌介导玉米幼胚的遗传转化
1.玉米幼胚的准备
将公司内部玉米自交系AX808种植于大田或温室中,取人工授粉后8-10天(夏季)/10-13天(秋季)的玉米作为幼胚来源。
2.农杆菌的准备
(1)取已转化鉴定好的甘油农杆菌在含有100mg/L kan(卡那霉素)和12mg/L tet(四环素)的YEP固体培养基上划线后于28℃暗培养2-3天;
(2)在灭菌的2ml离心管中加入1ml侵染培养基,取步骤(1)的农杆菌放入侵染培养基中,并用移液枪充分吹打混匀;
(3)另取一灭菌的2ml离心管,用侵染培养基调菌液浓度,使OD 660达到0.5-0.7。
3.玉米幼胚与农杆菌的共培养
(1)除去装幼胚离心管中的侵染培养基,加入1.5ml新鲜侵染培养基将胚清洗一次;
(2)除去侵染培养基,加入调好的农杆菌菌液;
(3)置于摇床最大转速震荡30s,室温放置5min;
(4)将胚倒到共培养基上,吸干液体;
(5)将胚平面朝上,盾面朝下放置;(6)将胚放到22℃暗培养2-3天。
4.愈伤的诱导和筛选
(1)共培养后的胚转到诱导愈伤培养基上,28℃培养箱中暗培养7-10天;
(2)将诱导好的愈伤转到筛选培养基上进行筛选培养,筛选压为5.0mM草甘膦,28℃暗培养2-3周;
(3)取第一次筛选存活的愈伤进行第二次筛选,筛选压为2.0mM草甘膦。
5.转化株系的再生与培养
(1)取筛选后长出的胚性愈伤放到预分化培养基上,28℃暗培养10-14天;
(2)取胚性愈伤到分化培养基上,28℃光培养10-14天,直到幼苗分化出来;
(3)将分化好的幼苗转到生根培养基上,28℃光培养,直到根发育完全;
(4)将长势良好的幼苗移栽至温室基质内生长。
待转基因植株开花结实后收种。将收获的种子播种在温室,植株长到4-6叶期时,采用PCR技术进行表达分析检测。
(三)转基因玉米的检测
1、用全式金公司2×EasyTaq PCR SuperMix(China,Beijing,Cat:AS111-11)普通PCR验证转入Cry1B.868基因的玉米植株。
PCR检测所用引物为:
引物1(CF1):atccagcgttactacgagcg(SEQ ID NO:7);
引物2(CR1):ggatgttaatgcccgcgaac(SEQ ID NO:8)。
片段大小:580bp。
PCR反应的条件:95℃30s,58℃30s,72℃40s,循环30次。
2、用qRT-PCR验证转入Cry1B.868基因的玉米植株
检测Cry1B.868基因拷贝数的具体方法如下:(1)分别取转入Cry1B.868核苷酸序列的玉米植株和野生型玉米植株的叶片各100mg,在研钵中用液氮研成匀浆,每个样品取3个重复;(2)使用EasyPure Plant Genomic DNA Kit(含RNase A)(Transgen,Beijing,China,Cat:EE111-01)提取上述样品的基因组DNA,具体方法参考其产品说明书;(3)用NanoDrop 2000(Thermo Scientific,USA)测定上述样品的基因组DNA浓度;(4)调整上述样品的基因组DNA浓度至同一浓度值,所述浓度值的范围为80-100ng/μl;(5)采用TransStartGreen荧光定量PCR方法鉴定样品的拷贝数,以经过鉴定已知拷贝数的样品作为标准品。同时以野生型玉米植株的样品为对照,每个样品3个重复,取平均值。
以下引物用来检测Cry1B.868核苷酸序列:
引物3(CF2):gctacagggcctgggaaac(SEQ ID NO:9);
引物4(CR2):gtcatccctgttctccaacca(SEQ ID NO:10);
探针1(CP1):6-FAM-cctttcgggcataccagcagtcactg-BHQ-2(SEQ ID NO:11)。
以下引物用来检测18S的核苷酸序列,用于内参调平。
引物5(CF3):ggatcagcgggtgttactaatagg(SEQ ID NO:12);
引物6(CR3):ccccggaacccaaagact(SEQ ID NO:13);
探针2(CP2):VIC-ccccgctggcaccttatgagaaatc-BHQ-2(SEQ ID NO:14)。
PCR反应体系为:
Figure BDA0003329645540000081
Green qPCR SuperMix(Transgen) 10μl
10μM Forward引物1μl
10μM Reverse引物1μl
Passive Reference Dye I(50X) 0.4μl
基因组DNA 2μl
水(ddH2O)5.6μl
PCR反应条件为:
步骤温度时间
Figure BDA0003329645540000082
重复步骤2-3,40次。
利用SDS2.3软件(Applied Biosystems)分析数据。
实验结果表明,Cry1B.868核苷酸序列己整合到所检测的玉米植株染色体组中,而且转入Cry1B.868核苷酸序列的玉米植株均获得了含有单拷贝Cry1B.868基因的转基因玉米植株。
实施例4转基因玉米植株的杀虫蛋白质检测
1、转基因玉米植株的杀虫蛋白质含量检测
本实施例涉及的溶液如下:
萃取缓冲液:8g/L NaCl,0.2g/L KH2PO4,2.9g/L Na2HPO4·12H2O,0.2g/L KCl,5.5ml/L吐温20(Tween-20),pH 7.4;
洗涤缓冲液PBST:8g/L NaCl,0.2g/L KH2PO4,2.9g/L Na2HPO4·12H2O,0.2g/LKCl,0.5ml/L吐温20(Tween-20),pH 7.4;
终止液:1M HCl。
取3mg转入Cry1B.868核苷酸序列的玉米植株的新鲜叶片作为样品,液氮研磨后加入800μl所述萃取缓冲液,4000rpm离心10min,取上清液用所述萃取缓冲液稀释40倍,取80μl稀释后的上清液用于ELISA检测。用ELISA(酶联免疫吸附测定法)试剂盒(ENVIRLOGIX公司)对样品中杀虫蛋白质(Cry1B.868蛋白)量占叶片鲜重的比例进行检测分析,具体方法参考产品说明书。
同时以野生型玉米植株和经荧光定量PCR鉴定为非转基因的玉米植株作为对照,按照上述方法进行检测分析。转入Cry1B.868核苷酸序列的共3个株系(S1、S2和S3),经荧光定量PCR鉴定为非转基因的(NGM)共1个株系,野生型的(CK)共1个株系;从每个株系选3株进行测试,每株重复6次。
转基因玉米植株的杀虫蛋白质(Cry1B.868蛋白)含量测定结果如表1所示。测得转入Cry1B.868核苷酸序列的玉米植株的新鲜叶片中杀虫蛋白(Cry1B.868蛋白)的平均表达量占叶片鲜重的比例(ng/g)为3241.7,这一结果表明Cry1B.868蛋白在玉米中均获得了较高的表达量和稳定性。
表1、转基因玉米植株的Cry1B.868蛋白表达量测定结果
Figure BDA0003329645540000091
实施例5转基因玉米植株的抗虫效果检测
将转入Cry1B.868核苷酸序列的玉米植株、野生型玉米植株和经PCR鉴定为非转基因的玉米植株对东方黏虫进行抗虫效果检测,具体步骤如下:
分别取转入Cry1B.868核苷酸序列的玉米植株、野生型玉米植株(WT)和经PCR鉴定为非转基因的玉米植株(NGM)(V3-V4期)的新鲜叶片,用无菌水冲洗干净并用滤纸将叶片上的水吸干,然后去除叶脉,剪成约3cm×1cm的长条状,取2片剪后的长条状叶片放入圆形塑料培养皿底部的滤纸上,所述滤纸用蒸馏水润湿,每个培养皿中放10头人工饲养的东方黏虫(初孵幼虫),虫试培养皿加盖后,在温度22-26℃、相对湿度70%-80%、光周期16h光照/8h黑暗的条件下放置3天后,统计死亡率。结果如表2和图3所示,转入Cry1B.868核苷酸序列的玉米转基因株系对东方黏虫具有良好抗性。
表2玉米离体叶片抗虫生测结果
WT NGM Cry1B.868
接虫数 50 50 50
3d死亡率 16%±5%a 18%±4%a 68%±8%b
4d死亡率 18%±4%a 22%±4%a 76%±5%b
注:表中数据为平均值±标准误;同行数字后的不同小写字母表示差异显著(P<0.05)。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
序列表
<110> 隆平生物技术(海南)有限公司
<120> 一种抑制或杀灭东方黏虫的方法及其应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3600
<212> DNA
<213> Bacillus thuringiensis
<400> 1
atgacgagca accggaagaa cgagaacgag atcatcaacg ccctctcgat ccctgctgtt 60
tcaaaccact ccgcgcagat gaacctgtcc accgacgcgc gcatcgagga ctccctctgc 120
atagccgagg gcaacaacat cgacccattc gtgtcggcca gcacggttca gaccggcatc 180
aacatcgcgg gccgtatcct cggcgtcctc ggtgtcccat tcgccggtca gatcgcgtcc 240
ttctactcgt tccttgtggg cgagctgtgg cctcgcggtc gtgacccgtg ggagatcttc 300
ctggagcatg tggagcagtt gatccggcag caagtcacgg agaacacccg cgatactgct 360
ctggccaggc tacagggcct gggaaactcc tttcgggcat accagcagtc actggaggac 420
tggttggaga acagggatga cgcgcgaaca cgctcggtac tctacaccca gtacatcgct 480
ctcgaactcg acttcctgaa cgctatgccg ctgttcgcca tcaggaacca ggaagttcca 540
ctccttatgg tgtacgccca ggccgccaac ttacatctgc tcctgctgcg ggacgccagc 600
ctgttcggct ccgagttcgg actcacatct caagaaatcc agcgttacta cgagcgccaa 660
gtggagaaga cccgtgagta cagtgactac tgcgctcgat ggtacaacac agggctcaac 720
aacctgcgcg gcaccaacgc tgagtcatgg ctccgttaca accagttccg ccgcgacttg 780
actttgggtg tcctagacct ggtggcgcta ttcccgtctt acgacacacg ggtgtaccca 840
atgaacacta gcgcgcaact cacgcgggag atctacacag acccaatcgg ccggacgaac 900
gcaccctccg gtttcgcatc cacgaattgg ttcaacaaca acgcaccctc cttctcggca 960
atcgaggccg ccgtcatccg ccctcctcac ctgctcgact ttcccgagca gctcacgatc 1020
ttctccgtgc tctcacgctg gtccaacaca cagtacatga actactgggt cgggcaccga 1080
ttggagagta ggacgatccg tggcagcttg agcaccagta cccacggcaa caccaacacc 1140
tccatcaacc cagttacgct acagttcacg agccgcgacg tttaccggac tgagtcgttc 1200
gcgggcatta acatccttct gacaacgccc gtcaacggcg tcccgtgggc ccggttcaac 1260
tggcgtaacc cgttgaactc cctgcgcggg tcattgctct acaccatcgg gtacacgggc 1320
gtcggcaccc agctcttcga cagtgaaact gagctgccgc ccgagaccac ggaacgcccg 1380
aactacgagt cctacagcca ccgcctgtcc aacatccggc tcatctctgg caacacgctg 1440
cgtgcgccgg tgtactcctg gacacaccgc agcgccgacc ggaccaacac gatctcttcc 1500
gactccatta accagatccc gctcgtgaag ggcttccgtg tgtggggtgg cacgagcgtc 1560
atcaccggtc cgggcttcac cggtggagac atactgcggc gcaacacttt cggcgacttc 1620
gtttcgttgc aagtgaacat caactcgccg atcacccagc gttaccgtct gaggttccgc 1680
tacgcttcaa gccgcgacgc gagggtcatt gtcctgaccg gagccgcgtc cacaggcgtg 1740
ggaggccaag tctcagtcaa catgcctctc cagaagacga tggagatagg cgagaacttg 1800
actagccgaa ccttccggta cactgatttc tcgaaccctt tctcattcag agcgaaccct 1860
gacatcattg ggatctccga gcaaccgctg ttcggtgctg gctccatcag ctctggcgaa 1920
ctgtacatcg acaagattga gatcatcctg gcggatgcga cgttcgaggc cgagtctgac 1980
ctggagcggg ctcagaaggc tgtcaacgaa ctgttcacca gcagcaacca gattgggctc 2040
aagaccgacg tcacggacta tcacattgac caagtgtcca accttgtgga gtgcctgtcc 2100
gacgagttct gcctcgacga gaagaaggag ctgtccgaga aggtcaaaca cgcgaagcgt 2160
ctgagtgacg agcggaattt gctccaggac ccgaacttcc gtggcatcaa ccgccagctc 2220
gaccgtggtt ggcgcgggag tacagacatc accatccagg gaggcgacga tgtgttcaag 2280
gagaactatg tgacgctgct cgggactttc gacgaatgct acccgacgta tctctaccag 2340
aagatagacg agagtaaatt gaaggcgtac acccgctacc agcttcgcgg gtacatcgag 2400
gatagtcagg acctggaaat ctacctgatc cgatacaacg ccaagcacga gacagtgaac 2460
gtgccaggca cgggctcact ttggccattg agcgctccct ctccaatcgg aaagtgcgct 2520
caccactcgc accacttctc tctggacatc gacgtgggct gcaccgacct caacgaggac 2580
ctgggtgtct gggttatctt caagattaag acccaggacg gacatgcccg cctcggcaac 2640
ctggagttcc ttgaggagaa gcctctcgtg ggcgaggccc tcgctcgtgt gaagcgcgcc 2700
gagaagaaat ggcgagacaa gcgggagaag ctggagtggg agaccaacat cgtgtacaag 2760
gaggccaagg agtcagtgga cgcactcttc gtcaacagcc agtacgaccg cctccaggct 2820
gacaccaaca tcgccatgat ccacgcggct gacaagcggg tccacagcat ccgtgaggcg 2880
tacctgcccg agctgtcagt gatccctggt gtgaacgcgg cgatcttcga ggaactggag 2940
ggccgcatct tcacagcatt cagcctgtac gatgccagga atgttattaa gaacggtgac 3000
ttcaacaacg ggctgagttg ctggaacgtc aagggccatg tggacgtcga ggagcagaac 3060
aaccaccggt ccgtgctggt cgtgccggag tgggaggcag aggtgagcca ggaggtccgc 3120
gtctgccctg gtcgcggcta catcctccgt gtgactgcgt acaaggaagg ctacggtgaa 3180
ggctgcgtga ctatccacga gatcgagaac aacaccgacg agctcaagtt ctcgaactgt 3240
gtggaggagg aggtgtaccc gaacaacacc gttacttgca acgactacac tgccacgcaa 3300
gaggagtacg agggcactta cacttcccgg aatcgcggct atgatggcgc gtacgagtcc 3360
aacagcagcg tgcctgcgga ttatgcgtcc gcttacgagg agaaggcgta caccgacgga 3420
cggagggaca acccttgcga gtccaaccgt ggctacggtg actacactcc gctgcccgcc 3480
gggtacgtca ccaaggagct ggagtacttc ccggagaccg acaaagtctg gatcgagatc 3540
ggcgagacgg agggcacttt catcgtggac tcggtcgagc tgctactgat ggaggagtga 3600
<210> 2
<211> 1199
<212> PRT
<213> Bacillus thuringiensis
<400> 2
Met Thr Ser Asn Arg Lys Asn Glu Asn Glu Ile Ile Asn Ala Leu Ser
1 5 10 15
Ile Pro Ala Val Ser Asn His Ser Ala Gln Met Asn Leu Ser Thr Asp
20 25 30
Ala Arg Ile Glu Asp Ser Leu Cys Ile Ala Glu Gly Asn Asn Ile Asp
35 40 45
Pro Phe Val Ser Ala Ser Thr Val Gln Thr Gly Ile Asn Ile Ala Gly
50 55 60
Arg Ile Leu Gly Val Leu Gly Val Pro Phe Ala Gly Gln Ile Ala Ser
65 70 75 80
Phe Tyr Ser Phe Leu Val Gly Glu Leu Trp Pro Arg Gly Arg Asp Pro
85 90 95
Trp Glu Ile Phe Leu Glu His Val Glu Gln Leu Ile Arg Gln Gln Val
100 105 110
Thr Glu Asn Thr Arg Asp Thr Ala Leu Ala Arg Leu Gln Gly Leu Gly
115 120 125
Asn Ser Phe Arg Ala Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn
130 135 140
Arg Asp Asp Ala Arg Thr Arg Ser Val Leu Tyr Thr Gln Tyr Ile Ala
145 150 155 160
Leu Glu Leu Asp Phe Leu Asn Ala Met Pro Leu Phe Ala Ile Arg Asn
165 170 175
Gln Glu Val Pro Leu Leu Met Val Tyr Ala Gln Ala Ala Asn Leu His
180 185 190
Leu Leu Leu Leu Arg Asp Ala Ser Leu Phe Gly Ser Glu Phe Gly Leu
195 200 205
Thr Ser Gln Glu Ile Gln Arg Tyr Tyr Glu Arg Gln Val Glu Lys Thr
210 215 220
Arg Glu Tyr Ser Asp Tyr Cys Ala Arg Trp Tyr Asn Thr Gly Leu Asn
225 230 235 240
Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp Leu Arg Tyr Asn Gln Phe
245 250 255
Arg Arg Asp Leu Thr Leu Gly Val Leu Asp Leu Val Ala Leu Phe Pro
260 265 270
Ser Tyr Asp Thr Arg Val Tyr Pro Met Asn Thr Ser Ala Gln Leu Thr
275 280 285
Arg Glu Ile Tyr Thr Asp Pro Ile Gly Arg Thr Asn Ala Pro Ser Gly
290 295 300
Phe Ala Ser Thr Asn Trp Phe Asn Asn Asn Ala Pro Ser Phe Ser Ala
305 310 315 320
Ile Glu Ala Ala Val Ile Arg Pro Pro His Leu Leu Asp Phe Pro Glu
325 330 335
Gln Leu Thr Ile Phe Ser Val Leu Ser Arg Trp Ser Asn Thr Gln Tyr
340 345 350
Met Asn Tyr Trp Val Gly His Arg Leu Glu Ser Arg Thr Ile Arg Gly
355 360 365
Ser Leu Ser Thr Ser Thr His Gly Asn Thr Asn Thr Ser Ile Asn Pro
370 375 380
Val Thr Leu Gln Phe Thr Ser Arg Asp Val Tyr Arg Thr Glu Ser Phe
385 390 395 400
Ala Gly Ile Asn Ile Leu Leu Thr Thr Pro Val Asn Gly Val Pro Trp
405 410 415
Ala Arg Phe Asn Trp Arg Asn Pro Leu Asn Ser Leu Arg Gly Ser Leu
420 425 430
Leu Tyr Thr Ile Gly Tyr Thr Gly Val Gly Thr Gln Leu Phe Asp Ser
435 440 445
Glu Thr Glu Leu Pro Pro Glu Thr Thr Glu Arg Pro Asn Tyr Glu Ser
450 455 460
Tyr Ser His Arg Leu Ser Asn Ile Arg Leu Ile Ser Gly Asn Thr Leu
465 470 475 480
Arg Ala Pro Val Tyr Ser Trp Thr His Arg Ser Ala Asp Arg Thr Asn
485 490 495
Thr Ile Ser Ser Asp Ser Ile Asn Gln Ile Pro Leu Val Lys Gly Phe
500 505 510
Arg Val Trp Gly Gly Thr Ser Val Ile Thr Gly Pro Gly Phe Thr Gly
515 520 525
Gly Asp Ile Leu Arg Arg Asn Thr Phe Gly Asp Phe Val Ser Leu Gln
530 535 540
Val Asn Ile Asn Ser Pro Ile Thr Gln Arg Tyr Arg Leu Arg Phe Arg
545 550 555 560
Tyr Ala Ser Ser Arg Asp Ala Arg Val Ile Val Leu Thr Gly Ala Ala
565 570 575
Ser Thr Gly Val Gly Gly Gln Val Ser Val Asn Met Pro Leu Gln Lys
580 585 590
Thr Met Glu Ile Gly Glu Asn Leu Thr Ser Arg Thr Phe Arg Tyr Thr
595 600 605
Asp Phe Ser Asn Pro Phe Ser Phe Arg Ala Asn Pro Asp Ile Ile Gly
610 615 620
Ile Ser Glu Gln Pro Leu Phe Gly Ala Gly Ser Ile Ser Ser Gly Glu
625 630 635 640
Leu Tyr Ile Asp Lys Ile Glu Ile Ile Leu Ala Asp Ala Thr Phe Glu
645 650 655
Ala Glu Ser Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Glu Leu Phe
660 665 670
Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr Asp Tyr His
675 680 685
Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp Glu Phe Cys
690 695 700
Leu Asp Glu Lys Lys Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg
705 710 715 720
Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Ile
725 730 735
Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile
740 745 750
Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Leu Gly
755 760 765
Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu
770 775 780
Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Ile Glu
785 790 795 800
Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His
805 810 815
Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Ala
820 825 830
Pro Ser Pro Ile Gly Lys Cys Ala His His Ser His His Phe Ser Leu
835 840 845
Asp Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp
850 855 860
Val Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn
865 870 875 880
Leu Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg
885 890 895
Val Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu
900 905 910
Trp Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala
915 920 925
Leu Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile
930 935 940
Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala
945 950 955 960
Tyr Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe
965 970 975
Glu Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala
980 985 990
Arg Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp
995 1000 1005
Asn Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn His Arg Ser
1010 1015 1020
Val Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg
1025 1030 1035 1040
Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu
1045 1050 1055
Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr
1060 1065 1070
Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Val Tyr Pro Asn
1075 1080 1085
Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Gln Glu Glu Tyr Glu
1090 1095 1100
Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr Glu Ser
1105 1110 1115 1120
Asn Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu Lys Ala
1125 1130 1135
Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr
1140 1145 1150
Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu
1155 1160 1165
Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu
1170 1175 1180
Gly Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu
1185 1190 1195
<210> 3
<211> 253
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60
atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120
atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180
gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240
atgttactag atc 253
<210> 4
<211> 552
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgtctccgg agaggagacc agttgagatt aggccagcta cagcagctga tatggccgcg 60
gtttgtgata tcgttaacca ttacattgag acgtctacag tgaactttag gacagagcca 120
caaacaccac aagagtggat tgatgatcta gagaggttgc aagatagata cccttggttg 180
gttgctgagg ttgagggtgt tgtggctggt attgcttacg ctgggccctg gaaggctagg 240
aacgcttacg attggacagt tgagagtact gtttacgtgt cacataggca tcaaaggttg 300
ggcctaggat ccacattgta cacacatttg cttaagtcta tggaggcgca aggttttaag 360
tctgtggttg ctgttatagg ccttccaaac gatccatctg ttaggttgca tgaggctttg 420
ggatacacag cccggggtac attgcgcgca gctggataca agcatggtgg atggcatgat 480
gttggttttt ggcaaaggga ttttgagttg ccagctcctc caaggccagt taggccagtt 540
acccagatct ga 552
<210> 5
<211> 1993
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60
agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120
tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180
tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240
gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300
ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360
gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420
agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480
taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540
aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600
cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660
cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720
acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780
ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840
gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900
ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960
cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020
ttctctagat cggcgttccg gtgcatggtt agggcccggt agttctactt ctgttcatgt 1080
ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140
ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200
gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260
agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320
atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380
tagatcggag tagatttctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440
tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500
aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560
cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620
gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680
acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740
gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800
tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860
tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920
catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980
gttacttctg cag 1993
<210> 6
<211> 195
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctgaaatcac cagtctctct ctacaaatct atctctctct ataataatgt gtgagtagtt 60
cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 120
cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 180
accaaaatcc agtgg 195
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atccagcgtt actacgagcg 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggatgttaat gcccgcgaac 20
<210> 9
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gctacagggc ctgggaaac 19
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gtcatccctg ttctccaacc a 21
<210> 11
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cctttcgggc ataccagcag tcactg 26
<210> 12
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggatcagcgg gtgttactaa tagg 24
<210> 13
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccccggaacc caaagact 18
<210> 14
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccccgctggc accttatgag aaatc 25

Claims (8)

1.一种抑制或杀灭东方黏虫的方法,其特征在于,所述方法包括:(1)提供基因组插入编码SEQ ID NO:2所示Cry1B.868蛋白的基因的植物,所述植物表达Cry1B.868蛋白,产生东方黏虫抗性;和(2)所述植物与东方黏虫接触,抑制或杀灭所述东方黏虫。
2.根据权利要求1所述的方法,其特征在于,所述表达Cry1B.868蛋白的基因具有如SEQID NO:1所示核苷酸序列。
3.根据权利要求1所述的方法,其特征在于,所述植物为玉米、小麦、水稻或高粱。
4.根据权利要求1所述的方法,其特征在于,所述表达Cry1B.868蛋白的基因以单拷贝插入所述植物的基因组。
5.根据权利要求1~4任一项所述的方法,其特征在于,所述植物的Cry1B.868蛋白表达量为3~4μg/g叶片。
6.一种检测植物基因组是否插入Cry1B.868基因的方法,其特征在于,取植物样本的基因组DNA进行PCR反应,根据PCR结果判断基因组DNA是否插入Cry1B.868基因;其中PCR反应使用具有如SEQ ID NO:7和SEQ ID NO:8所示序列的引物对,或使用SEQ ID NO:9和SEQ IDNO:10所示序列的引物对和SEQ ID NO:11所示的探针。
7.根据权利要求6所述的方法,其特征在于,所述Cry1B.868基因表达如SEQ ID NO:2所示的氨基酸序列。
8.根据权利要求7所述的方法,其特征在于,所述Cry1B.868基因具有如SEQ ID NO:1所示核苷酸序列。
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