CN113913457B - 一种抑制或杀灭桃蛀螟的方法及其应用 - Google Patents

一种抑制或杀灭桃蛀螟的方法及其应用 Download PDF

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CN113913457B
CN113913457B CN202111274181.3A CN202111274181A CN113913457B CN 113913457 B CN113913457 B CN 113913457B CN 202111274181 A CN202111274181 A CN 202111274181A CN 113913457 B CN113913457 B CN 113913457B
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李晓娇
马小伟
吕玉平
贾志伟
王建海
石小堰
李涛
李树秀
王杰
林黎彦
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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桃蛀螟的方法及其应用，具体涉及一种抑制或杀灭桃蛀螟的方法和检测植物基因组是否插入Cry1Da_7基因的方法。其中，所述抑制或杀灭桃蛀螟的方法包括：(1)提供基因组插入编码SEQ ID NO:2所示Cry1Da_7蛋白的基因的植物，所述植物表达Cry1Da_7蛋白，产生桃蛀螟抗性；和(2)所述植物与桃蛀螟接触，抑制或杀灭所述桃蛀螟。本发明发现将Cry1Da_7基因插入植物基因组DNA后，植物显示良好的桃蛀螟抗性，其对桃蛀螟的杀虫率达92％～100％。

Description

一种抑制或杀灭桃蛀螟的方法及其应用
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桃蛀螟的方法以及应用。
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，桃蛀螟(Dichocrocispunctiferalis(Guenee))，又称桃蛀野螟，属鳞翅目螟蛾科，为杂食性害虫，广泛分布于我国境内，危害玉米、高粱等作物以及桃、柿、板栗等果树。每年因桃蛀螟造成的粮食损失巨大。
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，目前还未见通过表达Cry1Da_7基因控制桃蛀螟害虫的相关报道。
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桃蛀螟的方法和检测植物基因组DNA是否插入Cry1Da_7基因的方法。
在第一方面，本发明提供一种抑制或杀灭桃蛀螟的方法，所述方法包括：(1)提供基因组插入编码SEQ ID NO:2所示Cry1Da_7蛋白的基因的植物，所述植物表达Cry1Da_7蛋白，产生桃蛀螟抗性；和(2)所述植物与桃蛀螟接触，抑制或杀灭所述桃蛀螟。
在一些具体的实施方式中，所述表达Cry1Da_7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、高粱、粟、向日葵、蓖麻、姜、棉花、桃、柿、核桃、板栗、无花果或松树。
在一些具体的实施方式中，所述表达Cry1Da_7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Cry1Da_7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Cry1Da_7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Cry1Da_7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，或使用如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Cry1Da_7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Cry1Da_7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Cry1Da_7基因插入植物(玉米)基因组DNA后，Cry1Da_7转基因植物(玉米)显示桃蛀螟抗性，并且在转基因植物(玉米)表达量较低(以单拷贝插入Cry1Da_7基因，Cry1Da_7蛋白表达量在3μg/g样品)的情况下，仍具有优良的抗桃蛀螟特性(杀虫率92％以上)。
附图说明
图1为本发明中含有Cry1Da_7核苷酸序列的重组克隆载体LP20-T构建流程图；
图2为本发明中含有Cry1Da_7核苷酸序列的重组表达载体LP-PT20构建流程图；
图3为本发明中转基因玉米植株接种桃蛀螟的叶片损伤图，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，Cry1Da_7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1Cry1Da_7基因的获得和合成
由南京金斯瑞生物科技公司合成Cry1Da_7基因(SEQ ID NO:1)，编码如SEQ IDNO:2所示的Cry1Da_7杀虫蛋白质的氨基酸序列(1166个氨基酸)。合成的所述Cry1Da_7核苷酸序列的5’端连接有NcoI酶切位点，3’端连接有EcoRI酶切位点。
实施例2载体构建
1、构建克隆载体
将合成的Cry1Da_7核苷酸序列分别连入克隆载体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LP20-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；Cry1Da_7为Cry1Da_7核苷酸序列(SEQ ID NO:1)。
将重组克隆载体LP20-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LP20-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LP20-T中插入的所述Cry1Da_7核苷酸序列为序列表中SEQ ID NO:1所示的核苷酸序列，即Cry1Da_7核苷酸序列正确插入。
2、构建含有Cry1Da_7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LP20-T和表达载体LP-BB1(载体骨架：pCAMBIA3301(CAMBIA机构提供))，将切下的Cry1Da_7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；Cry1Da_7：Cry1Da_7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(SEQ IDNO: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-PT20)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-PT20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Cry1Da_7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
对己经构建正确的重组表达载体LP-PT20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-PT20酶切后进行酶切验证，结果表明重组表达载体LP-PT20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Cry1Da_7基因的玉米植株。
PCR检测所用引物为：
F：ctgtccgagaaggtgaagca(SEQ ID NO:7)；
R：aagataacccacacgcccag(SEQ ID NO:8)。
片段大小：548bp。
PCR反应的条件：95℃30s,58℃30s,72℃40s，循环30次。
2、用qRT-PCR验证转入Cry1Da_7基因的玉米植株
检测Cry1Da_7基因拷贝数的具体方法如下：(1)分别取转入Cry1Da_7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NanoDrop2000(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TransStart Green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作为对照，每个样品3个重复，取其平均值。
以下引物用来检测Cry1Da_7核苷酸序列：
引物3(CF2)：acctgtacatccagtctggtgtgt(SEQ ID NO:9)；
引物4(CR2)：cgtactcggcctcgaacgt(SEQ ID NO:10)；
探针1(CP1)：6-FAM-atcgacaggatcgagttcatccccg-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：
PCR反应条件为：
步骤温度时间
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，Cry1Da_7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Cry1Da_7核苷酸序列的玉米植株均获得了含有单拷贝Cry1Da_7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Cry1Da_7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(Cry1Da_7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Cry1Da_7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(Cry1Da_7蛋白)含量测定结果如表1所示。测得转入Cry1Da_7核苷酸序列的玉米植株的新鲜叶片中杀虫蛋白(Cry1Da_7蛋白)的平均表达量占叶片鲜重的比例(ng/g)为3163.8，这一结果表明Cry1Da_7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Cry1Da_7蛋白表达量测定结果
实施例5转基因玉米植株的抗虫效果检测
将转入Cry1Da_7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桃蛀螟进行抗虫效果检测，具体步骤如下：
分别取转入Cry1Da_7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头人工饲养的桃蛀螟(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Cry1Da_7核苷酸序列的玉米转基因株系对桃蛀螟具有良好抗性。
表2玉米离体叶片抗虫生测结果
WT NGM Cry1Da_7
接虫数 50 50 50
3d死亡率 14％±5％a 16％±5％a 92％±4％b
4d死亡率 16％±5％a 20％±7％a 100％±0％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> 一种抑制或杀灭桃蛀螟的方法及其应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3501
<212> PRT
<213> Bacillus thuringiensis
<400> 1
Ala Thr Gly Gly Cys Thr Gly Ala Gly Ala Thr Cys Ala Ala Cys Ala
1 5 10 15
Ala Cys Cys Ala Gly Ala Ala Cys Cys Ala Gly Thr Gly Cys Gly Thr
20 25 30
Cys Cys Cys Gly Thr Ala Cys Ala Ala Cys Thr Gly Cys Cys Thr Gly
35 40 45
Ala Gly Cys Ala Ala Cys Cys Cys Thr Ala Ala Gly Gly Ala Gly Ala
50 55 60
Thr Cys Ala Thr Cys Cys Thr Gly Gly Gly Thr Gly Ala Gly Gly Ala
65 70 75 80
Ala Cys Gly Cys Cys Thr Gly Gly Ala Gly Ala Cys Cys Gly Gly Cys
85 90 95
Ala Ala Cys Ala Cys Cys Gly Thr Ala Gly Cys Cys Gly Ala Cys Ala
100 105 110
Thr Thr Ala Gly Cys Cys Thr Gly Gly Gly Cys Cys Thr Cys Ala Thr
115 120 125
Cys Ala Ala Cys Thr Thr Cys Cys Thr Cys Thr Ala Cys Ala Gly Cys
130 135 140
Ala Ala Cys Thr Thr Cys Gly Thr Gly Cys Cys Cys Gly Gly Cys Gly
145 150 155 160
Gly Thr Gly Gly Cys Thr Thr Cys Ala Thr Cys Gly Thr Gly Gly Gly
165 170 175
Cys Cys Thr Cys Cys Thr Gly Gly Ala Gly Cys Thr Thr Ala Thr Cys
180 185 190
Thr Gly Gly Gly Gly Cys Thr Thr Cys Ala Thr Cys Gly Gly Cys Cys
195 200 205
Cys Gly Thr Cys Cys Cys Ala Gly Thr Gly Gly Gly Ala Cys Ala Thr
210 215 220
Cys Thr Thr Cys Cys Thr Cys Gly Cys Cys Cys Ala Gly Ala Thr Cys
225 230 235 240
Gly Ala Gly Cys Ala Ala Cys Thr Gly Ala Thr Cys Ala Gly Cys Cys
245 250 255
Ala Gly Cys Gly Gly Ala Thr Cys Gly Ala Gly Gly Ala Gly Thr Thr
260 265 270
Cys Gly Cys Thr Ala Gly Gly Ala Ala Cys Cys Ala Gly Gly Cys Cys
275 280 285
Ala Thr Cys Thr Cys Cys Cys Gly Cys Cys Thr Gly Gly Ala Gly Gly
290 295 300
Gly Ala Cys Thr Cys Thr Cys Cys Ala Ala Cys Cys Thr Cys Thr Ala
305 310 315 320
Cys Ala Ala Gly Gly Thr Gly Thr Ala Cys Gly Thr Gly Cys Gly Cys
325 330 335
Gly Cys Gly Thr Thr Cys Ala Gly Cys Gly Ala Cys Thr Gly Gly Gly
340 345 350
Ala Gly Ala Ala Gly Gly Ala Cys Cys Cys Gly Ala Cys Cys Ala Ala
355 360 365
Cys Cys Cys Gly Gly Cys Cys Cys Thr Cys Cys Gly Cys Gly Ala Gly
370 375 380
Gly Ala Ala Ala Thr Gly Cys Gly Thr Ala Thr Cys Cys Ala Gly Thr
385 390 395 400
Thr Cys Ala Ala Cys Gly Ala Thr Ala Thr Gly Ala Ala Cys Thr Cys
405 410 415
Gly Gly Cys Cys Cys Thr Cys Ala Thr Cys Ala Cys Cys Gly Cys Cys
420 425 430
Ala Thr Cys Cys Cys Gly Cys Thr Cys Thr Thr Cys Cys Gly Cys Gly
435 440 445
Thr Gly Cys Ala Gly Ala Ala Cys Thr Ala Cys Gly Ala Gly Gly Thr
450 455 460
Gly Gly Cys Cys Cys Thr Cys Cys Thr Gly Thr Cys Cys Gly Thr Gly
465 470 475 480
Thr Ala Cys Gly Thr Thr Cys Ala Ala Gly Cys Cys Gly Cys Cys Ala
485 490 495
Ala Cys Cys Thr Cys Cys Ala Cys Cys Thr Cys Thr Cys Cys Ala Thr
500 505 510
Cys Cys Thr Cys Cys Gly Cys Gly Ala Cys Gly Thr Gly Ala Gly Cys
515 520 525
Gly Thr Gly Thr Thr Cys Gly Gly Cys Gly Ala Gly Cys Gly Cys Thr
530 535 540
Gly Gly Gly Gly Cys Thr Ala Cys Gly Ala Cys Ala Cys Cys Gly Cys
545 550 555 560
Cys Ala Cys Cys Ala Thr Cys Ala Ala Cys Ala Ala Cys Cys Gly Cys
565 570 575
Thr Ala Cys Thr Cys Cys Gly Ala Cys Cys Thr Cys Ala Cys Cys Thr
580 585 590
Cys Cys Cys Thr Cys Ala Thr Cys Cys Ala Cys Gly Thr Thr Thr Ala
595 600 605
Cys Ala Cys Cys Ala Ala Cys Cys Ala Cys Thr Gly Cys Gly Thr Gly
610 615 620
Gly Ala Cys Ala Cys Gly Thr Ala Cys Ala Ala Cys Cys Ala Gly Gly
625 630 635 640
Gly Cys Cys Thr Cys Cys Gly Cys Cys Gly Cys Cys Thr Gly Gly Ala
645 650 655
Gly Gly Gly Cys Cys Gly Cys Thr Thr Cys Cys Thr Cys Thr Cys Cys
660 665 670
Gly Ala Cys Thr Gly Gly Ala Thr Cys Gly Thr Gly Thr Ala Cys Ala
675 680 685
Ala Cys Cys Gly Cys Thr Thr Cys Cys Gly Cys Cys Gly Cys Cys Ala
690 695 700
Gly Cys Thr Cys Ala Cys Cys Ala Thr Cys Thr Cys Cys Gly Thr Cys
705 710 715 720
Cys Thr Gly Gly Ala Cys Ala Thr Cys Gly Thr Cys Gly Cys Cys Thr
725 730 735
Thr Cys Thr Thr Thr Cys Cys Cys Ala Ala Cys Thr Ala Cys Gly Ala
740 745 750
Cys Ala Thr Cys Cys Gly Cys Ala Cys Cys Thr Ala Cys Cys Cys Thr
755 760 765
Ala Thr Cys Cys Ala Gly Ala Cys Cys Gly Cys Cys Ala Cys Cys Cys
770 775 780
Ala Gly Cys Thr Cys Ala Cys Cys Cys Gly Cys Gly Ala Gly Gly Thr
785 790 795 800
Cys Thr Ala Cys Cys Thr Cys Gly Ala Cys Cys Thr Cys Cys Cys Gly
805 810 815
Thr Thr Cys Ala Thr Cys Ala Ala Cys Gly Ala Gly Ala Ala Cys Cys
820 825 830
Thr Cys Ala Gly Cys Cys Cys Gly Gly Cys Cys Gly Cys Cys Gly Thr
835 840 845
Cys Thr Ala Cys Cys Cys Gly Ala Cys Cys Thr Thr Cys Thr Cys Cys
850 855 860
Gly Cys Cys Gly Cys Thr Gly Ala Gly Thr Cys Cys Gly Cys Cys Ala
865 870 875 880
Thr Cys Ala Thr Thr Cys Gly Cys Ala Gly Cys Cys Cys Gly Cys Ala
885 890 895
Cys Cys Thr Cys Gly Thr Gly Gly Ala Cys Thr Thr Cys Cys Thr Cys
900 905 910
Ala Ala Cys Thr Cys Cys Thr Thr Cys Ala Cys Cys Ala Thr Cys Thr
915 920 925
Ala Cys Ala Cys Cys Gly Ala Cys Thr Cys Cys Cys Thr Cys Gly Cys
930 935 940
Cys Cys Gly Cys Ala Gly Cys Gly Cys Cys Thr Ala Cys Thr Gly Gly
945 950 955 960
Gly Gly Cys Gly Gly Thr Cys Ala Cys Cys Thr Cys Gly Thr Gly Ala
965 970 975
Ala Cys Thr Cys Cys Thr Thr Cys Cys Gly Cys Ala Cys Cys Gly Gly
980 985 990
Cys Ala Cys Cys Ala Cys Thr Ala Cys Cys Ala Ala Cys Cys Thr Cys
995 1000 1005
Ala Thr Cys Cys Gly Cys Ala Gly Cys Cys Cys Gly Cys Thr Cys Thr
1010 1015 1020
Ala Cys Gly Gly Cys Cys Gly Cys Gly Ala Gly Gly Gly Cys Ala Ala
1025 1030 1035 1040
Cys Ala Cys Cys Gly Ala Gly Cys Gly Cys Cys Cys Gly Gly Thr Gly
1045 1050 1055
Ala Cys Cys Ala Thr Cys Ala Cys Cys Gly Cys Cys Ala Gly Cys Cys
1060 1065 1070
Cys Gly Ala Gly Cys Gly Thr Gly Cys Cys Cys Ala Thr Cys Thr Thr
1075 1080 1085
Cys Cys Gly Cys Ala Cys Cys Cys Thr Cys Ala Gly Cys Thr Ala Cys
1090 1095 1100
Cys Cys Cys Ala Cys Cys Gly Gly Cys Cys Thr Gly Gly Ala Cys Ala
1105 1110 1115 1120
Ala Cys Ala Gly Cys Ala Ala Cys Cys Cys Thr Gly Thr Gly Gly Cys
1125 1130 1135
Gly Gly Gly Cys Ala Thr Cys Gly Ala Gly Gly Gly Cys Gly Thr Gly
1140 1145 1150
Gly Ala Gly Thr Thr Cys Cys Ala Gly Ala Ala Cys Ala Cys Cys Ala
1155 1160 1165
Thr Cys Thr Cys Cys Ala Gly Gly Ala Gly Cys Ala Thr Cys Thr Ala
1170 1175 1180
Cys Cys Gly Cys Ala Ala Gly Ala Gly Cys Gly Gly Cys Cys Cys Thr
1185 1190 1195 1200
Ala Thr Cys Gly Ala Cys Ala Gly Cys Thr Thr Cys Ala Gly Cys Gly
1205 1210 1215
Ala Gly Cys Thr Gly Cys Cys Thr Cys Cys Thr Cys Ala Gly Gly Ala
1220 1225 1230
Cys Gly Cys Cys Ala Gly Cys Gly Thr Gly Ala Gly Cys Cys Cys Thr
1235 1240 1245
Gly Cys Cys Ala Thr Cys Gly Gly Cys Thr Ala Cys Ala Gly Cys Cys
1250 1255 1260
Ala Cys Ala Gly Gly Cys Thr Gly Thr Gly Cys Cys Ala Cys Gly Cys
1265 1270 1275 1280
Cys Ala Cys Cys Thr Thr Cys Cys Thr Gly Gly Ala Gly Cys Gly Cys
1285 1290 1295
Ala Thr Cys Ala Gly Cys Gly Gly Cys Cys Cys Thr Cys Gly Cys Ala
1300 1305 1310
Thr Cys Gly Cys Gly Gly Gly Cys Ala Cys Cys Gly Thr Gly Thr Thr
1315 1320 1325
Cys Thr Cys Gly Thr Gly Gly Ala Cys Cys Cys Ala Cys Cys Gly Cys
1330 1335 1340
Ala Gly Cys Gly Cys Cys Thr Cys Thr Cys Cys Thr Ala Cys Gly Ala
1345 1350 1355 1360
Ala Cys Gly Ala Gly Gly Thr Gly Thr Cys Thr Cys Cys Thr Ala Gly
1365 1370 1375
Thr Cys Gly Cys Ala Thr Cys Ala Cys Cys Cys Ala Gly Ala Thr Cys
1380 1385 1390
Cys Cys Thr Thr Gly Gly Gly Thr Cys Ala Ala Gly Gly Cys Cys Cys
1395 1400 1405
Ala Cys Ala Cys Cys Cys Thr Gly Gly Cys Thr Ala Gly Thr Gly Gly
1410 1415 1420
Cys Gly Cys Thr Ala Gly Thr Gly Thr Cys Ala Thr Cys Ala Ala Gly
1425 1430 1435 1440
Gly Gly Cys Cys Cys Thr Gly Gly Cys Thr Thr Cys Ala Cys Cys Gly
1445 1450 1455
Gly Thr Gly Gly Thr Gly Ala Cys Ala Thr Cys Cys Thr Gly Ala Cys
1460 1465 1470
Cys Ala Gly Gly Ala Ala Cys Thr Cys Thr Ala Thr Gly Gly Gly Cys
1475 1480 1485
Gly Ala Gly Cys Thr Gly Gly Gly Cys Ala Cys Thr Cys Thr Gly Ala
1490 1495 1500
Gly Gly Gly Thr Cys Ala Cys Thr Thr Thr Cys Ala Cys Thr Gly Gly
1505 1510 1515 1520
Cys Cys Gly Cys Cys Thr Gly Cys Cys Thr Cys Ala Gly Thr Cys Thr
1525 1530 1535
Thr Ala Cys Thr Ala Cys Ala Thr Cys Cys Gly Cys Thr Thr Cys Cys
1540 1545 1550
Gly Cys Thr Ala Cys Gly Cys Thr Ala Gly Thr Gly Thr Cys Gly Cys
1555 1560 1565
Thr Ala Ala Cys Cys Gly Cys Thr Cys Thr Gly Gly Thr Ala Cys Thr
1570 1575 1580
Thr Thr Cys Cys Gly Cys Thr Ala Cys Thr Cys Thr Cys Ala Gly Cys
1585 1590 1595 1600
Cys Thr Cys Cys Gly Thr Cys Thr Thr Ala Cys Gly Gly Thr Ala Thr
1605 1610 1615
Cys Thr Cys Thr Thr Thr Cys Cys Cys Thr Ala Ala Gly Ala Cys Thr
1620 1625 1630
Ala Thr Gly Gly Ala Cys Gly Cys Thr Gly Gly Thr Gly Ala Gly Cys
1635 1640 1645
Cys Thr Cys Thr Gly Ala Cys Cys Ala Gly Thr Ala Gly Gly Ala Gly
1650 1655 1660
Cys Thr Thr Cys Gly Cys Thr Cys Ala Cys Ala Cys Thr Ala Cys Thr
1665 1670 1675 1680
Cys Thr Gly Thr Thr Cys Ala Cys Thr Cys Cys Thr Ala Thr Cys Ala
1685 1690 1695
Cys Thr Thr Thr Cys Thr Cys Thr Ala Gly Gly Gly Cys Thr Cys Ala
1700 1705 1710
Gly Gly Ala Gly Gly Ala Gly Thr Thr Cys Gly Ala Cys Cys Thr Gly
1715 1720 1725
Thr Ala Cys Ala Thr Cys Cys Ala Gly Thr Cys Thr Gly Gly Thr Gly
1730 1735 1740
Thr Gly Thr Ala Cys Ala Thr Cys Gly Ala Cys Ala Gly Gly Ala Thr
1745 1750 1755 1760
Cys Gly Ala Gly Thr Thr Cys Ala Thr Cys Cys Cys Cys Gly Thr Gly
1765 1770 1775
Ala Cys Cys Gly Cys Cys Ala Cys Gly Thr Thr Cys Gly Ala Gly Gly
1780 1785 1790
Cys Cys Gly Ala Gly Thr Ala Cys Gly Ala Cys Cys Thr Thr Gly Ala
1795 1800 1805
Gly Cys Gly Cys Gly Cys Cys Cys Ala Gly Ala Ala Gly Gly Thr Gly
1810 1815 1820
Gly Thr Gly Ala Ala Cys Gly Cys Cys Cys Thr Cys Thr Thr Cys Ala
1825 1830 1835 1840
Cys Thr Ala Gly Cys Ala Cys Thr Ala Ala Cys Cys Ala Gly Cys Thr
1845 1850 1855
Ala Gly Gly Cys Cys Thr Gly Ala Ala Gly Ala Cys Thr Gly Ala Cys
1860 1865 1870
Gly Thr Gly Ala Cys Cys Gly Ala Cys Thr Ala Cys Cys Ala Cys Ala
1875 1880 1885
Thr Cys Gly Ala Cys Cys Ala Ala Gly Thr Gly Ala Gly Cys Ala Ala
1890 1895 1900
Cys Cys Thr Ala Gly Thr Gly Gly Cys Cys Thr Gly Cys Cys Thr Cys
1905 1910 1915 1920
Thr Cys Cys Gly Ala Cys Gly Ala Gly Thr Thr Cys Thr Gly Cys Cys
1925 1930 1935
Thr Cys Gly Ala Cys Gly Ala Gly Ala Ala Gly Cys Gly Cys Gly Ala
1940 1945 1950
Gly Cys Thr Gly Thr Cys Cys Gly Ala Gly Ala Ala Gly Gly Thr Gly
1955 1960 1965
Ala Ala Gly Cys Ala Cys Gly Cys Cys Ala Ala Gly Cys Gly Cys Cys
1970 1975 1980
Thr Cys Thr Cys Cys Gly Ala Cys Gly Ala Gly Cys Gly Cys Ala Ala
1985 1990 1995 2000
Cys Cys Thr Gly Cys Thr Cys Cys Ala Gly Gly Ala Cys Cys Cys Cys
2005 2010 2015
Ala Ala Cys Thr Thr Cys Ala Gly Gly Gly Gly Cys Ala Thr Cys Ala
2020 2025 2030
Ala Cys Ala Gly Gly Cys Ala Gly Cys Cys Cys Gly Ala Cys Cys Gly
2035 2040 2045
Cys Gly Gly Cys Thr Gly Gly Cys Gly Cys Gly Gly Cys Thr Cys Cys
2050 2055 2060
Ala Cys Cys Gly Ala Cys Ala Thr Cys Ala Cys Cys Ala Thr Cys Cys
2065 2070 2075 2080
Ala Gly Gly Gly Cys Gly Gly Thr Gly Ala Cys Gly Ala Cys Gly Thr
2085 2090 2095
Ala Thr Thr Cys Ala Ala Gly Gly Ala Gly Ala Ala Cys Thr Ala Cys
2100 2105 2110
Gly Thr Thr Ala Cys Cys Cys Thr Cys Cys Cys Cys Gly Gly Cys Ala
2115 2120 2125
Cys Cys Thr Thr Cys Gly Ala Cys Gly Ala Gly Thr Gly Thr Thr Ala
2130 2135 2140
Cys Cys Cys Cys Ala Cys Cys Thr Ala Cys Cys Thr Cys Thr Ala Cys
2145 2150 2155 2160
Cys Ala Gly Ala Ala Gly Ala Thr Cys Gly Ala Cys Gly Ala Gly Thr
2165 2170 2175
Cys Cys Ala Ala Gly Cys Thr Gly Ala Ala Gly Gly Cys Cys Thr Ala
2180 2185 2190
Cys Ala Cys Cys Cys Gly Cys Thr Ala Cys Cys Ala Gly Cys Thr Cys
2195 2200 2205
Cys Gly Cys Gly Gly Cys Thr Ala Cys Ala Thr Cys Gly Ala Gly Gly
2210 2215 2220
Ala Cys Thr Cys Cys Cys Ala Gly Gly Ala Cys Cys Thr Gly Gly Ala
2225 2230 2235 2240
Ala Ala Thr Cys Thr Ala Cys Cys Thr Cys Ala Thr Cys Cys Gly Cys
2245 2250 2255
Thr Ala Cys Ala Ala Cys Gly Cys Cys Ala Ala Gly Cys Ala Cys Gly
2260 2265 2270
Ala Gly Ala Thr Cys Gly Thr Gly Ala Ala Cys Gly Thr Gly Cys Cys
2275 2280 2285
Thr Gly Gly Cys Ala Cys Cys Gly Gly Cys Ala Gly Cys Cys Thr Cys
2290 2295 2300
Thr Gly Gly Cys Cys Thr Cys Thr Cys Ala Gly Cys Gly Thr Gly Gly
2305 2310 2315 2320
Ala Gly Ala Ala Cys Cys Ala Gly Ala Thr Cys Gly Gly Cys Cys Cys
2325 2330 2335
Thr Thr Gly Cys Gly Gly Cys Gly Ala Gly Cys Cys Thr Ala Ala Cys
2340 2345 2350
Cys Gly Cys Thr Gly Cys Gly Cys Cys Cys Cys Thr Cys Ala Cys Cys
2355 2360 2365
Thr Cys Gly Ala Gly Thr Gly Gly Ala Ala Cys Cys Cys Thr Gly Ala
2370 2375 2380
Cys Cys Thr Cys Cys Ala Cys Thr Gly Cys Thr Cys Gly Thr Gly Cys
2385 2390 2395 2400
Ala Gly Gly Gly Ala Cys Gly Gly Cys Gly Ala Gly Ala Ala Gly Thr
2405 2410 2415
Gly Cys Gly Cys Cys Cys Ala Cys Cys Ala Thr Ala Gly Cys Cys Ala
2420 2425 2430
Cys Cys Ala Cys Thr Thr Cys Thr Cys Thr Cys Thr Gly Gly Ala Cys
2435 2440 2445
Ala Thr Cys Gly Ala Cys Gly Thr Gly Gly Gly Cys Thr Gly Cys Ala
2450 2455 2460
Cys Cys Gly Ala Cys Cys Thr Gly Ala Ala Cys Gly Ala Gly Gly Ala
2465 2470 2475 2480
Cys Cys Thr Gly Gly Gly Cys Gly Thr Gly Thr Gly Gly Gly Thr Thr
2485 2490 2495
Ala Thr Cys Thr Thr Cys Ala Ala Gly Ala Thr Cys Ala Ala Gly Ala
2500 2505 2510
Cys Cys Cys Ala Gly Gly Ala Cys Gly Gly Thr Cys Ala Cys Gly Cys
2515 2520 2525
Cys Ala Gly Gly Cys Thr Gly Gly Gly Thr Ala Ala Cys Cys Thr Gly
2530 2535 2540
Gly Ala Gly Thr Thr Cys Cys Thr Thr Gly Ala Gly Gly Ala Ala Ala
2545 2550 2555 2560
Ala Gly Cys Cys Thr Cys Thr Gly Cys Thr Gly Gly Gly Thr Gly Ala
2565 2570 2575
Gly Gly Cys Cys Cys Thr Gly Gly Cys Cys Ala Gly Gly Gly Thr Cys
2580 2585 2590
Ala Ala Gly Ala Gly Gly Gly Cys Thr Gly Ala Gly Ala Ala Gly Ala
2595 2600 2605
Ala Ala Thr Gly Gly Ala Gly Gly Gly Ala Thr Ala Ala Gly Ala Gly
2610 2615 2620
Gly Gly Ala Gly Ala Cys Cys Cys Thr Gly Cys Ala Gly Cys Thr Gly
2625 2630 2635 2640
Gly Ala Gly Ala Cys Cys Ala Cys Thr Ala Thr Cys Gly Thr Cys Thr
2645 2650 2655
Ala Cys Ala Ala Gly Gly Ala Gly Gly Cys Thr Ala Ala Gly Gly Ala
2660 2665 2670
Gly Thr Cys Thr Gly Thr Cys Gly Ala Thr Gly Cys Thr Cys Thr Gly
2675 2680 2685
Thr Thr Cys Gly Thr Cys Ala Ala Cys Thr Cys Thr Cys Ala Gly Thr
2690 2695 2700
Ala Cys Gly Ala Thr Ala Gly Ala Cys Thr Gly Cys Ala Ala Gly Cys
2705 2710 2715 2720
Thr Gly Ala Thr Ala Cys Cys Ala Ala Cys Ala Thr Cys Gly Cys Thr
2725 2730 2735
Ala Thr Gly Ala Thr Cys Cys Ala Cys Gly Cys Thr Gly Cys Gly Gly
2740 2745 2750
Ala Thr Ala Ala Gly Cys Gly Gly Gly Thr Cys Cys Ala Cys Cys Gly
2755 2760 2765
Gly Ala Thr Cys Cys Gly Gly Gly Ala Gly Gly Cys Thr Thr Ala Cys
2770 2775 2780
Cys Thr Thr Cys Cys Gly Gly Ala Gly Cys Thr Thr Thr Cys Thr Gly
2785 2790 2795 2800
Thr Cys Ala Thr Cys Cys Cys Gly Gly Gly Thr Gly Thr Cys Ala Ala
2805 2810 2815
Cys Gly Cys Thr Gly Cys Gly Ala Thr Cys Thr Thr Cys Gly Ala Gly
2820 2825 2830
Gly Ala Ala Cys Thr Thr Gly Ala Gly Gly Ala Ala Cys Gly Gly Ala
2835 2840 2845
Thr Cys Thr Thr Cys Ala Cys Thr Gly Cys Gly Thr Thr Thr Ala Gly
2850 2855 2860
Thr Cys Thr Thr Thr Ala Cys Gly Ala Thr Gly Cys Gly Cys Gly Gly
2865 2870 2875 2880
Ala Ala Cys Ala Thr Cys Ala Thr Cys Ala Ala Gly Ala Ala Cys Gly
2885 2890 2895
Gly Gly Gly Ala Cys Thr Thr Cys Ala Ala Cys Ala Ala Thr Gly Gly
2900 2905 2910
Thr Cys Thr Gly Cys Thr Gly Thr Gly Cys Thr Gly Gly Ala Ala Cys
2915 2920 2925
Gly Thr Cys Ala Ala Gly Gly Gly Thr Cys Ala Thr Gly Thr Cys Gly
2930 2935 2940
Ala Gly Gly Thr Cys Gly Ala Gly Gly Ala Ala Cys Ala Ala Ala Ala
2945 2950 2955 2960
Cys Ala Ala Thr Cys Ala Thr Cys Gly Thr Ala Gly Thr Gly Thr Cys
2965 2970 2975
Cys Thr Thr Gly Thr Cys Ala Thr Thr Cys Cys Thr Gly Ala Gly Thr
2980 2985 2990
Gly Gly Gly Ala Gly Gly Cys Gly Gly Ala Gly Gly Thr Cys Thr Cys
2995 3000 3005
Thr Cys Ala Ala Gly Ala Gly Gly Thr Cys Cys Gly Thr Gly Thr Thr
3010 3015 3020
Thr Gly Cys Cys Cys Gly Gly Gly Gly Cys Gly Thr Gly Gly Gly Thr
3025 3030 3035 3040
Ala Cys Ala Thr Thr Cys Thr Thr Cys Gly Thr Gly Thr Thr Ala Cys
3045 3050 3055
Thr Gly Cys Gly Thr Ala Cys Ala Ala Gly Gly Ala Gly Gly Gly Gly
3060 3065 3070
Thr Ala Cys Gly Gly Gly Gly Ala Gly Gly Gly Gly Thr Gly Cys Gly
3075 3080 3085
Thr Thr Ala Cys Thr Ala Thr Thr Cys Ala Thr Gly Ala Gly Ala Thr
3090 3095 3100
Thr Gly Ala Gly Ala Ala Cys Ala Ala Thr Ala Cys Thr Gly Ala Thr
3105 3110 3115 3120
Gly Ala Gly Cys Thr Thr Ala Ala Gly Thr Thr Cys Ala Ala Cys Ala
3125 3130 3135
Ala Thr Thr Gly Thr Gly Thr Thr Gly Ala Gly Gly Ala Gly Gly Ala
3140 3145 3150
Gly Gly Thr Thr Thr Ala Cys Cys Cys Gly Ala Ala Cys Ala Ala Thr
3155 3160 3165
Ala Cys Thr Gly Thr Thr Ala Cys Gly Thr Gly Cys Ala Thr Cys Ala
3170 3175 3180
Ala Cys Thr Ala Cys Ala Cys Gly Gly Cys Ala Ala Cys Gly Cys Ala
3185 3190 3195 3200
Ala Gly Ala Gly Gly Ala Ala Thr Ala Cys Gly Ala Gly Gly Gly Gly
3205 3210 3215
Ala Cys Gly Thr Ala Cys Ala Cys Cys Thr Cys Gly Cys Gly Thr Ala
3220 3225 3230
Ala Thr Ala Gly Ala Gly Gly Gly Thr Ala Thr Gly Ala Thr Gly Ala
3235 3240 3245
Gly Gly Cys Gly Thr Ala Cys Gly Gly Ala Ala Ala Cys Ala Ala Cys
3250 3255 3260
Cys Cys Gly Thr Cys Gly Gly Thr Thr Cys Cys Ala Gly Cys Ala Gly
3265 3270 3275 3280
Ala Thr Thr Ala Thr Gly Cys Cys Thr Cys Gly Gly Thr Thr Thr Ala
3285 3290 3295
Thr Gly Ala Gly Gly Ala Gly Ala Ala Gly Thr Cys Gly Thr Ala Cys
3300 3305 3310
Ala Cys Gly Gly Ala Thr Ala Gly Ala Cys Gly Ala Cys Gly Cys Gly
3315 3320 3325
Ala Gly Ala Ala Thr Cys Cys Ala Thr Gly Thr Gly Ala Gly Thr Cys
3330 3335 3340
Ala Ala Ala Thr Cys Gly Ala Gly Gly Ala Thr Ala Cys Gly Gly Ala
3345 3350 3355 3360
Gly Ala Thr Thr Ala Cys Ala Cys Ala Cys Cys Ala Thr Thr Ala Cys
3365 3370 3375
Cys Ala Gly Cys Ala Gly Gly Ala Thr Ala Cys Gly Thr Thr Ala Cys
3380 3385 3390
Ala Ala Ala Gly Gly Ala Gly Thr Thr Gly Gly Ala Ala Thr Ala Cys
3395 3400 3405
Thr Thr Cys Cys Cys Gly Gly Ala Ala Ala Cys Ala Gly Ala Thr Ala
3410 3415 3420
Ala Ala Gly Thr Thr Thr Gly Gly Ala Thr Thr Gly Ala Ala Ala Thr
3425 3430 3435 3440
Cys Gly Gly Ala Gly Ala Ala Ala Cys Ala Gly Ala Ala Gly Gly Ala
3445 3450 3455
Ala Cys Ala Thr Thr Cys Ala Thr Cys Gly Thr Cys Gly Ala Cys Thr
3460 3465 3470
Cys Ala Gly Thr Ala Gly Ala Ala Thr Thr Gly Thr Thr Gly Thr Thr
3475 3480 3485
Gly Ala Thr Gly Gly Ala Ala Gly Ala Ala Thr Gly Ala
3490 3495 3500
<210> 2
<211> 1166
<212> PRT
<213> Bacillus thuringiensis
<400> 2
Met Ala Glu Ile Asn Asn Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu
1 5 10 15
Ser Asn Pro Lys Glu Ile Ile Leu Gly Glu Glu Arg Leu Glu Thr Gly
20 25 30
Asn Thr Val Ala Asp Ile Ser Leu Gly Leu Ile Asn Phe Leu Tyr Ser
35 40 45
Asn Phe Val Pro Gly Gly Gly Phe Ile Val Gly Leu Leu Glu Leu Ile
50 55 60
Trp Gly Phe Ile Gly Pro Ser Gln Trp Asp Ile Phe Leu Ala Gln Ile
65 70 75 80
Glu Gln Leu Ile Ser Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala
85 90 95
Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Lys Val Tyr Val Arg
100 105 110
Ala Phe Ser Asp Trp Glu Lys Asp Pro Thr Asn Pro Ala Leu Arg Glu
115 120 125
Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Ile Thr Ala
130 135 140
Ile Pro Leu Phe Arg Val Gln Asn Tyr Glu Val Ala Leu Leu Ser Val
145 150 155 160
Tyr Val Gln Ala Ala Asn Leu His Leu Ser Ile Leu Arg Asp Val Ser
165 170 175
Val Phe Gly Glu Arg Trp Gly Tyr Asp Thr Ala Thr Ile Asn Asn Arg
180 185 190
Tyr Ser Asp Leu Thr Ser Leu Ile His Val Tyr Thr Asn His Cys Val
195 200 205
Asp Thr Tyr Asn Gln Gly Leu Arg Arg Leu Glu Gly Arg Phe Leu Ser
210 215 220
Asp Trp Ile Val Tyr Asn Arg Phe Arg Arg Gln Leu Thr Ile Ser Val
225 230 235 240
Leu Asp Ile Val Ala Phe Phe Pro Asn Tyr Asp Ile Arg Thr Tyr Pro
245 250 255
Ile Gln Thr Ala Thr Gln Leu Thr Arg Glu Val Tyr Leu Asp Leu Pro
260 265 270
Phe Ile Asn Glu Asn Leu Ser Pro Ala Ala Val Tyr Pro Thr Phe Ser
275 280 285
Ala Ala Glu Ser Ala Ile Ile Arg Ser Pro His Leu Val Asp Phe Leu
290 295 300
Asn Ser Phe Thr Ile Tyr Thr Asp Ser Leu Ala Arg Ser Ala Tyr Trp
305 310 315 320
Gly Gly His Leu Val Asn Ser Phe Arg Thr Gly Thr Thr Thr Asn Leu
325 330 335
Ile Arg Ser Pro Leu Tyr Gly Arg Glu Gly Asn Thr Glu Arg Pro Val
340 345 350
Thr Ile Thr Ala Ser Pro Ser Val Pro Ile Phe Arg Thr Leu Ser Tyr
355 360 365
Pro Thr Gly Leu Asp Asn Ser Asn Pro Val Ala Gly Ile Glu Gly Val
370 375 380
Glu Phe Gln Asn Thr Ile Ser Arg Ser Ile Tyr Arg Lys Ser Gly Pro
385 390 395 400
Ile Asp Ser Phe Ser Glu Leu Pro Pro Gln Asp Ala Ser Val Ser Pro
405 410 415
Ala Ile Gly Tyr Ser His Arg Leu Cys His Ala Thr Phe Leu Glu Arg
420 425 430
Ile Ser Gly Pro Arg Ile Ala Gly Thr Val Phe Ser Trp Thr His Arg
435 440 445
Ser Ala Ser Pro Thr Asn Glu Val Ser Pro Ser Arg Ile Thr Gln Ile
450 455 460
Pro Trp Val Lys Ala His Thr Leu Ala Ser Gly Ala Ser Val Ile Lys
465 470 475 480
Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Thr Arg Asn Ser Met Gly
485 490 495
Glu Leu Gly Thr Leu Arg Val Thr Phe Thr Gly Arg Leu Pro Gln Ser
500 505 510
Tyr Tyr Ile Arg Phe Arg Tyr Ala Ser Val Ala Asn Arg Ser Gly Thr
515 520 525
Phe Arg Tyr Ser Gln Pro Pro Ser Tyr Gly Ile Ser Phe Pro Lys Thr
530 535 540
Met Asp Ala Gly Glu Pro Leu Thr Ser Arg Ser Phe Ala His Thr Thr
545 550 555 560
Leu Phe Thr Pro Ile Thr Phe Ser Arg Ala Gln Glu Glu Phe Asp Leu
565 570 575
Tyr Ile Gln Ser Gly Val Tyr Ile Asp Arg Ile Glu Phe Ile Pro Val
580 585 590
Thr Ala Thr Phe Glu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Val
595 600 605
Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asp
610 615 620
Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Ala Cys Leu
625 630 635 640
Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val
645 650 655
Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro
660 665 670
Asn Phe Arg Gly Ile Asn Arg Gln Pro Asp Arg Gly Trp Arg Gly Ser
675 680 685
Thr Asp Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr
690 695 700
Val Thr Leu Pro Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr
705 710 715 720
Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Tyr Thr Arg Tyr Gln Leu
725 730 735
Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg
740 745 750
Tyr Asn Ala Lys His Glu Ile Val Asn Val Pro Gly Thr Gly Ser Leu
755 760 765
Trp Pro Leu Ser Val Glu Asn Gln Ile Gly Pro Cys Gly Glu Pro Asn
770 775 780
Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu His Cys Ser Cys
785 790 795 800
Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp
805 810 815
Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val
820 825 830
Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu
835 840 845
Glu Phe Leu Glu Glu Lys Pro Leu Leu Gly Glu Ala Leu Ala Arg Val
850 855 860
Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Thr Leu Gln Leu
865 870 875 880
Glu Thr Thr Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu
885 890 895
Phe Val Asn Ser Gln Tyr Asp Arg Leu Gln Ala Asp Thr Asn Ile Ala
900 905 910
Met Ile His Ala Ala Asp Lys Arg Val His Arg Ile Arg Glu Ala Tyr
915 920 925
Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu
930 935 940
Glu Leu Glu Glu Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg
945 950 955 960
Asn Ile Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Leu Cys Trp Asn
965 970 975
Val Lys Gly His Val Glu Val Glu Glu Gln Asn Asn His Arg Ser Val
980 985 990
Leu Val Ile Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val
995 1000 1005
Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly
1010 1015 1020
Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp
1025 1030 1035 1040
Glu Leu Lys Phe Asn Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn
1045 1050 1055
Thr Val Thr Cys Ile Asn Tyr Thr Ala Thr Gln Glu Glu Tyr Glu Gly
1060 1065 1070
Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Glu Ala Tyr Gly Asn Asn
1075 1080 1085
Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr
1090 1095 1100
Thr Asp Arg Arg Arg Glu Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly
1105 1110 1115 1120
Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu Tyr
1125 1130 1135
Phe Pro Glu Thr Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly
1140 1145 1150
Thr Phe Ile Val Asp Ser Val Glu Leu Leu Leu Met Glu Glu
1155 1160 1165
<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
ctgtccgaga aggtgaagca 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aagataaccc acacgcccag 20
<210> 9
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
acctgtacat ccagtctggt gtgt 24
<210> 10
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgtactcggc ctcgaacgt 19
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atcgacagga tcgagttcat ccccg 25
<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 (3)

1.一种抑制或杀灭桃蛀螟的方法，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Cry1Da_7蛋白的基因的植物，所述植物表达Cry1Da_7蛋白，产生桃蛀螟抗性；和(2)所述植物与桃蛀螟接触，抑制或杀灭所述桃蛀螟，所述植物为玉米。
2.根据权利要求1所述的方法，其特征在于，所述表达Cry1Da_7蛋白的基因以单拷贝插入所述植物的基因组。
3.根据权利要求1或2所述的方法，其特征在于，所述植物的Cry1Da_7蛋白表达量为3～4μg/g叶片。
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