CN111171118B - 一种植物抗虫基因mCry2Ab及其载体和应用 - Google Patents
一种植物抗虫基因mCry2Ab及其载体和应用 Download PDFInfo
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Abstract
本申请公开了一种人工合成用于转基因抗虫植物的Bt杀虫基因mCry2Ab以及用于植物抗虫的蛋白质,所述蛋白质的氨基酸序列为SEQ ID NO:1,其编码基因的核酸序列为SEQ ID NO 3。本申请还公开了相应的载体,以及蛋白质、基因在抗虫害中的应用。本申请改造后的mCry2Ab基因转入植物后,所获得的高表达mCry2Ab蛋白的转基因植物具有甜菜夜蛾和斜纹夜蛾的抗性,较Cry2Ab和经过核苷酸修饰的yCry2Ab抗性更好。
Description
技术领域
本申请涉及基因工程生物防治技术领域,具体而言涉及人工改造的抗虫基因mCry2Ab及其表达载体及应用。
背景技术
Bt基因编码杀虫晶体蛋白,来自苏云金芽孢杆菌(BacillusthuringHansis)。在其芽孢形成过程中会产生δ-内毒素的杀虫伴胞晶体蛋白,这些蛋白具有很高的杀虫活性。其作用原理为这种抗虫蛋白能被碱性肠液溶解,水解为更小的活性毒素片段-核心片段(Hofte和Whiteley,1989)。该活性片段能抗蛋白酶的进一步水解,被激活的蛋白质结合在昆虫肠道上的刷状小泡上,引起穿孔从而影响渗透平衡,细胞膨胀并溶解,靶标生物停止取食并最后死亡。研究表明许多靶标害虫的肠道上皮细胞都具有Bt蛋白高亲和性的结合位点(Hofte和Whiteley,1989)。在过去的几十年里,已确定数十种苏云金芽孢杆菌菌系及130多种它们编码的杀虫晶体蛋白。
生物防治是利用某些有益生物或生物代谢产物来控制害虫种群数量,以达到降低或消灭害虫的目的,如用赤眼蜂或白僵菌防治草地螟。其特点是对人、畜安全,对环境污染少,对某些害虫可达到长期控制的目的;但是效果常不稳定,并且不论草地螟发生轻重均需同样投资进行。
为了解决农业防治、化学防治、物理防治和生物防治在实际应用中的局限性,科学家们经过研究发现将编码杀虫蛋白的抗虫基因转入植物中,可获得一些抗虫转基因植物以防治植物虫害。
Cry2Ab杀虫蛋白是众多杀虫蛋白中的一种,1981年Schenpf和Whiteley从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆了第一个编码δ-内毒素的cry基因Cry1Aa1。1989年Widner W.R和Whiteley H.R从苏云金芽孢杆菌(Bacillus thuringiensis)中克隆了Cry2Ab基因。Cry2Ab蛋白被昆虫摄入进入中肠,毒蛋白原毒素被溶解在昆虫中肠的碱性pH环境下。蛋白N-和C-末端被碱性蛋白酶消化,将原毒素转变成活性片段;活性片段和昆虫中肠上皮细胞膜上表面上受体结合,插入肠膜,导致细胞膜出现穿孔病征,破坏细胞膜内外的渗透压变化及pH平衡等,扰乱昆虫的消化过程,最终导致其死亡。
孟山都在公开号为CN1332800A的中国专利申请中对Cry2Ab特定的核苷酸和氨基酸序列进行了保护,同时对该蛋白的细胞器定位和使用的信号肽进行保护。在申请日为2014-09-19公开号为CN104313036A的中国专利申请中中国农大赖锦盛教授对Cry2Ab核苷酸序列进行了修饰和调整,提高了其对东方黏虫的抗性。在申请日为2013-12-12公开号为CN103688974A的中国专利申请中,北京大北农生物技术有限公司针对原核苷酸序列和氨基酸序列开发了应用于国内一些新虫子的抗性的新用途,Cry2Ab基因和yCry2Ab基因在部分夜蛾科害虫的抗性上存在抗性不足的问题,例如对甜菜夜蛾和斜纹夜蛾抗性存在不足,针对该基因的上述技术问题本发明对该基因的核苷酸序列进行了调整,该调整造成了两个氨基酸序列的改变,本发明以孟山都公司Cry2Ab基因原序列和中国农大赖锦盛教授修饰了核苷酸序列的yCry2Ab基因为对照,构建了载体,进行了甜菜夜蛾和斜纹夜蛾的抗性测定,本发明经过氨基酸调整的mCry2Ab基因能够有效的提高甜菜夜蛾和斜纹夜蛾的抗性。
发明内容
针对上述技术问题,本申请的目的在于提供一种的mCry2Ab基因,改造后的mCry2Ab基因转入植物后,所获得的高表达mCry2Ab蛋白的转基因植物具有甜菜夜蛾和斜纹夜蛾的抗性,较原始Cry2Ab和仅对核苷酸修饰后的yCry2Ab抗性更好。
为实现上述目的,本申请提供一种用于植物抗虫的蛋白质,所述蛋白质的氨基酸序列为SEQ ID NO:1。表达该mCry2Ab蛋白的转基因植物具有甜菜夜蛾和斜纹夜蛾的抗性,较原始Cry2Ab和仅对核苷酸修饰后的Cry2Ab抗性更好。
本申请还公开了一种抗虫基因,所述抗虫基因包含编码上述的蛋白质的基因序列。
在根据本发明的一个实施方案中,所述抗虫基因的核苷酸序列为SEQ ID NO:3。改造mCry2Ab基因转入植物后,所获得的高表达mCry2Ab蛋白的转基因植物具有甜菜夜蛾和斜纹夜蛾的抗性,较原始Cry2Ab和仅对核苷酸修饰后的yCry2Ab抗性更好。
本发明还公开了一种含有上述抗虫基因的表达盒、重组载体、重组微生物或转基因细胞系。通过构建相应的表达盒、重组载体、重组微生物或转基因细胞系使该抗虫基因能够应用于实践中,有利于防止农业虫害。
本发明还公开了一种表达载体,所述表达载体包上述的抗虫基因。
在根据本发明的一个实施方案中,该表达载体,中依次包含以下基因结构:
花椰菜花叶病毒(CaMV)的35S启动子(pr35s);抗虫基因;胭脂碱合成酶的终止子(Nos);玉米泛素基因启动子(Ubi);编码磷丝菌素乙酰转移酶基因(PAT);自花椰菜花叶病毒(CaMV)的终止子(PAT)。
本发明进一步公开了用于植物抗虫的蛋白质,或者,所述抗虫基因,或,含有所述抗虫基因的表达盒、重组载体、重组微生物或转基因细胞系在植物抗虫中的应用,所述应用选自以下两者或两者之一:a)制备具有抗虫效果的药剂;b)培育具有或提高抗虫能力的转基因植物。
在根据本发明一个实施方案中,所述应用中防治的害虫选自甜菜夜蛾、斜纹夜蛾、玉米螟、棉铃虫和东方黏虫中的一种或多种。
本发明还进一步公开了一种培育具有或提升了抗虫能力的植物的方法,所述方法包括如下步骤:将上述抗虫基因导入受体植物中,得到转基因植物;所述转基因植物与所述受体植物相比,所述转基因植物的抗虫性增强。
在根据本发明的一个实施方案中所述植物选自单子叶植物、双子叶植物、禾本科植物中的一种或多种;优选为玉米。
本发明具有以下有益效果:
本发明提供的改造后的mCry2Ab基因转入植物后,所获得的高表达mCry2Ab蛋白的转基因植物具有甜菜夜蛾和斜纹夜蛾的抗性,较Cry2Ab和仅对核苷酸序列进行优化的基因抗性更好。
附图说明
图1为本发明控制害虫的方法的含有ZmCTP+mCry2Ab核苷酸序列的重组表达载体LP-PT02的载体图;
图2为本发明控制害虫的方法的含有ZmCTP+Cry2Ab核苷酸序列的重组表达载体LP-PT02CK的载体图;
图3为本发明控制害虫的方法的含有ZmCTP+yCry2Ab核苷酸序列的重组表达载体LP-PT02CKy的载体图
图4为本发明控制害虫的方法的转化体的含有mCry2Ab核苷酸序列PCR检测图,其中WT为野生型植株,PC为质粒对照,NC为水对照,1~20为20个阳性转化体;
图5为本发明控制害虫的方法的转化体的含有Cry2Ab核苷酸序列PCR检测图,其中WT为野生型植株,PC为质粒对照,NC为水对照,1~20为20个阳性转化体;
图6为本发明控制害虫的方法的转化体的含有yCry2Ab核苷酸序列PCR检测图,其中WT为野生型植株,PC为质粒对照,NC为水对照,1~20为20个阳性转化体;
图7为本发明控制害虫的方法的转化体的抗斜纹夜蛾,其中WT为野生型植株,Cry2Ab为转化了孟山都版本的Cry2Ab转化事件,yCry2Ab为优化了核苷酸序列的Cry2Ab的转化事件、mCry2Ab为转化了经过修饰的Cry2Ab转化事件。
具体实施方式
以下实施例用于说明本申请,但不用来限制本申请的范围。
下面将更详细地描述本申请的具体实施例。提供这些实施例是为了能够更透彻地理解本申请,并且能够将本申请的范围完整的传达给本领域的技术人员。
如在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语,故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式,然所述描述乃以说明书的一般原则为目的,并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
实施例1 mCry2Ab基因的获得和合成
1、获得mCry2Ab核苷酸序列
mCry2Ab杀虫蛋白质的氨基酸序列(634个氨基酸),如序列表中SEQ ID NO:1所示;编码相应于所述mCry2Ab杀虫蛋白质的氨基酸序列(634个氨基酸)的mCry2Ab核苷酸序列(1905个核苷酸),如序列表中SEQ ID NO:3所示。
yCry2Ab杀虫蛋白质的氨基酸序列(633个氨基酸),如序列表中SEQ ID NO:2所示;编码相应于所述yCry2Ab杀虫蛋白质的氨基酸序列(633个氨基酸)的Cry2Ab核苷酸序列(1902个核苷酸),如序列表中SEQ ID NO:4所示。
2、合成上述mCry2Ab核苷酸序列
所述mCry2Ab前面加上ZmCTP信号肽核苷酸序列(如序列表中SEQ ID NO:3所示)和所述ZmCTP信号肽Cry2Ab核苷酸序列(如序列表中SEQ ID NO:4所示)由南京金斯瑞生物科技公司合成;合成的所述ZmCTP+mCry2Ab核苷酸序列(SEQ ID NO:3)的5’端还连接有NcoI酶切位点,所述ZmCTP+mCry2Ab核苷酸序列(SEQ ID NO:3)的3’端还连接有EcoRI酶切位点;合成的所述ZmCTP+mCry2Ab核苷酸序列(SEQ ID NO:4)的5’端还连接有NcoI酶切位点,所述ZmCTP+mCry2Ab核苷酸序列(SEQ ID NO:4)的3’端还连接有EcoRI酶切位点。
实施例2载体构建
将合成的ZmCTP+mCry2Ab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LP02-T(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;pUCorigin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;mCry2Ab为mCry2Ab核苷酸序列(SEQ ID NO:3))。
然后将重组克隆载体LP02-T用热激方法转化大肠杆菌T1感受态细胞(Transgen,Beijing,China;Cat.No:CD501),其热激条件为:50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组克隆载体LP02-T),42℃水浴30秒;37℃水浴45分钟(200rpm转速下摇床摇动),涂布的氨苄青霉素(100毫克/升)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)中于温度37℃条件下培养过夜。碱法提取其质粒:将菌液在12000rpm转速下离心1min,去上清液,沉淀菌体用100μl冰预冷的溶液I(25mM Tris-HCl,10mM EDTA(乙二胺四乙酸),50mM葡萄糖,pH8.0)悬浮;加入150μl新配制的溶液II(0.2M NaOH,1%SDS(十二烷基硫酸钠)),将管子颠倒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(10mM Tris-HCl,1mM EDTA,PH8.0)溶解沉淀;于温度37℃下水浴30min,消化RNA;于温度-20℃保存备用。
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LP02-T中插入的所述ZmCTP+mCry2Ab核苷酸序列为序列表中SEQ ID NO:3所示的核苷酸序列,即ZmCTP+mCry2Ab核苷酸序列正确插入。
按照上述构建重组克隆载体LP02-T的方法,将合成的所述Cry2Ab、yCry2Ab核苷酸序列连入克隆载体pEASY-T5上,得到重组克隆载体LP02CK-T、PT02CKy-T,其中,Cry2Ab为Cry2Ab核苷酸序列(SEQ ID NO:4)。酶切和测序验证重组克隆载体LP02CK-T和PT02CKy-T中所述Cry2Ab核苷酸序列正确插入。
2、构建含有mCry2Ab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LP02-T和表达载体LP-BB(载体骨架:pCAMBIA3301(CAMBIA机构可以提供)),将切下的mCry2Ab核苷酸序列片段插到表达载体LP-BB的NcoI和EcoRI位点之间,利用常规的酶切方法构建载体是本领域技术人员所熟知的,构建成重组表达载体LP-PT02,其构建流程如图1所示(Kan:卡那霉素基因;RB:右边界;pr35s:指来自花椰菜花叶病毒(CaMV)的35S启动子(SEQ ID NO:8;mCry2Ab:mCry2Ab核苷酸序列(SEQ ID NO:3);Nos:胭脂碱合成酶的终止子(SEQ ID NO:6);Ubi:玉米Ubiquitin(泛素)基因启动子(SEQ ID NO:5);PAT:编码磷丝菌素乙酰转移酶基因(SEQ ID NO:7);35s:指来自花椰菜花叶病毒(CaMV)的终止子(SEQ ID NO:9);LB:左边界)。
将重组表达载体LP-PT02用热激方法转化大肠杆菌T1感受态细胞,其热激条件为:50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组表达载体LP-PT02),42℃水浴30秒;37℃水浴1小时(200rpm转速下摇床摇动);然后在含50mg/L卡那霉素(Kanamycin)的LB固体平板(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)上于温度37℃条件下培养12小时,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)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶NcoI和EcoRI酶切后鉴定,并将阳性克隆进行测序鉴定,结果表明重组表达载体LP-PT02在NcoI和EcoRI位点间的核苷酸序列为序列表中SEQ IDNO:3所示核苷酸序列,即mCry2Ab核苷酸序列。
按照上述构建重组表达载体LP-PT02的方法,将NcoI和EcoRI酶切重组克隆载体LP02-T切下的所述ZmCTP+Cry2Ab、ZmCTP+yCry2Ab核苷酸序列插入表达载体LP-BB,得到重组表达载体LP-PT02CK、LP-PT02CKy。酶切和测序验证重组表达载体LP-PT02CK、LP-PT02CKy在NcoI和EcoRI位点间即为所述ZmCTP+Cry2Ab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-PT02、LP-PT02CK和LP-PT02CKy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、3μL质粒DNA(重组表达载体);置于液氮中1分钟,37℃温水浴10分钟;将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时,涂于含50mg/L的利福平(Rifampicin)和50mg/L的卡那霉素(Kanamycin)的LB平板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-PT02、LP-PT02-CK和LP-PT02CKy酶切后进行酶切验证,结果表明重组表达载体LP-PT02、LP-PT02-CK和LP-PT02CKy结构完全正确。
转化具体步骤如下:
1.玉米幼胚的准备
将公司内部玉米自交系AX808种植于大田或是温室,取人工授粉后8-10天(夏季)/10-13天(秋季)的玉米作为幼胚的来源。
2.农杆菌的准备
(1)取已转化鉴定好的农杆菌甘油菌在添加有100mg/L kan和12mg/L tet的YEP固体培养基上划线,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,并用移液枪充分打散混匀;
(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)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,盾面朝上放置;
(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,筛选压同为2.0mM;
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。
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。
(二)转基因玉米植株的检测
1、用全式金公司2×EasyTaq PCR SuperMix(China,Beijing,Cat:AS111-11)普通PCR验证转入Cry2Ab、mCry2Ab基因的玉米植株
PCR的引物是:
Cry2Ab-128F:AGAAGAACAACCACAGCCTG(SEQ ID NO:10)
Cry2Ab-724R:TGTCGTGAAGCCTCGTATTG(SEQ ID NO:11)
片段大小:597bp
以下引物用来检测yCry2Ab核苷酸序列:
Cry2Ab F:GACCGAGTGGAAGAAGAACA(SEQ ID NO:16)
Cry2Ab R:ACTCGAACACGTTGAGGAAC(SEQ ID NO:17)
片段大小:654bp
PCR反应的条件是:30个循环,每个循环是95℃30’,58℃30’,72℃40’。
2、用qRT-PCR验证转入Cry2Ab基因的玉米植株
分别取转入mCry2Ab核苷酸序列的玉米植株和转入Cry2Ab核苷酸序列的玉米植株的叶片约100mg作为样品,用Transgen的EasyPure Plant Genomic DNA Kit(含RNase A)(Transgen,Beijing,China,Cat:EE111-01)提取其基因组DNA,通过TransStart Green荧光定量PCR方法检测mCry2Ab基因的拷贝数。同时以野生型玉米植株作为对照,按照上述方法进行检测分析。实验设3次重复,取平均值。
检测Cry2Ab基因拷贝数的具体方法如下:
步骤1、分别取转入mCry2Ab核苷酸序列的玉米植株和野生型玉米植株的叶片各100mg,分别在研钵中用液氮研成匀浆,每个样品取3个重复;
步骤2、使用Transgen的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;
步骤3、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度;
步骤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;
步骤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,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;荧光定量PCR引物和探针序列分别是:
以下引物用来检测mCry2Ab和Cry2Ab核苷酸序列:
引物1(CF2):TCTCCTTCATTCGTGACGTG如序列表中SEQ ID NO:12所示;
引物2(CR2):GCCGACTGGTAGGTGTTGAT如序列表中SEQ ID NO:13所示;
以下引物用来检测yCry2Ab核苷酸序列:
引物1(CF2y):CTACCGCGACTACCTGAAGA(SEQ ID NO:18)
引物1(CR2y):GTCCTGAACTCCAGCATGTC(SEQ ID NO:19)
以下引物用来检测18s的核苷酸序列,用于内参调平
18srRNA-F:CCATCCCTCCGTAGTTAGCTTCT(SEQ ID NO:14)
18srRNA-R:CCTGTCGGCCAAGGCTATATAC(SEQ ID NO:15)
PCR反应体系为:
PCR反应条件为:
重复步骤2-3,40次
利用SDS2.3软件(Applied Biosystems)分析数据。
图4~图6为PCR检测图,其中WT为野生型植株,PC为质粒对照,NC为水对照,1~20为20个阳性转化体。实验结果表明,mCry2Ab和Cry2Ab核苷酸序列己整合到所检测的玉米植株的染色体组中,而且转入mCry2Ab核苷酸序列的玉米植株均获得了含有单拷贝mCry2Ab、Cry2Ab和yCry2Ab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(Cry2Ab蛋白)的含量检测
本实验中涉及的溶液如下:
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。
分别取3mg转入mCry2Ab核苷酸序列的玉米植株和转入Cry2Ab、yCry2Ab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的转速下离心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,Cry2A试剂盒)对样品中杀虫蛋白质(Cry2A蛋白)量占叶片鲜重的比例进行检测分析,具体方法参考其产品说明书。
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mCry2Ab核苷酸序列的共3个株系(2A1、2A2和2A3),转入Cry2Ab核苷酸序列的共3个株系(2A4、2A5和2A6),转入yCry2Ab核苷酸序列的共3个株系(2A7、2A8和2A9)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(Cry2Ab蛋白)含量的实验结果如表1所示。分别测得转入mCry2Ab核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列以及yCry2Ab)的玉米植株的新鲜叶片中杀虫蛋白(Cry2Ab蛋白)平均表达量占叶片鲜重的比例(ng/g)分别为3052.8、2872.2和3007.7,这一结果表明Cry2Ab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Cry2A蛋白表达量测定平均结果
mCry2Ab蛋白的体外表达及纯化的步骤具体如下,Cry2Ab和yCry2Ab蛋白纯化步骤相同:
1、人工合成序列表中序列1所示的双链DNA分子
2、将步骤1合成的双链DNA分子与原核表达载体pEASY-E1连接,得到重组质粒pEASY-mCry2Ab。对重组质粒pEASY-mCry2Ab进行测序。测序结果表明,重组质粒pEASY-mCry2Ab中含有序列表中序列1所示的DNA分子,表达序列表中序列2所示的mCry2Ab蛋白。
3、将重组质粒pEASY-mCry2Ab导入大肠杆菌transetta,得到重组菌,将该重组菌命名为transetta-mCry2Ab。
4、取transetta-mCry2Ab的单克隆,接种至100mL LB液体培养基(含50μg/mL氨苄霉素),37℃、200rpm振荡培养12h,得到培养菌液。
5、取培养菌液,按体积比为1:100接种至50mL LB液体培养基(含50μg/mL氨苄霉素),37℃、200rpm振荡培养至OD600nm值为0.6,然后加入IPTG并使其浓度为1mM,28℃、220rpm振荡培养4h,4℃、10000rpm离心10min,收集菌体沉淀。
6、取菌体沉淀,加入100mL pH 8.0、100mM的Tris-HCl缓冲液,重悬后超声破碎(超声波功率600W,循环程序为:破碎4s,停6s,共20min),然后4℃、10000rpm离心10min,收集上清液甲。
7、取上清液甲,4℃、12000rpm离心10min,收集上清液乙。
8、采用GE公司生产的镍柱对上清液乙进行纯化(纯化的具体步骤参考镍柱的说明书),然后采用赛默飞世尔公司生产的蛋白定量试剂盒对mCry2Ab蛋白进行定量。
按照上述方法,将步骤1中mCry2Ab双链DNA分子替换为Cry2Ab的双链DNA分子,其它步骤均不变,得到Cry2Ab蛋白,用得到的抗虫蛋白进行害虫饲喂。
抗虫效果如表2所示
实施例5转基因玉米植株的抗虫效果检测
将转入mCry2Ab、Cry2Ab和yCry2Ab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斜纹夜蛾和甜菜夜蛾进行抗虫效果检测。
分别取转入mCry2Ab、Cry2Ab和yCry2Ab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(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,然后将玉米叶片去除叶脉,同时剪成约1cm×4cm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头人工饲养的甜菜夜蛾和斜纹夜蛾(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(光/暗)16:8的条件下放置3天后,统计死亡率。转入mCry2Ab核苷酸序列的共3个株系(2A1、2A2和2A3),转入Cry2Ab核苷酸序列的共3个株系(2A4、2A5和2A6),转入yCry2Ab核苷酸序列的共3个株系(2A7、2A8、2A9)经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。结果如表3、表4和图7所示。
5、技术效果
图7为本发明控制害虫的方法的转化体的抗斜纹夜蛾,其中WT为野生型植株,Cry2Ab为转化了未经修饰的Cry2Ab转化事件,mCry2Ab为转化了经过修饰的Cry2Ab转化事件。如图7、表2和表3所示,改造mCry2Ab基因转入植物后,所获得的高表达mCry2Ab蛋白的转基因植物具有甜菜夜蛾和斜纹夜蛾的抗性,较Cry2Ab和yCry2Ab抗性更好,在玉米螟、棉铃虫、东方黏虫的抗虫效果上没有明显差异。
表2
表3斜纹夜蛾离体叶片抗虫生测实验数据
mCry2Ab | Cry2Ab | yCry2Ab | |
接虫数 | 180 | 180 | 180 |
3d致死率 | 80%±9%b | 57%±7%a | 58%±7%a |
4d致死率 | 95%±4%b | 73%±8%a | 72%±8%a |
表4甜菜夜蛾离体叶片抗虫生测实验数据
mCry2Ab | Cry2Ab | yCry2Ab | |
接虫数 | 180 | 180 | 180 |
2d致死率 | 92%±7%b | 60%±6%a | 62%±6%a |
3d致死率 | 98%±2%b | 80%±7%a | 79%±7%a |
虽然,上文中已经用一般性说明及具体实施例对本申请作了详尽的描述,但在本申请基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本申请精神的基础上所做的这些修改或改进,均属于本申请要求保护的范围。
序列表
<110> 隆平生物技术(海南)有限公司
<120> 一种植物抗虫基因mCry2Ab及其载体和应用
<130> 201902
<141> 2019-12-19
<160> 19
<170> SIPOSequenceListing 1.0
<210> 1
<211> 634
<212> PRT
<213> BacillusthuringHansis
<400> 1
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370 375 380
Arg Glu Gly Val Ala Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu
385 390 395 400
Thr Thr Leu Gly Leu Arg Ser Gly Ala Phe Thr Ala Arg Gly Asn Ser
405 410 415
Asn Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu
420 425 430
Val Val Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Glu Ile
435 440 445
Arg Asn Ile Ala Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr
450 455 460
Met Val Ser Val His Asn Arg Lys Asn Asn Ile His Ala Val His Glu
465 470 475 480
Asn Gly Ser Met Ile His Leu Ala Pro Asn Asp Tyr Thr Gly Phe Thr
485 490 495
Ile Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe
500 505 510
Ile Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln
515 520 525
Asn Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr
530 535 540
Asn Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val
545 550 555 560
Thr Ile Asn Gly Arg Val Tyr Thr Ala Thr Asn Val Asn Thr Thr Thr
565 570 575
Asn Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn
580 585 590
Ile Gly Asn Val Val Ala Ser Ser Asn Ser Asp Val Pro Leu Asp Ile
595 600 605
Asn Val Thr Leu Asn Ser Gly Thr Gln Phe Asp Leu Met Asn Ile Met
610 615 620
Leu Val Pro Thr Asn Ile Ser Pro Leu Tyr
625 630
<210> 2
<211> 633
<212> PRT
<213> BacillusthuringHansis
<400> 2
Met Asn Ser Val Leu Asn Ser Gly Arg Thr Thr Ile Cys Asp Ala Tyr
1 5 10 15
Asn Val Ala Ala His Asp Pro Phe Ser Phe Gln His Lys Ser Leu Asp
20 25 30
Thr Val Gln Lys Glu Trp Thr Glu Trp Lys Lys Asn Asn His Ser Leu
35 40 45
Tyr Leu Asp Pro Ile Val Gly Thr Val Ala Ser Phe Leu Leu Lys Lys
50 55 60
Val Gly Ser Leu Val Gly Lys Arg Ile Leu Ser Glu Leu Arg Asn Leu
65 70 75 80
Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg Glu
85 90 95
Thr Glu Lys Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala Arg
100 105 110
Val Asn Ala Glu Leu Thr Gly Leu Gln Ala Asn Val Glu Glu Phe Asn
115 120 125
Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Arg Asn Ala Val Pro Leu
130 135 140
Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn Arg
145 150 155 160
Leu Pro Gln Phe Gln Met Gln Gly Tyr Gln Leu Leu Leu Leu Pro Leu
165 170 175
Phe Ala Gln Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp Val Ile
180 185 190
Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr Tyr
195 200 205
Arg Asp Tyr Leu Lys Asn Tyr Thr Arg Asp Tyr Ser Asn Tyr Cys Ile
210 215 220
Asn Thr Tyr Gln Ser Ala Phe Lys Gly Leu Asn Thr Arg Leu His Asp
225 230 235 240
Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr Val
245 250 255
Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser Gly
260 265 270
Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser Phe
275 280 285
Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn Ser
290 295 300
Asn Tyr Val Leu Asn Gly Phe Ser Gly Ala Arg Leu Ser Asn Thr Phe
305 310 315 320
Pro Asn Ile Val Gly Leu Pro Gly Ser Thr Thr Thr His Ala Leu Leu
325 330 335
Ala Ala Arg Val Asn Tyr Ser Gly Gly Ile Ser Ser Gly Asp Ile Gly
340 345 350
Ala Ser Pro Phe Asn Gln Asn Phe Asn Cys Ser Thr Phe Leu Pro Pro
355 360 365
Leu Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp Arg
370 375 380
Glu Gly Val Ala Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu Thr
385 390 395 400
Thr Leu Gly Leu Arg Ser Gly Ala Phe Thr Ala Arg Gly Asn Ser Asn
405 410 415
Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu Val
420 425 430
Val Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Glu Ile Arg
435 440 445
Asn Ile Ala Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr Met
450 455 460
Val Ser Val His Asn Arg Lys Asn Asn Ile His Ala Val His Glu Asn
465 470 475 480
Gly Ser Met Ile His Leu Ala Pro Asn Asp Tyr Thr Gly Phe Thr Ile
485 490 495
Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe Ile
500 505 510
Ser Glu Lys Phe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln Asn
515 520 525
Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr Asn
530 535 540
Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val Thr
545 550 555 560
Ile Asn Gly Arg Val Tyr Thr Ala Thr Asn Val Asn Thr Thr Thr Asn
565 570 575
Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn Ile
580 585 590
Gly Asn Val Val Ala Ser Ser Asn Ser Asp Val Pro Leu Asp Ile Asn
595 600 605
Val Thr Leu Asn Ser Gly Thr Gln Phe Asp Leu Met Asn Ile Met Leu
610 615 620
Val Pro Thr Asn Ile Ser Pro Leu Tyr
625 630
<210> 3
<211> 1905
<212> DNA
<213> BacillusthuringHansis
<400> 3
atggacaact ccgtcctgaa ctctggtcgc accaccatct gcgacgccta caacgtcgcg 60
gcgcatgatc cattcagctt ccagcacaag agcctcgaca ctgttcagaa ggagtggacg 120
gagtggaaga agaacaacca cagcctgtac gtggaccccg tcgtcggcac ggtggccagc 180
ttccttctca agaaggtcgg ctctctcgtc gggaagcgca tcctctcgga actccgcaac 240
ctgatctttc catctggctc caccaacctc atgcaagaca tcctcaggga gaccgagaag 300
tttctcaacc agcgcctcaa cactgatacc cttgctcgcg tcaacgctga gctgacgggt 360
ctgcaagcaa acgtggagga gttcaaccgc caagtggaca acttcctcaa ccccaaccgc 420
aatgcggtgc ctctgtccat cacttcttcc gtgaacacca tgcaacaact gttcctcaac 480
cgcttgcctc agttccagat gcaaggctac cagctgctcc tgctgccact ctttgctcag 540
gctgccaacc tgcacctctc cttcattcgt gacgtgatcc tcaacgctga cgagtggggc 600
atctctgcag ccacgctgag gacctaccgc gactacctga agaactacac cagggactac 660
tccaactatt gcatcaacac ctaccagtcg gccttcaagg gcctcaatac gaggcttcac 720
gacatgctgg agttcaggac ctacatgttc ctgaacgtgt tcgagtacgt cagcatctgg 780
tcgctcttca agtaccagag cctgctggtg tccagcggcg ccaacctcta cgccagcggc 840
tctggtcccc aacaaactca gagcttcacc agccaggact ggccattcct gtattcgttg 900
ttccaagtca actccaacta cgtcctcaac ggcttctctg gtgctcgcct ctccaacacc 960
ttccccaaca ttgttggcct ccccggctcc accacaactc atgctctgct tgctgccaga 1020
gtgaactact ccggcggcat ctcgagcggc gacattggtg catcgccgtt caaccagaac 1080
ttcaactgct ccaccttcct gccgccgctg ctcaccccgt tcgtgaggtc ctggctcgac 1140
agcggctccg accgcgaggg cgtggccacc gtcaccaact ggcaaaccga gtccttcgag 1200
accacccttg gcctccggag cggcgccttc acggcgcgtg gaaattctaa ctacttcccc 1260
gactacttca tcaggaacat ctctggtgtt cctctcgtcg tccgcaacga ggacctccgc 1320
cgtccactgc actacaacga gatcaggaac atcgcctctc cgtccgggac gcccggaggt 1380
gcaagggcgt acatggtgag cgtccataac aggaagaaca acatccacgc tgtgcatgag 1440
aacggctcca tgatccacct ggcgcccaat gattacaccg gcttcaccat ctctccaatc 1500
cacgccaccc aagtgaacaa ccagacacgc accttcatct ccgagaagtt cggcaaccag 1560
ggcgactccc tgaggttcga gcagaacaac accaccgcca ggtacaccct gcgcggcaac 1620
ggcaacagct acaacctgta cctgcgcgtc agctccattg gcaactccac catcagggtc 1680
accatcaacg ggagggtgta cacagccacc aatgtgaaca cgacgaccaa caatgatggc 1740
gtcaacgaca acggcgcccg cttcagcgac atcaacattg gcaacgtggt ggccagcagc 1800
aactccgacg tcccgctgga catcaacgtg accctgaact ctggcaccca gttcgacctc 1860
atgaacatca tgctggtgcc aactaacatc tcgccgctgt actga 1905
<210> 4
<211> 1902
<212> DNA
<213> BacillusthuringHansis
<400> 4
atgaactccg tcctcaacag cggccgcacc accatctgcg acgcctacaa cgtggccgcc 60
cacgacccct tctccttcca acacaagtcc ctggacaccg tccagaagga gtggaccgag 120
tggaagaaga acaaccacag cctgtacctc gaccccatcg tcggcaccgt ggcctccttc 180
ctgctgaaga aggtcggctc cctcgtcggc aagcgtatcc tgtccgagct gcgcaacctc 240
atcttcccca gcggcagcac caacctgatg caggacatcc tgcgcgagac cgagaagttc 300
ctcaaccaga ggctgaacac cgacaccctg gctcgcgtga acgccgagct gaccggcctc 360
caggccaacg tcgaggagtt caaccgccag gtggacaact tcctgaaccc caaccgtaac 420
gccgtccccc tctccatcac ctcctccgtc aacaccatgc agcagctgtt cctgaaccgc 480
ctcccccagt tccagatgca gggctaccag ctgctcctgc tgcccctctt cgcccaggct 540
gccaacctgc acctgtcctt catcagggac gtcatcctca acgccgacga gtggggcatc 600
agcgccgcca ccctgcgcac ctaccgcgac tacctgaaga actacacccg cgactactcc 660
aactactgca tcaacaccta ccagagcgct ttcaagggcc tcaacacccg tctgcacgac 720
atgctggagt tcaggaccta catgttcctc aacgtgttcg agtacgtgtc catctggtcc 780
ctgttcaagt accagagcct gctcgtctcc tccggcgcca acctgtacgc cagcggctcc 840
ggcccccagc agacccagag cttcacctcc caggactggc ccttcctgta ctccctcttc 900
caggtcaact ccaactacgt cctgaacggc ttcagcggcg cccgcctgag caacaccttc 960
cccaacatcg tcggcctccc cggctccacc accacccacg ccctgctggc tgcccgcgtg 1020
aactactccg gcggcatctc ctccggcgac atcggcgcca gccccttcaa ccagaacttc 1080
aactgctcca ccttcctccc ccccctgctg acccccttcg tgcgctcctg gctcgactcc 1140
ggctccgacc gcgagggcgt cgccaccgtc accaactggc agaccgagag cttcgagacc 1200
accctgggcc tgaggtccgg cgccttcacc gctcgtggca acagcaacta cttccccgac 1260
tacttcatcc gcaacatctc cggcgtcccc ctcgtcgtgc gcaacgagga cctgcgcagg 1320
cccctgcact acaacgagat ccgcaacatc gcctccccca gcggcacccc cggcggcgcc 1380
cgtgcctaca tggtgtccgt ccacaaccgc aagaacaaca tccacgccgt ccacgagaac 1440
ggctccatga tccacctcgc tccaaacgac tacaccggct tcaccatcag ccccatccac 1500
gccacccaag tcaacaacca gacccgcacc ttcatctccg agaaattcgg caaccagggc 1560
gacagcctga ggttcgagca gaacaacacc accgcccgct acaccctgcg cggcaacggc 1620
aactcctaca acctctacct gcgtgtgtcc tccatcggca acagcaccat ccgcgtcacc 1680
atcaacggca gggtgtacac cgccaccaac gtcaacacca ccaccaacaa cgacggcgtc 1740
aacgacaacg gcgcccgctt cagcgacatc aacatcggca acgtggtcgc ttcctccaac 1800
tccgacgtcc ccctggacat caacgtgacc ctcaactccg gcacccagtt cgacctgatg 1860
aacatcatgc tggtccccac caacatcagc cccctctact aa 1902
<210> 5
<211> 1993
<212> DNA
<213> Zea mays L.
<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> 253
<212> DNA
<213> Agrobacterium tumefaciens
<400> 6
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> 7
<211> 552
<212> DNA
<213> Zea mays L.
<400> 7
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> 8
<211> 1134
<212> DNA
<213> CaMV
<400> 8
ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag gtggctccta 60
caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg 120
tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac 180
gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg acgcacaatc 240
ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac 300
acgctgacaa gctgactcta gcagatctac cgtcttcggt acgcgctcac tccgccctct 360
gcctttgtta ctgccacgtt tctctgaatg ctctcttgtg tggtgattgc tgagagtggt 420
ttagctggat ctagaattac actctgaaat cgtgttctgc ctgtgctgat tacttgccgt 480
cctttgtagc agcaaaatat agggacatgg tagtacgaaa cgaagataga acctacacag 540
caatacgaga aatgtgtaat ttggtgctta gcggtattta tttaagcaca tgttggtgtt 600
atagggcact tggattcaga agtttgctgt taatttaggc acaggcttca tactacatgg 660
gtcaatagta tagggattca tattataggc gatactataa taatttgttc gtctgcagag 720
cttattattt gccaaaatta gatattccta ttctgttttt gtttgtgtgc tgttaaattg 780
ttaacgcctg aaggaataaa tataaatgac gaaattttga tgtttatctc tgctccttta 840
ttgtgaccat aagtcaagat cagatgcact tgttttaaat attgttgtct gaagaaataa 900
gtactgacag tattttgatg cattgatctg cttgtttgtt gtaacaaaat ttaaaaataa 960
agagtttcct ttttgttgct ctccttacct cctgatggta tctagtatct accaactgac 1020
actatattgc ttctctttac atacgtatct tgctcgatgc cttctcccta gtgttgacca 1080
gtgttactca catagtcttt gctcatttca ttgtaatgca gataccaagc ggcc 1134
<210> 9
<211> 195
<212> DNA
<213> CaMV
<400> 9
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> 10
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 10
agaagaacaa ccacagcctg 20
<210> 11
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 11
tgtcgtgaag cctcgtattg 20
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 12
tctccttcat tcgtgacgtg 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 13
gccgactggt aggtgttgat 20
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 14
ccatccctcc gtagttagct tct 23
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 15
cctgtcggcc aaggctatat ac 22
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 16
gaccgagtgg aagaagaaca 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 17
actcgaacac gttgaggaac 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 18
ctaccgcgac tacctgaaga 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 19
gtcctgaact ccagcatgtc 20
Claims (10)
1.一种用于植物抗虫的蛋白质,其特征在于,所述蛋白质的氨基酸序列为SEQ ID NO:1。
2.一种抗虫基因,其特征于,所述抗虫基因包含编码如权利要求1所述的蛋白质的基因序列。
3.如权利要求2所述的抗虫基因,其特征在于,所述抗虫基因的核苷酸序列为SEQ IDNO:3。
4.一种含有如权利要求2或3所述抗虫基因的表达盒、重组载体、重组微生物或转基因细胞系。
5.一种表达载体,其特征在于,所述表达载体包含如权利要求2所述的抗虫基因。
6.如权利要求5所述的表达载体,其特征在于,所述表达载体中依次包含以下基因结构:
花椰菜花叶病毒的35S启动子;抗虫基因;胭脂碱合成酶的终止子;玉米泛素基因启动子;编码磷丝菌素乙酰转移酶基因;自花椰菜花叶病毒的终止子。
7.如权利要求1所述的用于植物抗虫的蛋白质或者权利要求2或3所述的抗虫基因或含有权利要求2或3所述抗虫基因的表达盒、重组载体、重组微生物或转基因细胞系在植物抗虫中的应用,其特征在于,所述应用为:a)制备具有抗虫效果的药剂;b)培育具有或提高抗虫能力的转基因植物;所述应用中防治的害虫选自甜菜夜蛾、斜纹夜蛾、玉米螟、棉铃虫、草地贪夜蛾和东方黏虫中的一种或多种。
8.一种培育具有或提升了抗虫能力的植物的方法,其特征在于,所述方法包括如下步骤:将权利要求2或3所述抗虫基因导入受体植物中,得到转基因植物。
9.如权利要求8所述的方法,其特征在于:所述植物选自单子叶植物或双子叶植物。
10.如权利要求8或9所述的方法,其特征在于:所述植物为玉米。
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