CN107058376B - 一种防治农作物半翅目害虫的方法 - Google Patents
一种防治农作物半翅目害虫的方法 Download PDFInfo
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- CN107058376B CN107058376B CN201710358776.4A CN201710358776A CN107058376B CN 107058376 B CN107058376 B CN 107058376B CN 201710358776 A CN201710358776 A CN 201710358776A CN 107058376 B CN107058376 B CN 107058376B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明公开了一种防治农作物半翅目害虫的方法,所述方法是在农作物中表达抗虫蛋白,所述抗虫蛋白为Bt抗虫蛋白Cry30Ca。对于抗虫基因cry30Ca,利用合适的启动子,可以保证在高效表达该抗虫基因的同时,提高植物体内抗虫蛋白表达的稳定性;cry30Ca基因所编码的抗虫蛋白Cry30Ca,具有抗半翅目昆虫,特别是飞虱科的活性,将其转入农作物,能够使转基因植物获得抗半翅目,特别是飞虱科昆虫的能力,减少农业生产中的产量损失、降低农业生产成本。
Description
(一)技术领域
本发明涉及一种防治农作物半翅目害虫的方法,特别涉及在水稻中表达抗虫蛋白、使水稻获得抗飞虱科昆虫能力,防治水稻飞虱科害虫的方法。
(二)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,但是农药的残留会对人体健康和环境带来不良影响。利用生物技术方法培育含有抗虫基因的转基因抗虫农作物,可以大幅度降低化学杀虫剂的使用,有效保护农作物免受害虫为害,目前全球范围内,商业化的转基因抗虫玉米、大豆、棉花等已经大面积推广种植。
在田间,半翅目害虫的种类非常多,如蝽类、螨类、飞虱科等。半翅目害虫靠刺吸植物组织汁液来获取营养,危害多种农作物、对农业生产造成巨大损失。比如烟粉虱危害番茄、辣椒、烟草、棉花等农作物,是一种世界性害虫;褐飞虱是水稻上最主要的害虫之一,并且具有长距离迁飞的特性;蚜虫是一种危害非常广泛的害虫。半翅目害虫很难防治,其繁殖力强、虫体小,有些害虫具有迁飞能力;其刺吸式的取食方式使得传统农药对其收效甚微。利用植物转基因技术,将抗虫基因转入农作物中,是对抗半翅目害虫的可行方法。
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,比较常见的是Bt杀虫晶体蛋白,如Cry1Ab、Cry1C等,它们已被大量运用于商业化的转基因抗虫作物。Bt杀虫晶体蛋白的种类多达300余种,其中部分蛋白具有杀虫活性。Cry30Ca是Bt杀虫晶体蛋白中的一种,具有抗飞虱科昆虫的活性。将Cry30Ca转入植物中,可以使植物获得抗飞虱科昆虫的潜力。然而,外源基因在植物体内的表达需要合适的条件,并非任意的方法都能达到稳定高效表达外源蛋白的效果。利用植物内源性启动子构建基因表达框、在水稻中启动编码Cry30Ca蛋白基因的表达的转基因水稻,能够获得抗飞虱科昆虫的活性。该方法获得的转基因水稻具备抗虫能力,可以减少田间飞虱科的害虫对水稻产量造成的损失,具有重要的应用前景。
(三)发明内容
本发明目的是提供一种防治农作物半翅目害虫的方法,特别是防治水稻飞虱科害虫的方法,该方法将cry30Ca基因导入水稻中、表达Cry30Ca蛋白,使水稻获得抗飞虱科害虫的能力。
本发明采用的技术方案是:
本发明提供一种防治农作物半翅目害虫的方法,所述方法是在农作物中表达抗虫蛋白,所述抗虫蛋白为Bt抗虫蛋白Cry30Ca。
进一步,所述Bt抗虫蛋白Cry30Ca的氨基酸序列为SEQ ID NO.2所示,所述Bt抗虫蛋白Cry30Ca编码基因的核苷酸序列为SEQ ID NO.1所示。
进一步,所述抗虫蛋白的表达采用植物内源性启动子,所述植物内源性启动子包括但不限于水稻肌动蛋白1启动子pOsActin1或玉米泛素启动子pZmUbi。
进一步,所述表达抗虫蛋白的方法为:将含有启动子、抗虫蛋白编码基因和终止子的表达框导入载体中,构建T-DNA载体;然后将T-DNA载体转化农杆菌,再转入植物中,从而促进抗虫蛋白在植物中的表达,使农作物获得抗半翅目昆虫的能力,减少半翅目害虫对农作物的危害;所述终止子为玉米磷酸烯醇式丙酮酸羧化酶终止子PEPC-ter。
进一步,所述农作物半翅目害虫为飞虱科昆虫,所述植物为水稻、玉米、小麦、高粱、大豆、油菜或棉花。
本发明所涉及植物内源性启动子,其来源于植物体自身;原核生物如细菌、真菌和病毒的启动子不在其列。植物内源性启动子有许多种,常见的有泛素启动子Ubi、肌动蛋白启动子Actin等。所有植物内源性启动子启动cry30Ca基因的表达,都应视为本发明所提供的方法。
本行业的技术人员均知道以下理论:
(1)Cry蛋白家族种类繁多,但不是所有蛋白都具有杀虫活性,而且每种抗虫蛋白对应的抗虫谱有很大差异。Cry蛋白目前已经发现了300多种,其中很多是具有杀虫活性的,比如对鳞翅目具有很高活性的Cry1Ab/1Ac、对鞘翅目具有活性的Cry3C等。不同的Cry蛋白,其抗虫谱是有显著差异的,所对应的抗虫种类不尽相同;甚至只相差几个氨基酸,都能影响到抗虫蛋白的活性。启动子的选择对抗虫基因的表达起到至关重要的作用。目前商业化种植的转基因农作物中,很多品系采用了来自原核生物的启动子,如花椰菜花叶病毒启动子CaMV 35s、木薯叶脉花叶病毒启动子Csv等。并不是任意启动子都能高效且稳定地表达特定的外源基因,只有利用合适的启动子才能使外源基因在植物体内稳定表达,而不被植物自身所甲基化、基因剪切等。同时,单子叶植物与双子叶植物对启动子有不同的喜好;因此,对于不同的农作物,启动子的选择不一而同。
(2)不是所有的Cry基因转入农作物(优选水稻)后,都能使其获得对抗半翅目(优选飞虱科)害虫的能力。一方面,农作物(优选转基因水稻)获得抗虫能力依赖于外源的抗虫蛋白对半翅目(优选飞虱科)害虫的活性;另一方面,农作物(优选转基因水稻)的抗虫性能取决于外源抗虫蛋白在农作物中的表达量。
与现有技术相比,本发明方法有益效果主要体现在:对于抗虫基因cry30Ca,利用合适的启动子,可以保证在高效表达该抗虫基因的同时,提高植物体内抗虫蛋白表达的稳定性;cry30Ca基因所编码的抗虫蛋白Cry30Ca,具有抗半翅目昆虫,特别是飞虱科的活性,将其转入农作物,能够使转基因植物获得抗半翅目,特别是飞虱科昆虫的能力,减少农业生产中的产量损失、降低农业生产成本。
(四)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
本发明以下实施例中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 MolecularBiology,和J.Sambrook等编写的Cold Spring Harbor Laboratory Press(2001)出版的Molecular Cloning:A Laboratory Manual,3rd ED.等文献均有详细的说明。
实施例1、构建一个农杆菌转化水稻表达抗虫基因的载体
由上海生工人工合成抗虫基因Cry30Ca(核苷酸序列为SEQ ID NO.1,编码蛋白的氨基酸序列为SEQ ID NO.2所示,其5’端被设计上BamHI位点,3’端被设计上SacI位点,以BamHI-SacI双酶切可以得到基因片段)。对于水稻,选用水稻肌动蛋白1启动子(pOsActin1,Genbank:NC_008398)来启动cry30Ca基因的表达,其5’端被设计上HindIII位点,3’端被设计上BamHI位点,HindIII-BamHI片段;选用玉米磷酸烯醇式丙酮酸羧化酶终止子(PEPC-ter,Genebank:X15239)作为该基因的终止子,其5’端被设计上SacI位点,3’端被设计上KpnI位点,SacI-KpnI片段。水稻启动子pOsActin1从水稻的基因组中通过PCR获得,使用的引物分别是:pAct-F(5’AAGCTTAGGTCATTCATATGCTTGAGAAGAGTC);pAct-R(5’GGATCCTCGGCGTCAGCCATCTTCTAC)。
农杆菌转化T-DNA载体是基于pCambia 1300(NCBI序列编号AF234296)载体而构建的。将pCambia 1300以HindIII和KpnI酶切、回收,作为载体片段;将pActin1、cry30Ca、PEPC-ter分别酶切后得到插入片段,并连接到载体片段中,得到最终载体pCambia1300-Actin1-cry30Ca。将载体转入农杆菌菌株中,得到含有该T-DNA载体的水稻转化农杆菌。
实施例2、转基因水稻的获得
转基因水稻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)。选取成熟饱满的水稻种子去壳,诱导产生愈伤组织为转化材料。取含有目的基因的农杆菌(实施例1制备的pCambia1300-Actin1-cry30Ca)划板,挑单克隆菌落接种准备转化农杆菌。将待转化的水稻愈伤组织放入OD600在0.3-0.4之间的农杆菌液中(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组织,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组织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转移到分化培养基,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到温室,作为鉴定材料。
实施例3、转基因农作物抗虫能力的测定
利用褐飞虱和白背飞虱测定实施例2获得的10个转基因水稻株系的抗虫活性,测定方法是:将褐飞虱和白背飞虱的初孵若虫接到转基因水稻和同一生长期的非转基因水稻上,每个株系置于单独的养虫笼中,在恒温25℃、恒湿85%的培养室里培养5至7天,记录数据。测试的10个转基因株系中有8个株系未受到昆虫的危害,昆虫全部死亡;非转基因对照组上的昆虫全部成活,且水稻受害严重。
最后,还需要注意的是,以上列举的仅是本发明的若干实施例。显然,本发明不限于以上实施例,还可以有许多延伸和拓展。本领域的普通技术人员能从本发明公开的内容直接到导出或联想到的所有延伸,均应认为是本发明的保护范围。
SEQUENCE LISTING
<110> 浙江大学
<120> 一种防治农作物半翅目害虫的方法
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Asn Pro Glu Asn Lys Pro Ala Phe Leu Ile Leu Tyr Ala Gln Thr Ala
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Gly Ala Leu Asp Leu Ala Ala Leu Phe Pro Asn Tyr Asp Ile Cys Ile
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Thr Phe Tyr Lys Ser Asp Tyr Asn Gly Asn Ala Pro Thr Thr Gln Thr
435 440 445
Tyr Gln Ala Gly Arg Asn Ser Asn Asn Phe Ile Asn Thr Phe Met Asn
450 455 460
Gly Pro Gln Glu Ala Ser Ser Ser Asn Asn Ile Ser Ile Lys Gln Thr
465 470 475 480
Asn His Ile Leu Ser Asp Ile Lys Met Ile Tyr Ser Arg Thr Gly Gly
485 490 495
Thr Tyr Pro Ser Tyr Asp Phe Gly Tyr Ser Phe Ala Trp Thr His Thr
500 505 510
Ser Val Asn Pro Asp Asn Leu Ile Val Pro Asn Arg Ile Thr Gln Ile
515 520 525
Pro Ala Val Lys Ala Asp Tyr Leu Thr Ser Pro Ala Lys Val Ile Ala
530 535 540
Gly Pro Gly His Thr Gly Gly Asp Leu Val Ala Leu Leu Asn Ala Ala
545 550 555 560
Thr Gln Ala Gly Arg Met Gln Ile Gln Cys Lys Thr Gly Ser Phe Thr
565 570 575
Gly Ala Ser Arg Arg Tyr Gly Ile Arg Ile Arg Tyr Ala Ala Asn Asn
580 585 590
Ala Leu Thr Val Ser Leu Ser Tyr Thr Val Gln Gly Gly Asn Thr Met
595 600 605
Ser Thr Thr Phe Ile Thr Glu Arg Thr Phe Leu Arg Pro Asn Asn Thr
610 615 620
Ile Pro Thr Asp Leu Lys Tyr Glu Glu Phe Lys Tyr Lys Glu Tyr Asn
625 630 635 640
Gln Ile Ile Thr Met Thr Ala Pro Gln Asn Thr Ile Val Thr Ile Ala
645 650 655
Ile Gln Gln Leu Asn Ala Phe Pro Asn Asp Gln Leu Ile Ile Asp Arg
660 665 670
Ile Glu Phe Tyr Pro Met Asp Gln Gly Val Val Pro Cys Thr Val Asn
675 680 685
Claims (6)
1.一种防治农作物半翅目害虫的方法,其特征在于所述方法是在农作物中表达抗虫蛋白,所述抗虫蛋白为Bt抗虫蛋白Cry30Ca;所述Bt抗虫蛋白Cry30Ca的氨基酸序列为SEQ IDNO.2所示;所述半翅目害虫为褐飞虱或白背飞虱。
2.如权利要求1所述的方法,其特征在于所述Bt抗虫蛋白Cry30Ca编码基因的核苷酸序列为SEQ ID NO.1所示。
3.如权利要求1所述的方法,其特征在于所述抗虫蛋白的表达采用植物内源性启动子。
4.如权利要求3所述的方法,其特征在于所述植物内源性启动子为水稻肌动蛋白1启动子pOsActin1或玉米泛素启动子pZmUbi。
5.如权利要求1所述的方法,其特征在于所述方法为:将含有启动子、抗虫蛋白编码基因和终止子的表达框导入载体中,构建T-DNA载体;然后将T-DNA载体转化农杆菌,再转入植物中,从而促进抗虫蛋白在植物中的表达;所述终止子为玉米磷酸烯醇式丙酮酸羧化酶终止子PEPC-ter。
6.如权利要求1所述的方法,其特征在于所述农作物为水稻、玉米、小麦、高粱、大豆、油菜或棉花。
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