CN101492686A - 苏云金芽胞杆菌杀线虫晶体蛋白基因cry1518-35及应用 - Google Patents
苏云金芽胞杆菌杀线虫晶体蛋白基因cry1518-35及应用 Download PDFInfo
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Abstract
本发明属于微生物基因工程技术领域。公开了一种来自于苏云金芽胞杆菌的杀线虫晶体蛋白基因的分离、克隆及其对线虫的生物活性。本发明从对北方根结线虫有活性的苏云金芽胞杆菌YBT-1518菌株中分离得到一个新的杀虫晶体蛋白基因cry1518-35;该基因的编码产物为一种新的杀虫晶体蛋白Cry1518-35;该蛋白Cry1518-35对北方根结线虫有很高的杀虫活性。本发明还包括表达杀虫晶体蛋白基因cry1518-35的大肠杆菌重组菌EMB0225的制备,该重组大肠杆菌菌已保藏在中国典型培养物保藏中心(CCTCC),其保藏编号为CCTCC NO:M209009。
Description
技术领域
本发明属于农业生物防治和农业微生物基因工程领域。具体涉及一个具有杀线虫活性的杀虫晶体蛋白基因的分离、克隆以及它编码的蛋白在微生物杀虫剂中的应用。
背景技术
苏云金芽胞杆菌(Bacillus thuringiensis,Bt)是一类广泛存在于土壤中的革兰氏阳性细菌,在其生长发育过程中,能够形成内生芽胞,因其伴随芽胞形成而产生对昆虫具有特异毒性的杀虫晶体蛋白(InsecticidalCrystal Proteins,ICPs),而有别于分泌肠毒素的蜡状芽胞杆菌(Bacillus cereus)和引起炭疽病的炭疽芽胞杆菌(Bacillus anthracis)。研究表明,苏云金芽胞杆菌产生的晶体蛋白可以对鳞翅目、双翅目、鞘翅目、膜翅目、同翅目等昆虫纲10个目500多种昆虫以及原生动物、线形动物门、扁形动物门中某些有害种类也有特异的生物活性(Schnepf H E,Crickmore N,Rie J V,Lereclus D,Baum J,Feitelson J,Zeigler D R,Dean D H.1998.Bacillus thuringiensis and its pesticidal crystal proteins.Microbiol Mol Biol Rev,62:775-806)。Aroian的实验室用实验充分证明了苏云金芽胞杆菌对线虫的特异活性(Wei*J.Z.,Hale*K.,Carta L.,Platzer E.,WongC.,Fang S.C.,and Aroian R.V.(2003).Bacillus thuringiensis Crystal proteins that target nematodes.Proc.Natl.Acad.Sci.100:2760-2765)。
苏云金芽胞杆菌野生菌株一般含有多个编码杀虫晶体蛋白的基因。自1981年Schnepf和Whiteley从库斯塔克亚种(subsp.kurstaki)菌株HD-1中克隆得到第一个杀虫晶体蛋白基因以来,陆续发现并鉴定了许多新的杀虫晶体蛋白基因。此后,新的杀虫晶体蛋白基因的克隆一直是苏云金芽胞杆菌研究领域中的热点。因此在1995年的无脊椎病理学年会上成立了由Crickmore等学者组成的杀虫晶体蛋白基因命名委员会,并于1996年正式提出了苏云金芽胞杆菌杀虫晶体蛋白按氨基酸序列同源性进行分类的新的分类系统,规定了命名规则和分类原则。其规定:cry基因是来自苏云金芽胞杆菌编码伴胞晶体蛋白的杀虫基因或者与已知cry基因有明显的序列相似性的基因,cyt基因是编码具有溶细胞活性的伴胞晶体蛋白基因或与已知编码Cyt蛋白有明显的序列相似性的基因。根据全长基因推导的氨基酸序列的同源性分为四个等级。级与级之间的界限为同源性95%、78%和45%。截至2008年12月,苏云金芽胞杆菌杀虫晶体蛋白基因已累计达到55类436种cry基因和2类27个cyt基因(Crickmore N,Zeigler DR,Feitelson J,Schnepf E,van Rie J,Lereclus D,Baum J,Dean DH.1998.Revision of the nomenclature for the Bacillus thuringiensis pesticidalcrystal proteins.Microbiol.Mol.Biol.Rev.62:807-813)。
根结线虫(Meloidogyne)是一种重要的植物寄生线虫,主要寄生于被感染植物的块根、块茎、鳞茎或球茎上,从寄主体内吸取营养,造成寄主植物的生长发育不良、畸形、矮化甚至死亡,每年都给农业生产带来重大损失。它们可以寄生于110多种植物,包括大豆、花生、烟草、黄瓜、牡丹等,而茄科、葫芦科、十字花科等植物的受害尤其严重(刘维志,植物病原线虫学,北京:中国农业出版社,2000)。由于其生活史短,分布面广,目前已经成为农业生产上最重要的病原生物之一(Widmer T L,Abawi G S.Mechanism ofsuppression of Meloidogyne hapla and its damage by a green manure of Sudan grass.Plant Disease,2000,85(5):562-568.)。同时又由于根结线虫的侵入往往会使真菌和细菌更易侵染植物,是诱发植物病害的重要原因之一(汪来发等.根结线虫生物防治研究进展.南京林业大学学报(自然科学版),2002,26(1):64-68.)。
传统的防治根结线虫的方法主要包括轮作、休耕和化学防治。但是在许多的发展中国家,人们必须依靠多年生作物和作物连作,轮作和休耕都很难用于防治根结线虫;而化学防治的方法又会给环境带来很大污染。因此,通过生物的方法防治根结线虫逐渐成为该领域的研究热点之一。关于苏云金芽胞杆菌对植物寄生线虫防治的研究,最早的报道为1972年Prasad等首次发现该菌的β-外毒素即苏云金素对根结线虫的卵和幼虫有较高的毒杀作用(Prasad S S V,Tilak K V B R,Gollakota K G.Role of Bacillus thuringiensis var.thuringiensis on the larval survivability and egg hatching of Meloidogyne spp.,the causative agent of root-knotdisease.J.Invertebr.Pathol.,1972,20:377-378.)。随后的研究又发现苏云金素可以明显降低线虫对植物的侵染(Ignoffo C M,Dropkin V H.Deleterious effects of thermostable toxin of Bacillus toxin of Bacillusthuringiensis on species of soil-inhabiting myceliophagus,and plant-parasitic nematodes.J.Kans.Entomol.Soc.,1977,50:394-398.;Devidas P,Cibulski R J,Rehberger L.Evaluation of Bacillus thuringiensis exotoxin fornematode control.Nematologica,1988,34(3):249-301.)。上个世纪80年代Bone等人的研究首次证实了苏云金杆菌晶体蛋白对线虫的杀虫活性(Bone L W,Bottjer K P,Gill S S.Trichostrongylus colubriformis:Egglethality due to Bacillus thuringiensis crystal toxin.Exp.Parasitol.,1985,60:314-322.)。之后,美国Mycogen公司的大量研究工作也进一步证实了苏云金芽胞杆菌伴胞晶体对线虫的作用(Bradfish G A(1992).Processfor controlling lepidopteran pests.EP19920920639.)。此外他们还克隆了多个具有线虫活性的毒素蛋白的编码基因并申请了相关专利,而我们所能看到的关于苏云金芽胞杆菌对线虫的防治的报道也多见于这些专利中的描述。
目前,已经有越来越多的抗线虫的苏云金杆菌制剂在田间应用。Sharma等用苏云金亚种(subsp.thuringiensis)和以色列亚种(subsp.israelensis)菌剂处理大麦根际土壤对南方根结线虫(Meloidogyneincognita)起到了一定的防效;此外,他们还发现,用以色列亚种菌株Bti-H14的毒素蛋白处理大豆和玉米种子,能够使大豆胞囊线虫的寄生率显著降低(Sharma R D.Bacillus thuringiensis:a biocontrol agent ofMeloidogyne incognica on barely.Nematologia Brasilera,1994,18:79-84.)。Ivanova则报道某种苏云金芽胞杆菌制剂能够用于防治黄瓜和番茄上的根结线虫;而Ensard则用苏云金杆菌制剂在防治香蕉穿孔线虫方面也取得了显著效果(Ivanova T S.Efficiency of biopreparation in control of gall nematode in protected soil.Agrokhimiya,1996,3:101-106.;Ensard J.Effects of three microbial broth cultures on growth and populations offree living and plant-parasitic nematodes on banana.European Journal of Plant Pathology,1998,104(5):457-463.)。然而,随着Bt杀虫剂应用的推广和使用强度的不断增加,目标害虫的抗性现象陆续被科学家们所报道。研究人员从目标害虫对Bt杀虫剂的抗性机制的研究中发现,昆虫对Bt杀虫剂的抗性的产生与受体的识别与结合密切相关。因此,新的杀虫晶体蛋白基因的克隆和应用已经成为预防与控制目标害虫对Bt杀虫剂产生抗性的关键途径,是各种防治策略的核心内容。而目前杀线虫晶体蛋白基因资源十分有限,因此,寻找与克隆新的杀线虫晶体蛋白基因已经成为苏云金芽胞杆菌研究领域中最活跃的部分之一。苏云金芽胞杆菌YBT-1518菌株是由申请人所在的农业微生物国家重点实验室分离并保存的无鞭毛菌株(文献见实施例),它能够在其芽胞形成过程中形成米粒状的伴胞晶体,生物测定表明,它的晶体蛋白对秀丽小杆线虫(Caenorhabditis elegans)和北方根结线虫(Meloidgyne hapla)具有较高的活性。在进一步的基因克隆中在该菌株中发现了2个杀线虫活性的晶体蛋白基因cry5B和cry6Aa2,但是该菌中是否还存在着其他的具有杀虫活性的基因,前人的研究还不清楚。
发明内容
本发明的目的在于从苏云金芽胞杆菌中分离、克隆一个新的具有杀线虫活性的晶体蛋白基因,利用其编码的蛋白应用于微生物杀虫剂中。本发明从报道的苏云金芽胞杆菌YBT-1518中分离并克隆了一种新型的杀线虫晶体蛋白基因cry1518-35,验证它对北方根结线虫具有高毒力,并揭示了该晶体蛋白在防治根结线虫领域的应用。
本发明是这样实现的:
申请人从报道的苏云金芽胞杆菌菌株YBT-1518(菌株来源参见实施例1)中分离到一个新的晶体蛋白基因cry1518-35。经过测序发现本发明的晶体蛋白基因cry1518-35其编码区由861个碱基组成,具有序列表SEQ ID NO:1所示的核苷酸序列。经过序列分析发现本发明的cry1518-35基因编码的蛋白Cry1518-35是由286个氨基酸残基组成,具有序列表SEQ ID NO:1所示的氨基酸序列,预计分子量大小为34kDa。通过原核表达和室内生物测定证实本发明的基因cry1518-35在微生物中表达的Cry1518-35蛋白,能够对北方根结线虫有毒杀作用,这就预示着该基因可以用于构建抗线虫的转基因植物。
本发明提供的上述基因序列是一种新的杀线虫晶体蛋白基因,该基因可以应用于转化微生物和植物,使之表达出Cry1518-35,使受体生物表现出对线虫的毒杀活性,在寄生线虫的生物防治方面具有广泛的应用价值。
更详细的技术方案参考实施例部分的描述。
附图说明
序列表SEQ ID NO:1是本发明分离克隆的苏云金芽胞杆菌杀线虫晶体蛋白基因的核苷酸序列和其编码的蛋白质序列;
图1:是本发明实施例中杀线虫晶体蛋白基因cry1518-35所在的BAC克隆子的物理图谱;
图2:是本发明实施例中克隆载体pUC18-cry1518-35的构建图及其物理图谱;
图3:是本发明实施例中超量表达载体pEMB0225的构建图及其物理图谱;
图4:是本发明实施例中杀线虫晶体蛋白基因cry1518-35在重组菌EMB0225中表达纯化的SDS-PAGE电泳分析;图中:
M:蛋白质分子量标准;
1:纯化后的Cry1518-35杀线虫晶体蛋白。
具体实施方案
以下叙述是根据本发明实施方案的实施例。应该说明的是,本发明的实施例对于本发明只有说明作用,而没有限制作用。有关DNA的标准操作方法和所使用的药品均参考《分子克隆实验指南》所描述的内容(参见萨姆布鲁克和拉塞尔,2001,分子克隆实验指南,第三版,金冬雁等(译),科学出版社,北京)。本发明中所涉及的其他各种实验操作,均为本领域的常规技术,文中没有特别说明的部分,本领域的普通技术人员可以参照本发明申请日之前的各种常用工具书、科技文献或相关的说明书、手册等加以实施。
实施例1苏云金芽胞杆菌YBT-1518中杀线虫晶体蛋白基因cry1518-35的克隆
本发明以苏云金芽胞杆菌YBT-1518作为杀线虫晶体蛋白基因cry1518-35的来源菌株,该菌株的来源见文献:Yu,Z.,P.Bai,W.Ye,F.Zhang,L.Ruan,Z.Yu,and M.Sun.A Novel Negative Regulatory Factor forNematicidal Cry Protein Gene Expression in Bacillus thuringiensis.J.Microbiol.Biotechnol.18(6):1033-1039。该菌株无鞭毛,能形成典型的长米粒状晶体,对根结线虫和秀丽小杆线虫具有高毒力。
1.苏云金芽胞杆菌YBT-1518总质粒的抽提
将苏云金芽胞杆菌菌株YBT-1518过夜活化,按1/100(v/v)的接种量转接至5mL新鲜的LB培养基中(LB培养基配方:胰蛋白胨1%,酵母提取物0.5%,氯化钠0.5%,pH 7.0),28℃,200rpm培养至对数生长中期。STE(10mM Tris·HCl,1mM EDTA pH 8.0)洗涤菌体1次;加入90μL溶液I(50mM蔗糖,25mM Tris·HCl(pH 8.0),10mM EDTA)悬浮菌体,再加100mg/mL的溶菌酶(在溶液I中加溶菌酶)10μL,冰浴2h以上;加入200μL新鲜配制的溶液II(0.2M NaOH,1%十二烷基硫酸钠,即SDS),轻缓混匀后置冰上冰浴5~10min;加150μL溶液III(5M KAc 60mL,冰乙酸11.5mL,定容至100mL),混匀后置冰上放5min;以12,000rpm离心5min,吸取上清液到一个新的离心管,苯酚/氯仿/异戊醇溶液(体积比为25∶24∶1,v∶v∶v)抽提2次;上清液中加入2倍体积的无水乙醇或等体积的异丙醇,于室温静置5~10min;12,000离心5min,弃上清,加入70%乙醇,离心洗涤沉淀1次;真空干燥沉淀,用30μL含有20μg/mLRNase的TE溶液(1mM EDTA,10mM Tris-HCl,pH 8.0)溶解沉淀。
2.苏云金芽胞杆菌YBT-1518总质粒文库的构建
将上步制备的质粒加入适量HindIII进行不完全酶切,回收5-12kb片段连接于载体pHT304(载体来源见:Arantes O and Lereclus D.1991.Construction of cloning vectors for Bacillus thuringiensis.Gene 108:115-119)转化大肠杆菌DH5α,并涂布氨苄青霉素抗性平板(含有100μg/mL的氨苄青霉素,100μg/mL IPTG和80μg/mL的X-gal),抗性平板置于37℃培养箱中培养14h后,挑取白斑于新鲜的LB平板(含有100μg/mL的氨苄青霉素),即得菌株YBT-1518的质粒文库,随机挑取1000个克隆保存于-80℃冰箱,从中随机挑取12个克隆抽取质粒之后进行HindIII酶切和0.8%琼脂糖电泳检测得该文库的平均插入片段大小为8kb,1000个克隆大约可以覆盖整个质粒基因组的2~3倍(假设质粒基因组总大小为300kb,每个质粒的拷贝数为3个)。
3.苏云金芽胞杆菌YBT-1518中杀线虫晶体蛋白基因cry1518-35的克隆
利用基因cry6A的特异片段为探针,从上述构建好的总质粒文库中筛选到了一个阳性克隆子EMB0229,对该克隆子的测序(由Invitrogen公司完成)表明该重组子中所含有一个大小为10kb左右的质粒pBMB0229(见图1)。对该质粒进行序列分析发现该质粒上除了编码有基因cry6Aa2外,在这个基因的下游还存在一个同cry6Aa2高度相似的基因。申请人依据该基因的特异序列并在两端引入酶切位点BamHI和HindIII设计上游引物P1(引物序列为:5’-CGCGGATCCATGAATAATATTAATAAGAAG-3’)和下游引物P2(引物序列为:5’-CCCAAGCTTCTAAGATATATAAGCATTTAAAG-3’),并以重组质粒pBMB0229为模板进行PCR扩增以获得目标基因,PCR反应的体系和程序如下:
25μL反应体系包含:2.5μL 10×PCR反应缓冲液,1μL dNTP(各2.5mM),0.5μL特异性上游引物P1(20mM),0.5μL特异性下游引物P2(20mM),0.2μL模板(质粒pBMB0229),0.25μL Ex Taq酶,加灭菌去离子水至25μL。PCR反应参数和程序为:94℃,5min,1个循环;94℃,20s,52℃,20s,72℃,1.5min,30个循环;72℃,10min,1个循环。
将扩增到的目标片段通过PCR产物回收试剂盒(购自Omega生物技术公司)纯化回收后通过T/A克隆连接到T载体pMD18-T Simple Vector上(购自TaKaRa公司),得到含有cry1518-35全长序列的重组质粒pUC18-cry1518-35(见图2).。之后将该重组质粒转化大肠杆菌(E.coli)DH5α,并涂布氨苄青霉素抗性平板(AmpR,终浓度为100μg/mL)。将抗性平板置于37℃培养箱中培养12-16h,待转化子长到一定大小以后,通过质粒快检(大肠杆菌质粒快检方法参考:张桂敏等,一种简便快速筛选重组子的方法,湖北大学学报(自然科学版),2005,27(3):280-281.)对转化子进行筛选。将筛选到的转化子用5mL的液体LB培养基活化培养,然后抽提质粒进行酶切验证,酶切片段大小与预期大小一致。最后将筛选到的阳性转化子用5mL的液体LB培养基活化培养,取1mL的过夜培养物送样测序(由Invitrogen公司完成)。测序结果显示该基因具有如序列表SEQ ID NO:1中所示的核苷酸序列,申请人将该基因命名为cry1518-35(在本发明中,被命名的基因以斜体小写表示)。通过软件分析预测此段编码序列可编码一个如序列表SEQ ID NO:1中所示的由286个氨基酸组成的多肽片段,预测其分子量为34kDa,申请人将其命名为Cry1518-35(在本发明中,被命名的蛋白以正体大写表示)。
4.苏云金芽胞杆菌YBT-1518中杀线虫晶体蛋白基因cry1518-35的序列分析
对cry1518-35的进一步序列分析表明,该基因由861个核苷酸组成,编码一个由286个氨基酸组成的多肽,其理论分子量为34-kDa,在GenBank上进行的BlastP分析发现,同该多肽最相似的蛋白质为Cry6Aa2蛋白,其氨基酸序列的一致性为29.7%,相似性为40.4%,依据晶体蛋白基因的命名原则(参见文献(CrickmoreN,Zeigler DR,Feitelson J,Schnepf E,van Rie J,Lereclus D,Baum J,Dean DH.1998.Revision of thenomenclature for the Bacillus thuringiensis pesticidal crystal proteins.Microbiol.Mol.Biol.Rev.62:807-813),该基因将被归为I类新型cry基因。
实施例2杀线虫晶体蛋白基因cry1518-35在大肠杆菌JM109中的表达纯化及生物活性测定
1.杀线虫晶体蛋白基因cry1518-35在大肠杆菌中超量表达载体的构建
将实施例1中获得的含有基因cry1518-35的重组质粒pUC18-cry1518-35经过BamHI和HindIII双酶切,同时将质粒载体pQE30进行相同的双酶切,然后将上述载体和外源的酶切产物通过0.8%的琼脂糖凝胶分离后,切下目标片段并通过DNA凝胶回收试剂盒(购自Omega生物技术公司)纯化回收。之后将两者通过T4DNA连接酶(购自TaKaRa生物技术公司)连接,从而获得了含有基因cry1518-35全长ORF的重组表达质粒pEMB0225(见图3),将该重组表达质粒转化大肠杆菌E.coli DH5α(购自Tiangen公司),并抽提质粒分别进行BamHI和HindIII双酶切验证,酶切片段大小与预期大小一致。进一步的测序结果表明cry1518-35基因被正确地插入了到了表达载体pQE30中。
2.杀线虫晶体蛋白基因cry1518-35在大肠杆菌JM109中的表达和纯化
为了大量表达Cry1518-35蛋白,中请人将上述携带有其编码序列的重组表达质粒pEMB0225转化到大肠杆菌JM109(购自Tiangen公司)中,得到了重组菌JM109/pEMB0225。将该重组菌株接种于5mL LB液体培养基中(另外附加终浓度为100μg/mL氨苄青霉素),过夜活化。以1∶100(v/v)转接至50mL LB液体培养基中,置于37℃摇床培养至OD600为0.5-0.8左右以后,加入1.0mmol/L的异丙基-B-D-硫代半乳糖苷(即IPTG,购自sigma公司)于37℃诱导培养3h。将上述50mL重组菌JM109/pEMB0225的3h诱导培养物经过12000rpm离心30s收集菌体,利用超声波(技术参数:功率400W,破碎30s,间歇30s)将细胞破碎后12000rpm离心15min取上清。然后将上清液过Ni-IDA亲和层析柱(His镍柱)(购自Novagen公司)提纯该特异蛋白Cry1518-35(具体的纯化步骤按照试剂盒操作说明书进行)。对最终的纯化产物进行SDS-PAGE电泳检测,结果如图4所示,提纯的蛋白与蛋白分子量标准比对,估算分子量为35kDa,与预计的晶体蛋白Cry1518-35分子量大小35.2kDa基本吻合,证明本发明的杀线虫晶体蛋白Cry1518-35在大肠杆菌JM109中得到了成功的表达。
申请人将上述的表达杀线虫晶体蛋白基因的cry1518-35的重组大肠杆菌命名为(Escherichia coli)EMB0225,于2009年1月8日将该重组大肠杆菌送交湖北省武汉市武汉大学内的中国典型培养物保藏中心(CCTCC)保藏,其保藏编号为:CCTCC NO:M209009。
重组大肠杆菌EMB0225的生物学及遗传学特性:
①生物学特性:菌体直杆状,营养体呈链状或单生,长1-3微米,宽0.4-0.7微米,革兰氏染色阴性,无芽胞,在牛肉膏蛋白胨培养基上菌落边缘整齐,表面有光泽、湿润、光滑、呈灰色,在生理盐水中容易分散。生长温度范围为15-46℃,最适生长温度为37℃。兼性厌氧,在有氧条件下生长良好。在含0.5%NaCl的牛肉膏蛋白胨培养基中生长,最适生长pH为6.8-8.0。在葡萄糖、麦芽胞、甘露醇、木胶糖、阿拉伯胶等培养基中产酸产气,甲基红反应阳性。
②遗传学特性:本发明是以大肠杆菌Escherichia coli Bl21为出发菌株得到的基因工程菌,含有本发明得到的杀线虫晶体蛋白基因cry1518-35。经继代培养,杀线虫晶体蛋白基因cry1518-35能够在工程菌中稳定存在,表达正常,对受体菌的生长无重大影响。
3.杀线虫晶体蛋白Cry1518-35对北方根结线虫生物活性的测定
以北方根结线虫(Meloidogyne hapla)的二龄幼虫作为靶标线虫检测Cry蛋白对线虫的生物活性。取被北方根结线虫感染的番茄根,用自来水冲洗干净。从根部取下根结线虫卵块,用0.5%NaClO消毒2次,然后25℃孵化3~5d,孵化出的线虫即作为生物测定的靶标。生物测定在96孔板上进行,每孔吸入40头2龄幼虫(具体方法参考文献:余子全等,苏云金芽胞杆菌伴胞晶体蛋白对植物寄生线虫生物测定方法的建立和高毒力菌株的筛选,农业生物技术学报,2007(15):867-871)。整个实验设5~7个浓度梯度,每个浓度设3个重复,用20μg/mL BSA作为阴性对照,纯化的Cry6Aa2蛋白作为阳性对照。5天后统计死亡率,将死亡率校正后换算成几率值,蛋白浓度换算成对数值,求出二者之间回归方程计算LC50,结果见表1。
从表1中可以看出,本发明得到的杀线虫晶体蛋白Cry1518-35对北方根结线虫显示出了很高的杀虫活性,半致死浓度为7.43μg/mL,而且与已知的杀线虫晶体蛋白Cry6Aa2的杀线虫活性相当。这些结果证明本发明得到的杀线虫晶体蛋白Cry1518-35对北方根结线虫具有高活性。
表1:杀线虫晶体蛋白Cry1518-35和Cry6Aa2对北方根结线虫的活性检测
本发明发现的晶体蛋白Cry1518-35对北方根结线虫具有高毒力,为北方根结线虫的生物防治提供了一种新的基因资源。
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Claims (6)
1、一个来自于苏云金芽胞杆菌的杀线虫晶体蛋白基因cry1518-35,它的核苷酸序列如序列表SEQ ID NO:1所示。
2、一个来自于苏云金芽胞杆菌的杀线虫晶体蛋白基因cry1518-35的编码蛋白,它的氨基酸序列如序列表SEQ ID NO:1所示。
3、一株转杀线虫晶体蛋白基因cry1518-35的重组大肠杆菌(Escherichia coli)EMB0225,保藏在中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO:M209009。
4、权利要求2所述的编码蛋白在制备微生物杀虫剂中的应用。
5、权利要求2所述的编码蛋白在转基因微生物或转基因植物中的应用。
6、权利要求3所述的重组大肠杆菌EMB0225在制备微生物杀虫剂中的应用。
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CN109182209B (zh) * | 2018-10-12 | 2021-06-18 | 沈阳农业大学 | 一种用于防治植物根结线虫的肠杆菌及应用 |
CN115057915A (zh) * | 2021-12-28 | 2022-09-16 | 四川农业大学 | 一种大肠杆菌表达蛋白Cry5B的制备及抗大熊猫蛔虫活性 |
CN115057915B (zh) * | 2021-12-28 | 2024-04-26 | 四川农业大学 | 一种大肠杆菌表达蛋白Cry5B在制备抗大熊猫蛔虫成虫和L4期幼虫活性药物中的应用 |
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