CN102781955B - Bt蛋白Cry4Cb2、其编码基因及应用 - Google Patents
Bt蛋白Cry4Cb2、其编码基因及应用 Download PDFInfo
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Abstract
本发明提供了一种新的Bt蛋白Cry4Cb2及其编码基因,所述蛋白是1)SEQ ID No.2所示的氨基酸序列组成的蛋白质,或2)SEQ ID No.2所示的氨基酸序列经取代、缺失和/或增加一个或几个氨基酸且具有同等活性的由1)所述蛋白质衍生的蛋白质。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种新的Bt蛋白及其编码基因和应用。
背景技术
在人类生产过程中,虫害是造成农业生产损失及影响人类健康的重要因素,据FAO统计,全世界农业生产每年因虫害造成的经济损失高达14%,病害损失达12%,草害损失达11%。损失额高达1260亿美元,相当于中国农业总产值的一半,英国的4倍多。另外,蚊媒病在预防医学中占有重要位置,其中登革热和黄热病等蚊媒病传播力强、流行面广、发病率高、危害性大。据WHO统计,全世界每年感染登革热人数多达8000万,我国的海南省在1980和1986年曾经暴发过两次登革热,发病分别达到437469例和113589例。登革热和黄热病主要由埃及伊蚊传播。
为了减少这些损失,多年来,对农作物害虫及蚊虫普遍采用化学防治手段进行防治,但由于化学农药的长期、大量使用,造成了对环境的污染,农副产品中农药残留量增加,给人类的生存和健康带来了危害。此外,化学农药在杀灭害虫的同时,也杀伤了天敌及其它有益物,破坏了生态平衡。与化学防治相比,生物防治具有安全、有效、持久的特点。并且避免了化学防治带来的一系列问题。因此,生物防治技术成了人们研究的热点。在生物杀虫剂中,苏云金芽孢杆菌是目前世界上用途最广、产量最大的一类微生物杀虫剂。
苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)是一种革兰氏阳性细菌,它的分布极为广泛,在芽孢形成的同时可形成具有杀虫活性的由蛋白质组成的伴胞晶体,又名杀虫晶体蛋白(Insectididal crystal proteins,简称ICPs),ICPs是由cry基因编码的,对敏感昆虫有强烈毒性,而对高等动物和人无毒性。近几十年来,Bt已广泛应用于控制多种鳞翅目、双翅目、鞘翅目等害虫。此外,Bt还对膜翅目、同翅目、直翅目、食毛目等多种害虫及植物病原线虫、螨类、原生动物有控害作用。目前在农田害虫、森林害虫及卫生害虫的防治中Bt已成为化学合成农药的有力替代品,Bt还是转基因抗虫工程植物重要的基因来源。
自1981年Schnepf从菌株HD-1Dipel中克隆了第一个能表达杀虫活性的基因以来(Adang M.J et al,Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp.kurstaki HD-73 and their toxicity to Manduca sexta,Gene,1985,36(3):289~300.),人们已经分离克隆了390多种编码杀虫晶体蛋白的基因,根据编码的氨基酸序列同源性它们被分别确定为不同的群、亚群、类和亚类(Crickmore N,Zeigler D R,Feitelson J,et al.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.Microbiol Mol Biol Rev,1998,62:807-813;http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/)。一般而言,Cry1,Cry2和Cry9等毒蛋白对鳞翅目害虫有效;其中研究的最多的是Cry1和Cry9类蛋白,它们编码的杀虫晶体蛋白分子量为130-140kD,许多基因目前已被广泛应用于植物的鳞翅目害虫的防治(Kozie,M.G.,Beland,G.L.,Bowman,C.,et al.Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis.Bio/Technology,1993,11:194-200;Perlak,F.J.,Deaton,R.W.,Armstrong,T.A.,et al.Insect resistant cotton plants.bio/technology,1990;8:939-943;Van Frankenhuyzen,K.,Gringorten,L.,and Gauhier,D.1997.Cry9Ca1 toxin,a Bacillus thuringiensis insecticidal crystal protein with high activity against the spruce bud worm(Choristoneura fnniferana).Appl.Environ,Microbviol.63:4132-4134;王飞,2001,苏云金芽孢杆菌特异菌株生物学特性及cry9新基因的研究,硕士论文,南开大学)。苏云金芽胞杆菌以色列亚种(B.thuringiensis subsp.israelensis,简称Bti)产生的毒素蛋白对蚊虫具有很好杀虫活性,被广泛运用于蚊虫的防治(Goldberg L J,and Margalit J,1977.A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii,Uranotaenia unguiculata,Culex univitattus,Aedes aegypti,and Culex pipiens.Mosqito News,37:355-358;)。同时,Cyt蛋白具有溶细胞性,对某些Cry蛋白具有增效作用及延缓昆虫的抗性(Wu,D.,Johnson,J.J.,and Federici,B.A.1994.Synergism of mosquitocidal toxicity between CytA and CryIVD Proteins using inclusion sproduced from cloned genes of Bacillus thuringiensis.Mol.Microbiol.13:965-972;Wirth,M.C.,Georghiou,G.P.,and Federeci,B.A.1997.CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito,Culex quinquefasciatus.Proc.Natl.Acad.Sci.94:10536-10540)
自本世纪初发现苏云金芽胞杆菌至今已有100多年的历史,在农作物和园艺植物害虫、森林害虫以及卫生害虫的防治方面得到广泛的应用,也起到良好的效果。但是,由于大规模和反复使用苏云金芽胞杆菌,许多昆虫种群已相继在不同程度上对杀虫晶体蛋白产生了抗性。以Bt杀虫晶体蛋白为基础的杀虫剂的使用已有50多年的历史,最初一直没有检测到昆虫对Bt的抗性,但是,上世纪80年中期开始,抗性问题不断在实验室及田间试验中得到证实(McGaughey,W.H.1985.Insect resistance to the biological insecticide Bacillus thuringiensis.Science.229:193-195),原因主要是持续使用单品种及亚致剂量的Bt以及Bt转基因抗虫植物的应用造成昆虫种群 长期受到杀虫剂的选择压力。1985年,McGaughey报道仓库谷物害虫印度谷螟(Plodia interpunctella)在Dipel(Bt subsp.kurstaik HD-1的商品制剂)的选择压力下,繁殖15代后,抗性增加97倍;在高剂量选择压力下,抗性可增加250倍。1990年,在夏威夷首次证实大田中的小菜蛾对Bt杀虫剂产生了明显的抗性(Tabashnik,B.E.,Finson,N.,Groeters,F.R.,et al.1994.Reversal of resistance to Bacillus thuringiensisin Plutella xylostella.Proc.Natl.Acad.Sci.USA.91:4120-4124),上世纪90年代以来,在我国应用Bt杀虫剂时间较长的深圳、广州、上海等地,发现Bt杀虫剂对小菜蛾防治效果明显下降,意味着抗性已经形成(冯夏.1996.广东小菜蛾对苏云金杆菌的抗性研究.昆虫学报,39(3):238-244;Hofte,H.,Van Rie,J.,Jansens,S.,Van Houtven,A.,Vanderbruggen,H.,and Vaeck,M.,1988.Monoclonal antibody analysis and insecticidal spectrum of three types of lepidopteran-specific insecticidal crystal proteins of Bacillus thuringiensis.Appl.Environ.Microbiol.54:2010-2017)。目前发现在实验室及田间至少有十几种昆虫对Bt及其杀虫晶体蛋白产生了抗性,用选择压力数学模型预测到,在Bt转基因抗虫植物选择压力的条件下,昆虫将会产生抗性(Schnepf,E.,Crickmore,N.,Van Pie,J.,et al.1998.Bacillus thuringiens is and its pesticidal Crystal proteins.Microbiol.Mol.Biol.Rev.65(3):775-806)。另外,有研究证明Bti在大田的使用中尚未发现抗性问题(Regis L,et al.,2000.The use of bacterial larvicides in mosquito and black fly control programsin Brazil.Mem.Instituto Oswaldo Cruz,95:207-210.),但是蚊虫对其抗性问题不断在实验室中得到证实,这种情况也可能会在大田中出现(Georghiou G P,and Wirth M C,1997.Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp.israelensis on development of resistance in the mosquito Culex quinquefasciatus(Diptera:Culicidae). Applied and Environmental Microbiology,63:1095-1101.)。
为避免抗性昆虫所造成的损失,寻找新的高毒力基因资源是解决这个问题的有效途径,这对我国的生物防治有着十分重要的意义。
发明内容
本发明的第一个目的在于针对上述不足提供一种新的BT毒力蛋白资源。
本发明的第二个目的在于提供编码所述蛋白的基因。
本发明的目的还在于提供上述蛋白及基因的应用。
本发明从四川省成都平原土壤中分离得到的苏云金芽孢杆菌(Bacillus thuringiensis)新菌株YWC2-8。通过对YWC2-8的毒力测试表明,YWC2-8对鞘翅目害虫、鳞翅目害虫、双翅目害虫等等,均具有极高的毒力。
根据Cry4类基因保守序列设计1对特异引物,扩增其基因组DNA,结果表明该菌株存在Cry4类基因,进一步设计其全长基因引物,克隆得到Cry4Cb基因,其核苷酸序列如序列表SEQ ID No.1所示,序列SEQ ID No.1的全长为3474bp,分析表明,GC含量为35.90%,编码1157个氨基酸组成的蛋白。经测定,其氨基酸序列如SEQ ID No.2所示。在softberry网站采用bacterial sigma7.0promoter程序对全序列进行预测表明,在基因编码区上游含有RNA聚合酶活化位点的序列,将该基因命名为cry4Cb2。本发明进一步分析了Cry4Cb2蛋白的氨基酸组成(见表1)。
表1Cry4Cb2蛋白的氨基酸组成
氨基酸 | 百分比% | 氨基酸 | 百分比% |
Ala(A): | 5.96 | Met(M): | 2.07 |
Cys(C): | 1.12 | Asn(N): | 7.78 |
Asp(D): | 5.53 | Pro(P): | 3.98 |
Glu(E): | 5.01 | Gln(Q): | 4.67 |
Phe(F): | 3.28 | Arg(R): | 3.37 |
Gly(G): | 5.96 | Ser(S): | 6.91 |
His(H): | 2.68 | Thr(T): | 7.95 |
Ile(I): | 6.40 | Val(V): | 5.70 |
Lys(K): | 5.70 | Trp(W): | 1.30 |
Leu(L): | 8.82 | Tyr(Y): | 5.79 |
应当理解,本领域技术人员可根据本发明公开的氨基酸序列,在不影响其活性的前提下,取代、缺失和/或增加一个或几个氨基酸,得到所述蛋白的突变序列。例如在非活性区段,将第760位的Tyr替换为Asn。因此,本发明Bt蛋白还包括SEQ ID No.2所示氨基酸序列经取代、替换和/或增加一个或几个氨基酸,具有Cry4Cb2蛋白同等活性的由Cry4Cb2衍生得到的蛋白质。本发明基因包括编码所述蛋白的核酸序列。
此外,应理解,考虑到密码子的简并性以及不同物种密码子的偏爱性,本领域技术人员可以根据需要使用适合特定物种表达的密码子。
本发明的基因和蛋白质可以从菌株YWC2-8中克隆或分离得到,或者通过DNA或肽合成的方法得到。
可将本发明基因与表达载体可操作地连接,得到能够表达本发明蛋白的重组表达载体,进而可以通过诸如农杆菌介导法、基因枪法、花粉管通道法等转基因方法,将所述表达载体导入宿主,得到转Cry4Cb2基因的转化体,例如农作物或者果树等植物,使其具备抗虫活性。
此外,还可以通过发酵本发明菌株YWC2-8,得到含有Cry4Cb2蛋白的发酵液,将其制备成杀虫剂,用于农作物害虫的防治。本领域技术人员还可以将上述基因转化细菌或真菌,通过大规模发酵生产本发明Bt蛋白。
本领域技术人员还可以根据本发明公开的基因,将其转化棉花、玉米、水稻、蔬菜等农作物,使其具备相应的抗虫活性。从而降低农药的使用量,减少环境污染,具有重要的经济价值和应用前景。
附图说明
图1显示的是cry4Cb2全长基因克隆,其中M,marker;1.cry4Cb2基因。
图2显示的是重组质粒pET-4Cb的酶切鉴定图谱,其中1.重组质粒pET-4Cb;2.用Nde I+EcoR I双酶切pET-30a;3.Nde I+EcoR I双酶切pET-4Cb;4.插入的DNA;M1、M2为Marker。
图3显示的是在E.coli BL21(DE3)中表达Cry4Cb2的SDS-PAGE检测,其中M为蛋白marker;1.阴性对照(E.coiiBL21(DE3)(pET-30a));2.裂解上清;3.Cry4Cb2包涵体。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1Cry4Cb2基因的克隆
本发明从四川省成都平原土壤中分离得到的苏云金芽孢杆菌(Bacillus thuringiensis)新菌株,该菌株已于2008年10月21日在中国微生物菌种保藏管理委员会普通微生物中心(地址:北京市朝阳区大屯路甲3号,中国科学院微生物研究所,邮编100101)保藏,分类命名为苏云金芽孢杆菌(Bacillus thuringiensis),保藏号为CGMCC No.2860。
本例通过如下方法克隆得到Cry4Cb2基因的全长序列。
采用基因组DNA纯化试剂盒(购自赛百盛公司)提取菌株HS18-1的总DNA。设计引物序列如下:
P1:5’ATGTCTAATCGTTATCAACGGTACCC 3’
P2:5’TCACTCGTTCATACAAATCAACTCGA 3’
PCR反应体系:
热循环反应:94℃预变性5min;94℃变性1min,52℃退火,72℃延伸2min,30个循环;72℃延伸5min;4℃停止反应。扩增反应产物在1%琼脂糖凝胶上电泳,置凝胶成像系统中观察PCR扩增结果。结果如图1所示,通过扩增得到了约为3.5kb的序列,将该序列进行测序,其核苷酸序列如SEQ ID No.1所示,与目的序列一致。
实施例2Cry4Cb2基因的表达及杀虫活性测定
根据cry4Cb2基因开放阅读框两端序列,设计并合成一对特异性引物cry4F:5′-GCGCATATG(NdeI) ATGTCTAATCGTTATCAACGGTACCC-3′;cry4R:5′-CGGAATTC(EcoR I)TCACTCGTTCATACAAATCAACTCGA-3′,分别在5’端引物Nde I和EcoR I酶切位点。以BtMC28质粒为模板进行扩增,扩增的产物采用Nde I和EcoR I进行双酶切,酶切产物与同样进行双酶切后的载体pET-30a(+)连接,转化E.coli DH5α感受态细胞,提取其质粒酶切电泳验证了插入片断大小符合预期目的后(图2),再转入受体菌E.coli.BL21(DE3)。将重组质粒命名为pET-4Cb,含重组质粒的重组子命名为E.coli.BL21(4Cb)。SDS-PAGE分析表明cry4Cb2基因的表达产物在菌体超声破碎后的沉淀中(图3),分子量约为130kDa左右,与预测的蛋白分子量相符。cry4Cb2基因表达产物分别对甜菜夜蛾,棉铃虫及伊蚊的生测结果表明:表达产物对这三种虫都具有较好的杀虫活性。对棉铃虫杀虫活性最高,LC50为15.06μg/mL;对伊 蚊的LC50为17.41μg/mL;对甜菜夜蛾杀虫活性最低,LC50为20.29μg/mL。蛋白对鳞翅目杀虫活性的的测定方法参见(Song FP,Zhang J,Gu AX,et al.,2003.Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene.Appl.Environ.Microbiol 69:5207-5211),蛋白对双翅目杀虫活性的的测定方法参见(Ibarra JE,del Rincón MC,Sergio Ordúz,et al.,2003.Diversity of Bacillus thuringienisis Strains from Latin America with Insecticidal Activity against Different Mosquito Species.Appl Environ Microbiol 69:5269-5274).
工业实用性
本发明蛋白可以用于制备Bt杀虫剂,所述基因可以转化棉花、玉米、水稻、蔬菜等农作物,使其具备相应的抗虫活性,从而降低农药的使用量,减少环境污染,具有重要的经济价值和应用前景。
序列表
Claims (5)
1.一种Bt蛋白Cry4Cb2,其是:
SEQ ID No.2所示的氨基酸序列组成的蛋白质。
2.编码权利要求1所述Bt蛋白Cry4Cb2的基因。
3.如权利要求2所述的基因,其核苷酸序列如SEQ ID No.1所示。
4.含有权利要求2或3所述基因的表达载体。
5.含有权利要求1所述Bt蛋白Cry4Cb2的杀虫剂。
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Title |
---|
周钢.苏云金菌杀蚊新基因和菌株的筛选.《职业与健康》.2005,第21卷(第2期),161-167. |
四川盆地生态区苏云金芽胞杆菌cry基因的鉴定及新型模式cry基因的克隆;朱军等;《微生物学报》;20090304;第49卷(第3期);324-330 * |
朱军等.四川盆地生态区苏云金芽胞杆菌cry基因的鉴定及新型模式cry基因的克隆.《微生物学报》.2009,第49卷(第3期),324-330. |
苏云金菌杀蚊新基因和菌株的筛选;周钢;《职业与健康》;20050228;第21卷(第2期);161-167 * |
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