CN1952151A - 苏云金芽胞杆菌的杀虫晶体蛋白基因cry7Bal - Google Patents
苏云金芽胞杆菌的杀虫晶体蛋白基因cry7Bal Download PDFInfo
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- CN1952151A CN1952151A CNA2005100195960A CN200510019596A CN1952151A CN 1952151 A CN1952151 A CN 1952151A CN A2005100195960 A CNA2005100195960 A CN A2005100195960A CN 200510019596 A CN200510019596 A CN 200510019596A CN 1952151 A CN1952151 A CN 1952151A
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Abstract
本发明公开了一种来自于苏云金芽胞杆菌杀虫晶体蛋白基因的分离、克隆及其应用。本发明从苏云金芽胞杆菌华中亚种YBt-978菌株中分离得到一个新的杀虫晶体蛋白基因cry7Ba1,该基因的编码产物为一种新的杀虫晶体蛋白Cry7Ba1,对鳞翅目昆虫具有杀虫活性。Cry7Ba1蛋白由1154个氨基酸残基组成,它与现有公开发表的蛋白质同源性不超过80%,并且在该蛋白靠近N-末端一半的氨基酸序列与现有公开发表蛋白质同源性不超过50%。本发明还公开了这种新的杀虫晶体蛋白基因序列,通过合适的表达载体转化微生物表达了该基因的编码产物Cry7Ba1蛋白,使之表现出对鳞翅目害虫的杀虫活性。
Description
技术领域
本发明属微生物基因工程技术领域。具体地说,涉及一种来自于苏云金芽胞杆菌杀虫晶体蛋白基因的分离与克隆,与生物农药基因工程有关。
背景技术
苏云金芽胞杆菌(Bacillus thuringiensis)是一种杆状,革兰氏染色阳性反应,能形成内生芽胞的细菌,广泛存在于各种生态环境。在其芽胞期能形成而产生对昆虫具有特异毒性的由杀虫晶体蛋白(InsecticidalCrystal Proteins,ICPs)组成的伴胞晶体,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 andits pesticidal crystal proteins.Microbiol Mol Biol Rev,62:775-806)。
在苏云金芽胞杆菌野生菌株中广泛存在且一般含有多个编码杀虫晶体蛋白的基因。Schnepf等人于1981年从苏云金芽胞杆菌库斯塔克亚种HD1中克隆出第一个杀虫晶体蛋白基因,并于1985年根据DNA的碱基序列推导出第一个苏云金芽胞杆菌杀虫晶体蛋白的氨基酸序列(Schnepf HE,Wong HC,Whiteley HR1985.The amino acid sequence of a crystal protein from Bacillus thuringiensis deduced from the DNA basesequence.J. Biol.Chem.260(10):6264-6272)。此后,新的杀虫晶体蛋白基因的一直成为苏云金芽胞杆菌研究领域中的热点,陆续有大量的杀虫晶体蛋白基因被鉴定和克隆测序。因此在1995年的无脊椎病理学会年会上成立了由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%。杀虫晶体蛋白基因的4级分类。截至2005年8月,苏云金芽胞杆菌杀虫晶体蛋白基因已累计达到48类319种(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 pesticidal crystal proteins.Microbiol.Mol.Biol.Rev.62:807-813;
http://www.lifesci.sussex.ac.uk/home/Neil Crickmore/Bt/index.html)。
苏云金芽胞杆菌杀虫剂最初都是使用筛选的野生型菌株进行生产,随着分子生物学的发展,人们逐渐利用基因工程的手段对野生型菌株进行改造。同时人们不断将苏云金芽胞杆菌的杀虫晶体蛋白基因转入植物中,构建转基因植物,用于农业害虫的防治。
然而,随着B.t杀虫剂应用的不断推广和使用强度的不断增强,目标害虫的抗性现象不断被科学家所发现。关于目标害虫对B.t杀虫剂的抗性机制,研究人员进行了深入研究。B.t的杀虫晶体蛋白需要经过晶体的溶解与原毒素的激活、毒素片段与中肠上皮细胞膜上受体的结合、在膜上的插入和孔的形成等过程才能产生杀虫效应。其中毒素片段与昆虫中肠上皮细胞膜上特异受体的识别和相互作用在很大程度上决定了杀虫晶体蛋白的活性谱和毒性大小,而昆虫对B.t杀虫剂的抗性的产生与受体的识别与结合密切相关。因此,新的尤其是新异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B.t杀虫剂抗性的关键途径,是各种防治策略的核心内容。近年来,寻找与克隆新的杀虫晶体蛋白基因一直是苏云金芽胞杆菌研究领域中最活跃的部分之一。本发明的意义即在于此。我国,于1996年分离并鉴定出一株苏云金芽胞杆菌新亚种YBt-978菌株,鞭毛抗原血清型为H40,分类上定为华中亚种(Dai J et al.1996.Bacillusthuringiensis subspecies huazhongensis,serotype H40,isolated from soils in the People′s Republic of China.Letters in Applied Microbiology.22(1):42-45)。通过提取伴胞晶体蛋白并用于生物测定,发现该伴胞晶体蛋白对小菜蛾等昆虫具有高效杀虫活性。
发明内容
本发明的目的在于从苏云金芽胞杆菌中分离并克隆具有高毒力杀虫晶体蛋白基因及其应用。
本发明的技术路线如附图1所示。
为了实现上述目的,本发明从苏云金芽胞杆菌华中亚种(Bacillus thuringiensis subsp.huazhongensis,serotype H40)YBt-978菌株(菌株来源见:Dai J et al.1996.Bacillus thuringiensis subspecies huazhongensis,serotype H40,isolated from soils in the People′s Republic of China.Letters in Applied Microbiology.22(1):42-45)中分离到一个新的杀虫晶体蛋白基因cry7Ba1,其编码区由3465个碱基组成,具有序列表SEQ IDNO:1所示的核苷酸序列。本发明的cry7Ba1基因编码Cry7Ba1蛋白,它由1154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。本发明的基因cry7Ba1在微生物中表达的Cry7Ba1蛋白,对鳞翅目昆虫具有杀虫活性。
本发明的Cry7Ba1蛋白与所有公开发表的蛋白质的同源性最高的为58.2%,不超过80%。而且,在所有公开发表的蛋白质中,与本发明的Cry7Ba1蛋白的N-末端一半的部分序列(SEQ ID NO:1中1至658个氨基酸序列)同源性最高的为37.1%,不超过50%。
本发明提供的上述基因序列是一种具有杀虫活性的新的杀虫晶体蛋白基因,该基因可以应用于转化微生物和/或植物,使之表现出对鳞翅目害虫的杀虫活性,并克服、延缓害虫对基因工程杀虫菌剂和转基因植物的抗药性。
更详细的技术方案由下列描述和实施例揭示:
需要说明的是,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J.萨姆布鲁克等,2002,分子克隆实验指南,第三版,金冬雁等(译),科学出版社,北京)。另外,本发明所克隆的基因cry7Ba1的命名用斜体字表示;本发明的cry7Ba1基因编码Cry7Ba1蛋白的命名用正体字表示,这是本技术领域的命名的惯例。
1、苏云金芽胞杆菌YBt-978中晶体蛋白Cry7Ba1的N-末端氨基酸序列的测序
本发明以苏云金芽胞杆菌YBt-978作为杀虫晶体蛋白Cry7Ba1的来源菌株,该菌株是苏云金芽胞杆菌血清型H40的标准菌株,该菌株的来源见文献:Dai J et al.,Bacillus thuringiensis subspecies huazhongensis,serotype H40,isolated from soils in the People′s Republic of China.Letters in Applied Microbiology.22(1).1996.42-45。该菌株能形成典型的菱形晶体,主要的晶体蛋白成分Cry7Ba1的分子量约为130千道尔顿(kDa)。对Cry7Ba1的N-末端氨基酸序列的测序具体步骤如下:
(1)菌株的培养和培养物的处理
将苏云金芽胞杆菌YBt-978用接种环通过无菌操作接种于5mL的LB(1%的NaCl,1%的蛋白胨,0.5%的酵母抽提物,pH7.0),置于30℃、200rpm的摇床中培养过夜;然后通过无菌操作将350uL的培养物转移至35rnL的ICPM(0.6%的蛋白胨,0.5%的葡萄糖,0.1%的CaCO3,0.1%MgSO4.7H2O,0.05%的KH2PO4,pH7.0)培养基中,置于30℃、200rpm的摇床中培养至芽胞和伴胞晶体完全脱落,将培养物收集,8000rpm离心1min,去掉上清;分别用0.5%NaCl溶液和灭菌去离子水将沉淀洗涤三遍。
(2)晶体蛋白的SDS-PAGE电泳和转PVDF膜
将洗涤后的沉淀按照沉淀:去离子水=1∶5的体积比重新在去离子水中混匀,加入等体积的2×上样缓冲液,混匀后,沸水浴中处理3-5min,12000rpm离心5min,以上清液进行SDS-PAGE电泳。其中2×上样缓冲液的配方和SDS-PAGE电泳操作步骤均参见Laemmli描述的方法(Laemmli,UK.1970.Cleavage ofstructural proteins during the assembly of the head of bacteriophage T4.Nature(London)227:680-685)。
然后按照标准的方法(见文献:Cook RG.1994.Amino acid sequence analysis of blotted proteins,p,207-220.In B.S.DunBar(ed.),Protein Blotting:A practical approach.Oxford University Press,New York.),利用半湿型转膜仪(BIO-RED),将凝胶上的蛋白质样品转移到PVDF膜。
3)晶体蛋白N-末端氨基酸序列的测定
通过蛋白质N-末端氨基酸序列仪(型号:ABI 491 Protein Sequencer,ABI Procise)测N-末端氨基酸序列,测序结果为:N端-MDIRNQNKYEVVYPA-C端
2.杀虫晶体蛋白基因cry7Ba1的5’末端DNA片段序列的获得
采用接头连接PCR(Adaptor-ligation PCR)法进行扩增。其原理和步骤参见Slebert P.D.等人的描述(Slebert P D et al.1995.An improved PCR method for walking in uncloned genomic DNA.Nucleic AcidsResearch 23:1087-1088)。具体步骤如下:
1)苏云金芽胞杆菌YBt-978总DNA的抽提
将苏云金芽胞杆菌YBt-978用接种环通过无菌操作接种于5mL的LB培养基中,置于30℃、200rpm的摇床中培养过夜;然后通过无菌操作将50μL的培养物转移至5mL的新鲜LB培养基中,同样条件培养至对数生长期;然后5000rpm,3min离心收集菌体,用STE1mL洗涤一次,加100μL溶液I和10μL溶菌酶(50mg/mL),37℃作用30min以上;加入200μL 2%SDS于50℃-60℃水浴30min;加入200μL 5mol/LNaCl混和,加入500μL苯酚/氯仿/异戊醇(按体积比:25∶24∶1),离心12000rpm,5min,吸取上清液,重复抽提1-2次;上层DNA溶液转移至Eppendorf管,加入等体积95%乙醇,室温静置5min后离心12000rpm,5min,沉淀用200μL 70%乙醇洗涤一次,冷冻抽干后溶于50μL TE溶液中。
2)总DNA的限制性内切酶酶切和接头的连接
(1)将事先准备好的苏云金芽胞杆菌的总DNA用HpaI酶于37℃酶切过夜(酶切体系为50μL,含有约1μg总DNA);(2)50μL酶切产物加入250μL苯酚∶氯仿∶异戊醇(体积比25∶24∶1),离心12000rpm,5min,将上层DNA溶液转移至Eppendorf管,加入等体积95%乙醇,室温静置5min后离心12000rpm,5min,沉淀用200μL 70%乙醇洗涤一次,冷冻抽干后溶于50μL灭菌水中;(3)将接头长短链(50μM)(接头长链序列为:5’-GCTCGAGTCGACCGTGGTACGCGTGTGTGAGCTCCCGGGATCCGGT-3’;接头短链序列为:5’-ACCGGATC-NH2-3’)按等体积混合成为4μL溶液,70℃保温10min;(4)4μL的接头溶液(25μM),10μL总DNA酶切产物溶液(约含0.5μgDNA),8U T4连接酶,2μL T4连接酶Buffer,加水配为20μL体系,16℃连接12h;(5)连接体系于60℃加热5mins后可作为PCR扩增的模板使用;
3)巢式PCR以及测序
用上述的连接产物,进行两轮PCR扩增。
根据已测得的N-末端氨基酸序列,设计合成基因特异引物简称为130P1、130P2;同时根据接头长链序列,设计合成接头引物简称为AP1、AP2。各引物序列如下:
130P1:5’-ATGGATATHMGNAATCARAAYAARTAYG-3’*
130P2:5’-AATAAATATGARGTWGTNTAYCCNGC-3’*
AP1:5’-GCTCGAGTCGACCGTGGTA-3’
AP2:5’-GTACGCGTGTGTGAGCTCC-3’
(*H=A,C和T,;M=A和C,;N=A,G,C和T,;R=A和G,;Y=C和T,;W=A和T)
(A)第一轮PCR。25μL反应体系包含:2.5μL 10×PCR反应缓冲液,1μL dNTP(各2.5mM),0.5μL接头引物AP1(20mM),0.5μL特异引物130P1(20mM),1μL模板(上述连接产物),0.5μL ExTaq酶,加灭菌去离子水至25μL。PCR反应参数为:94℃,1min,1个循环;94℃,1min,52℃,1min,72℃,1.5min,25个循环;72℃,5min,1个循环。将第一轮PCR产物稀释50倍,作为第二轮PCR扩增模板。
(B)第二轮PCR。50μL反应体系包含:5μL 10×PCR反应缓冲液,2μL dNTP(各2.5mM),1μL接头引物AP2(20mM),1μL特异引物130P2(20mM),1μL模板(第一轮PCR产物),1μL ExTaq酶,加灭菌去离子水至50μL。PCR反应参数为:除退火温度为56℃,其与同上。
(C)第二轮PCR产物通过PCR产物回收试剂盒(购自Veta-Gene公司产品)纯化回收后连接到T-载体上,转化大肠杆菌E.DH5α。以AP2和130P2为引物,以单菌落菌体为模板,通过PCR扩增对转化子进行筛选,并对阳性转化子T-载体中的外源片段进行测序。测序结果为SEQ ID NO:1中第19至491个核苷酸之间的序列。
根据上述测序结果,设计了特异引物简称为130S1-2、130A1-2,用于后面所述的通过PCR筛选包含晶体蛋白基因的BAC基因组文库和各种阳性转化子的过程中,扩增产物大小为434bp。引物序列为:
130S1-2:TTCTAATACAACATCAAAGTATCCACTC
130A1-2:GGTATCGTTCCAATACTAATTCTAAAC
3.苏云金芽胞杆菌YBt-978的杀虫晶体蛋白基因cry7Ba1的克隆
1)苏云金芽胞杆菌YBt-978菌株基因组BAC文库的构建
参照Luo和Wing(2003)的方法(参见:Luo and Wing.2003.An improved method for plant BAC libraryconstruction,p.3-19.In Grotewold,Erich,Plant functional genomic:methods and protocols. Scientific andmedical publishers,/Humana Press,Totowa,USA),利用BAC文库载体pBeloBAC11(参见图2所示)构建苏云金芽胞杆菌YBt-978菌株的基因组BAC文库。即:将苏云金芽胞杆菌YBt-978在LB培养基中生长到对数生长中期,10000rpm离心1分钟收集菌体于干净灭过菌的50ml离心管中;收集的菌体先加1ml TE缓冲液(1mM EDTA,10mM Tris base,pH 8.0))放到振荡器上轻轻震散,然后再加约40ml TE缓冲液静置5分钟后10000rpm离心1分钟收集菌体;将装有菌体的50ml离心管静置在50℃温水中,加入1.5ml的TE25S(0.3M蔗糖,25mM EDTA,25mM Tris Base,pH 8.0),混匀后再加入1.5ml温热的2%的胶(专门做包埋块的低熔点胶,称取0.1g溶于5ml TE25S中),用枪混匀后,逐一加到专门做包埋块的模子中,放于4℃冰箱中让包埋块冷凝。其余步骤,包括HindIII对包埋块中YBt-978基因组的部分酶切和酶切产物的回收、BAC文库载体pBeloBAC11的制备、酶切产物与载体的连接与转化DH10B、BAC文库质量检验,与Luo andWing(2003)报道的方法相同。
2)BAC基因组文库的筛选和杀虫晶体蛋白基因cry7Ba1的克隆
(A)BAC基因组文库的筛选
以130S1和130A1为引物,以BAC文库单菌落菌体为模板进行PCR扩增。反应体系为25μL,配方同上述;反应条件中退火温度为55℃,其余同上述。筛选出阳性克隆子EMB0491。参照J.萨姆布鲁克等的方法(J.萨姆布鲁克等,2002,分子克隆实验指南,第三版,金冬雁等(译),科学出版社,北京),抽提EMB0491中重组质粒pBMB0491。酶切结果表明pBMB0491包含约60kb的YBt-978基因组片段。
(B)杀虫晶体蛋白基因cry7Ba1的获得
以HindIII对阳性克隆子中YBt-978基因组片段进行完全酶切,酶切产物与同样被HindIII完全酶切的克隆载体pUC18(见图3所示)连接,转化E.coli DH5α。同样以编号为130S1-2和130A1-2的因物为引物,以转化子单菌落菌体为模板进行PCR扩增,筛选出阳性转化子EMB0493。抽提EMB0493中重组质粒pBMB0492。酶切结果表明pBMB0492其中包含约16kb的YBt-978基因组片段。进一步分析表明杀虫晶体蛋白基因cry7Ba1位于5kb XhoI片段中。
(C)杀虫晶体蛋白基因cry7Ba1和杀虫晶体蛋白Cry7Ba1的序列分析
分析杀虫晶体蛋白基因cry7Ba1所在的5kb XhoI片段的核苷酸序列,得到5235bp序列,其中包括3465bp的编码,编码序列见SEQ ID NO:1。
对此段编码序列可编码由1154个氨基酸组成的多肽,即杀虫晶体蛋白Cry7Ba1,推导其分子量为130558道尔顿(Da)。
比较此多肽与其它已知的Cry与Cyt蛋白的氨基酸序列,在GenBank基因库中(http://www.ncbi.nlm.nih.gov/)进行搜索和比较,发现在现有公开发表的所有蛋白中,与杀虫晶体蛋白Cry7Ba1最相似的蛋白质为杀虫晶体蛋白Cry7Ab2(GenBank注册号:U04368)、Cry7Ab1(GenBank注册号:U04367)和Cry7Aa1(GenBank注册号:M64478),相似性分别达到了58.2%、57.9%、57.1%。其中,在N-末端一半的部分序列(SEQ ID NO:1中1至658个氨基酸序列)相似度很低,相似性分别为37.1%、37.0%、36.4%,这部分是杀虫的活性部分,也是表现杀虫活性的必需和充分部分(SchnepfH E,CrickmoreN,Rie J V, Lereclus D,Baum J,Feitelson J,Zeigler D R,Dean D H.1998.Bacillus thuringiensis and itspesticidal crystal proteins.Microbiol Mol Biol Rev,62:775-806)。由此看出该杀虫基因确实为一个新的杀虫基因。因此该杀虫晶体蛋白基因被国际苏云金芽胞杆菌杀虫基因命名委员会命名为cry7Ba1,确定为cry7B基因的模式基因。另外,与这些已知蛋白在其C-末端一半的序列(SEQ ID NO:1中659至1154个氨基酸序列)非常相似,相似性分别达到了87.9%、86.9%、87.9%,这部分序列是形成伴胞晶体的功能结构域,与杀虫活性无关。
从以上序列分析可以看出,在现有所有公开的蛋白质中,与本发明的杀虫晶体蛋白Cry7Ba1同源性最高的为58.2%,不超过80%。当某个蛋白质的氨基酸序列与杀虫晶体蛋白Cry7Ba1的同源性超过80%时,则视为在本发明的杀虫晶体蛋白Cry7Ba1的覆盖范围之内。同样,在现有所有公开的蛋白质中,与本发明的杀虫晶体蛋白Cry7Ba1 N-末端一半的部分序列(SEQ ID NO:1中1至658个氨基酸序列)同源性最高的为37.1%,不超过50%。当某个蛋白质的氨基酸序列与本发明的杀虫晶体蛋白Cry7Ba1的N-末端一半的部分序列(SEQ ID NO:1中1至658个氨基酸序列)同源性超过50%时,则视为在本发明的杀虫晶体蛋白Cry7Ba1的覆盖范围之内。
4.苏云金芽胞杆菌YBt-978的杀虫晶体蛋白Cry7Ba1的杀虫活性
将cry7Ba1基因所在的5kb XhoI片段转载在大肠杆菌-苏云金芽胞杆菌穿梭载体pHT304(见图4,载体来源见:Arantes O and Lereclus D.1991.Construction of cloning vectors for Bacillus thuringiensis.Gene108:115-119)上,获得重组质粒pBMB0495(见图5)。然后通过成熟的电转化方法(吴岚,孙明,喻子牛.利用苏云金芽胞杆菌转座子Tn4430构建含crylAc10基因的解离载体.微生物学报,2000,40:264-269)将其转移到苏云金芽胞杆菌无晶体突变CryB菌株中(菌株来源见:Wu D,Federici BA.1993.A 20-kilodaltonprotein preserves cell viablility and promotes CytA crystal formation during sporulation in Bacillus thuringiensisJ.Bacteriol.,175:5276-5280),获得重组菌BMB0502。
(1)杀虫晶体蛋白Cry7Ba1的表达
将BMB0502在LB培养基(需要时加入终浓度为20μg/mL的红霉素)中过夜活化后,转移至ICPM培养基(需要时加入终浓度为20μg/mL的红霉素)中,培养至芽胞完全成熟脱落,收集菌体,分别用0.5%NaCl和灭菌去离子水洗涤三遍后,按照菌体:灭菌去离子水=1∶5的体积比重新混匀菌体,加入等体积的2×上样缓冲液,混匀后,沸水浴中处理3-5min,12000rpm离心5min,以上清液进行SDS-PAGE电泳。其中2×上样缓冲液的配方和SDS-PAGE电泳操作步骤均参见Laemmli描述的方法(Laemmli,UK.1970.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature(London)227:680-685)。如图6所示,BMB0502能形成和出发菌株YBt-978大小相同的130kDa晶体蛋白,而阴性对照BMB0503(只含有穿梭载体pHT304)无此对应条带。
(2)杀虫晶体蛋白Cry7Ba1形成的晶体形态
将BMB0502在LB培养基(使用时加入终浓度为20μg/mL的红霉素)中过夜活化后,转移至ICPM培养基(该培养基成分及用量见前面“发明内容”所述,使用时加入终浓度为20μg/mL的红霉素)中,培养至芽胞即将成熟脱落的阶段,收集菌体,用灭菌去离子水洗涤一遍后,按照菌体∶灭菌去离子水=1∶5的体积比重新混匀菌体,制备透射电镜(TEM)样品,用Epon812包埋,超薄切片,于80kv进行透射观察拍照。BMB0502能形成双金字塔型的伴胞晶体,如图7所示。
(3)杀虫晶体蛋白Cry7Ba1的杀虫效果评价
以小菜蛾(Plutella xylostella)3龄幼虫为对象测定杀虫晶体蛋白Cry7Ba1的杀虫效果。将BMB0502在LB培养基(使用时加入终浓度为20μg/mL的红霉素)中过夜活化后,转移至ICPM培养基(该培养基的组分及用量参见前面“发明内容”中“菌株的培养和培养物的处理一节所述”使用时加入终浓度为20μg/mL的红霉素)中,培养至芽胞即将成熟脱落的阶段,收集菌体,用灭菌去离子水洗涤一遍后,获得伴胞晶体混合物,按照标准测定程序(参见:沈鞠群,王锦举,喻子牛.1990.苏云金杆菌制剂的标准化程序和方法.生物防治通报,(增刊):12-16)进行生物测定。每个样品的生物测定重复一次,计算出LC50。结果表明本发明的杀虫晶体蛋白Cry7Ba1对小菜蛾表现出很高的毒性(如表1所示),表明本发明克隆的Cry7Ba1蛋白对鳞翅目昆虫具有很高的杀虫活性。
表1本发明克隆的杀虫晶体蛋白Cry7Ba1对小菜蛾生物测定实验结果
时间(天) | 材料 | 回归方程 | 回归系数(R2值) | LC50值(95%可信限)(ng/mL) |
35 | BMB0502BMB0502 | y=0.9928x+3.0334y=1.0554x+3.3103 | 0.99460.9901 | 95.7(93.9-97.5)39.9(38.2-41.6) |
附图说明
序列表SEQ ID NO:1是本发明的基因序列和编码序列
图1:是本发明的技术流程图
图2:是本发明实施例中BAC载体pBleoBAC11构建图
图3:是本发明的构建的克隆载体pUC18构建图
图4:是本发明实施例构建的穿梭载体构建图
图5:是本发明实施例构建的重组质粒pBMB0495
图6:是本发明克隆的苏云金芽胞杆菌Cry7Ba1伴胞晶体蛋白SDS-PAGE电泳分析
M:蛋白质分子量标准;
1:YBt-978菌株的伴胞晶体蛋白:
2:BMB0502菌株的伴胞晶体蛋白:
3:对照BMB0503菌株的处理。
图7:是本发明涉及的苏云金芽胞杆菌YBt-978和BMB0502菌株产生的伴胞晶体形态(内标尺为1μm)
A,B:YBt-978菌株的伴胞晶体形态;
C,D:BMB0502菌株的伴胞晶体形态。
具体实施方案
以卜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。本发明中所涉及的各种实验操作,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-978的杀虫晶体蛋白基因cry7Ba1的克隆
参照Luo和Wing(2003)报道的方法(参见:Luo and Wing.2003.An improved method for plant BAClibrary construction,p.3-19.In Grotewold,Erich,Plant functional genomic:methods and protocols.Scientific andmedical publishers,/Humana Press,Totowa,USA),利用BAC文库载体pBeloBACll构建苏云金芽胞杆菌YBt-978菌株(该菌株的来源见文献:Dai J et al.1996.Bacillus thuringiensis subspecies huazhongensis,serotypeH40.isolated from soils in the People′s Republic of China.Letters in Applied Microbiology.22(1):42-45,根据中国专利法和实施细则的有关规定,本申请人已经提出自本专利申请日开始至20年内向公众发放该YBt-978菌株的证明)的基因组BAC文库。以编号为130S1-2和130A1-2引物为引物,以BAC文库单菌落菌体为模板进行PCR扩增。筛选出阳性克隆子EMB0491。以HindIII对阳性克隆子中YBt-978基因组片段进行完全酶切,酶切产物与同样被HindIII完全酶切的克隆载体pUC18连接,转化E.coli DH5α。同样以130S1-2和130A1-2为引物,以转化子单菌落菌体为模板进行PCR扩增,筛选出阳性转化子EMB0493。抽提EMB0493中重组质粒pBMB0492。酶切结果表明pBMB0492其中包含约16kb的YBt-978基因组片段。进一步分析表明杀虫晶体蛋白基因cry7Ba1位于5kb XhoI片段中。
实施例2苏云金芽胞杆菌YBT-978的杀虫晶体蛋白基因cry7Ba1的序列分析
分析杀虫晶体蛋白基因cry7Ba1所在的5kb XhoI片段的核苷酸序列,得到5235bp序列,其中包括3465bp的编码,编码序列见SEQ ID NO:1。
对此段编码序列可编码由1154个氨基酸组成的多肽,推导其分子量为130558道尔顿(Da)。
比较此多肽与其它已知的Cry与Cyt蛋白的氨基酸序列,发现它与Cry7Ab2、Cry7Ab1、Cry7Aa1最相似,相似性分别达到了58.2%、57.9%、57.1%。其中,而N-末端一般的部分序列(SEQ ID NO:1中1至658个氨基酸序列)相似度很低,相似性分别为37.1%、37.0%、36.4%。由于与Cry7A杀虫晶体蛋白相似,此多肽已被杀虫晶体蛋白基因命名委员会建议命名为Cry7Ba1。
实施例3杀虫晶体蛋白基因cry7Ba1的表达
将cry7Ba1所在的5kb XhoI片段转载在大肠杆菌-苏云金芽胞杆菌穿梭载体pHT304(载体来源见:Arantes O and Lereclus D.1991.Construction of cloning vectors for Bacillus thuringiensis.Gene 108:115-119)上,获得重组质粒pBMB0495。然后通过成熟的电转化方法(吴岚,孙明,喻子牛.利用苏云金芽胞杆菌转座子Tn4430构建含crylAc10基因的解离载体.微生物学报,2000,40:264-269)将其转移到苏云金芽胞杆菌无晶体突变株CryB菌株(菌株来源见:Wu D and Federici BA.1993.A 20-kilodalton protein preservescell viablility and promotes CytA crystal formation during sporulation in Bacillus thuringiensis.J. Bacteriol.,175:5276-5280)中,获得重组菌命名为BMB0502。
将上述命名为BMB0502重组菌在LB培养基(使用时加入终浓度为20μg/mL的红霉素)中过夜活化后,转移至ICPM培养基(使用时加入终浓度为20μg/mL的红霉素)中,培养至芽胞完全成熟脱落,收集菌体,分别用0.5%NaCl和灭菌去离子水洗涤三遍后,按照菌体∶灭菌去离子水=1∶5的体积比重新混匀菌体,加入等体积的2倍上样缓冲液,混匀后,沸水浴中处理3-5min,12000rpm离心5min,以上清液进行SDS-PAGE电泳。其中2×上样缓冲液的配方和SDS-PAGE电泳操作步骤均参见Laemmli描述的方法(Laemmli,UK.1970.Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature(London) 227:680-685)。如图6所示,BMB0502能形成和出发菌株YBt-978大小相同的130kDa晶体蛋白,而阴性对照BMB0503(只含有穿梭载体pHT304)无此对应条带。
实施例4杀虫晶体蛋白Cry7Ba1的杀虫效果
以小菜蛾3龄幼虫为对象测定杀虫晶体蛋白Cry7Ba1的杀虫效果。将BMB0502在LB培养基(使用时加入终浓度为20μg/mL的红霉素)中过夜活化后,转移至ICPM培养基(使用时加入终浓度为20μg/mL的红霉素)中,培养至芽胞即将成熟脱落的阶段,收集菌体,用灭菌去离子水洗涤一遍后,获得伴胞晶体混合物,按照标准测定程序(见:沈鞠群,王锦举,喻子牛.苏云金杆菌制剂的标准化程序和方法.生物防治通报,1990,(增刊):12-16)进行生物测定。每个样品的生物测定重复一次,计算出LC50。结果表明杀虫晶体蛋白Cry7Ba1对小菜蛾表现出极高的毒性,说明Cry7Ba1蛋白溶液具有很高的鳞翅目杀虫活性,其实施效果参见表1所示。
序列表
SEQUENCE LISTING
<110>华中农业大学
<120>苏云金芽胞杆菌的杀虫晶体蛋白基因cry7Ba1
<130>
<141>2005-10-13
<160>2
<170>PatentIn version 3.1
<210>1
<211>3465
<212>DNA
<213>苏云金芽胞杆菌(Bacillus thuringiensis)
<220>
<221>gene
<222>(1)..(3465)
<223>
<220>
<221>CDS
<222>(1)..(3465)
<223>
<400>1
atg gat ata aga aat caa aat aaa tat gaa gta gta tac cca gca ata 48
Met Asp Ile Arg Asn Gln Asn Lys Tyr Glu Val Val Tyr Pro Ala Ile
1 5 10 15
aat gaa aca act tct aat aca aca tca aag tat cca ctc gca agt gat 96
Asn Glu Thr Thr Ser Asn Thr Thr Ser Lys Tyr Pro Leu Ala Ser Asp
20 25 30
cca atc aaa caa tat caa aat atg aat tat aaa gat agt ttg aat ata 144
Pro Ile Lys Gln Tyr Gln Asn Met Asn Tyr Lys Asp Ser Leu Asn Ile
35 40 45
att gag ggg aat aac gta atc act cca gta tct gga act gct gtt ttg 192
Ile Glu Gly Asn Asn Val Ile Thr Pro Val Ser Gly Thr Ala Val Leu
50 55 60
gca act gca agg aaa att ggt ggt aag atc gtt aag gct ata ggg gaa 240
Ala Thr Ala Arg Lys Ile Gly Gly Lys Ile Val Lys Ala Ile Gly Glu
65 70 75 80
caa atc ttg tct aaa atc ttg aaa gag att ctt gat tat tta tgg ccg 288
Gln Ile Leu Ser Lys Ile Leu Lys Glu Ile Leu Asp Tyr Leu Trp Pro
85 90 95
tct tca agc tcg tcc aat tca tgg gaa gag atg atg aag gaa gta gag 336
Ser Ser Ser Ser Ser Asn Ser Trp Glu Glu Met Met Lys Glu Val Glu
100 105 110
tat ctt att gat aaa aaa ata gag gaa tat gca aga aat aag gca ctt 384
Tyr Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Arg Asn Lys Ala Leu
115 120 125
gcg gtt ttg gaa gga ata gga aat gct gta gaa tcc tat tat agt gca 432
Ala Val Leu Glu Gly Ile Gly Asn Ala Val Glu Ser Tyr Tyr Ser Ala
130 135 140
tta gaa gcg tgg gaa tta gaa tct agt gaa cgt agt tta gaa tta gta 480
Leu Glu Ala Trp Glu Leu Glu Ser Ser Glu Arg Ser Leu Glu Leu Val
145 150 155 160
ttg gaa cga tat cag ttt gcg gtg cag ttt gca aga agt tca atg cca 528
Leu Glu Arg Tyr Gln Phe Ala Val Gln Phe Ala Arg Ser Ser Met Pro
165 170 175
tca ttt gcg att ata aat tat gaa att ccc tta tta gca aca tat gca 576
Ser Phe Ala Ile Ile Asn Tyr Glu Ile Pro Leu Leu Ala Thr Tyr Ala
180 185 190
aat gct gca aat gtt cat tta ctt tta atg aga gat ata caa ata tac 624
Asn Ala Ala Asn Val His Leu Leu Leu Met Arg Asp Ile Gln Ile Tyr
195 200 205
ggg gat aga tgg gga ata tct caa aat gat atg aat ctc ttc tta aaa 672
Gly Asp Arg Trp Gly Ile Ser Gln Asn Asp Met Asn Leu Phe Leu Lys
2l0 215 220
gaa caa gaa ata tac acg tct gaa tat tcg gaa cat tgc gta aag tgg 720
Glu Gln Glu Ile Tyr Thr Ser Glu Tyr Ser Glu His Cys Val Lys Trp
225 230 235 240
tat aat gag gga tta aat caa ttg aaa act aaa ggt ggc gca agt ggt 768
Tyr Asn Glu Gly Leu Asn Gln Leu Lys Thr Lys Gly Gly Ala Ser Gly
245 250 255
tta gtt tgg gag aat tat aac agt ttc cgt aca gaa atg aca att atg 816
Leu Val Trp Glu Asn Tyr Asn Ser Phe Arg Thr Glu Met Thr Ile Met
260 265 270
gta tta gat ctt gta gct ata ttt cca gcc tac aat atg agc aaa tat 864
Val Leu Asp Leu Val Ala Ile Phe Pro Ala Tyr Asn Met Ser Lys Tyr
275 280 285
cct ata gaa tca aca gta gaa tta aca aga aca att tat aca gat cca 912
Pro Ile Glu Ser Thr Val Glu Leu Thr Arg Thr Ile Tyr Thr Asp Pro
290 295 300
ctt ggt tac aca ggg tat agc aat gat gaa cat ccc aca tat tat tct 960
Leu Gly Tyr Thr Gly Tyr Ser Asn Asp Glu His Pro Thr Tyr Tyr Ser
305 310 315 320
tct gca aaa cca ttt tca tca ata gag agt aga gcc gta cta gca ccc 1008
Ser Ala Lys Pro Phe Ser Ser Ile Glu Ser Arg Ala Val Leu Ala Pro
325 330 335
tca tta ttc aaa tgg atc act caa ctt gaa gta tat aca aaa aaa tac 1056
Ser Leu Phe Lys Trp Ile Thr Gln Leu Glu Val Tyr Thr Lys Lys Tyr
340 345 350
agc tac tct tcc caa tat act acg ttg tgg act gga cta aga gtg att 1104
Ser Tyr Ser Ser Gln Tyr Thr Thr Leu Trp Thr Gly Leu Arg Val Ile
355 360 365
gct cag cct act aaa gat ttt act gat act gta tat gat tac gga agt 1152
Ala Gln Pro Thr Lys Asp Phe Thr Asp Thr Val Tyr Asp Tyr Gly Ser
370 375 380
tct tcg ggt tct gag aac aag gat gtc ttt gac ctt tat ggc aat gat 1200
Ser Ser Gly Ser Glu Asn Lys Asp Val Phe Asp Leu Tyr Gly Asn Asp
385 390 395 400
gta tat gac aca caa agt gtt gtt tca tcg tat aag cct aca ggt ggt 1248
Val Tyr Asp Thr Gln Ser Val Val Ser Ser Tyr Lys Pro Thr Gly Gly
405 410 415
ggc cat ttt ggg gtt cct cag ttt aga tta ttc tgg att act aaa tct 1296
Gly His Phe Gly Val Pro Gln Phe Arg Leu Phe Trp Ile Thr Lys Ser
420 425 430
aat ggg cta aga gaa caa att ttt aat tat gcg aat aat atg ggt tct 1344
Asn Gly Leu Arg Glu Gln Ile Phe Asn Tyr Ala Asn Asn Met Gly Ser
435 440 445
tac agt gcg tat agg ttt agt aag gac gaa tta cca ata gaa ttg ttg 1392
Tyr Ser Ala Tyr Arg Phe Ser Lys Asp Glu Leu Pro Ile Glu Leu Leu
450 455 460
cag cca cct ctt ttt gga gat ata gag gaa tac agt cat agg tta agt 1440
Gln Pro Pro Leu Phe Gly Asp Ile Glu Glu Tyr Ser His Arg Leu Ser
465 470 475 480
cac gtt tca gag gta att aaa gat tat ggt gaa gga atc att cct gta 1488
His Val Ser Glu Val Ile Lys Asp Tyr Gly Glu Gly Ile Ile Pro Val
485 490 495
tta ggt tgg aca cat gta agt gta act cgt gac aat aga att tat cca 1536
Leu Gly Trp Thr His Val Ser Val Thr Arg Asp Asn Arg Ile Tyr Pro
500 505 510
gat aag att aca caa ctt cca gcg gta aaa atg tat gag tta cta agc 1584
Asp Lys Ile Thr Gln Leu Pro Ala Val Lys Met Tyr Glu Leu Leu Ser
515 520 525
tca gcc gtt gtt gta aaa gga cct gga ttt aca ggt gga gat tta gtt 1632
Ser Ala Val Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Leu Val
530 535 540
aag aga acg ggc aat ggt ggc att gga cat ttt aat gtt agt gta gag 1680
Lys Arg Thr Gly Asn Gly Gly Ile Gly His Phe Asn Val Ser Val Glu
545 550 555 560
tcc cct ggt act cag agg tat cgc ctg aga ata cgt tat agt tca gag 1728
Ser Pro Gly Thr Gln Arg Tyr Arg Leu Arg Ile Arg Tyr Ser Ser Glu
565 570 575
gtt agt gga gta ttt cat atg caa att aac gat ata gaa act att cag 1776
Val Ser Gly Val Phe His Met Gln Ile Asn Asp Ile Glu Thr Ile Gln
580 585 590
gga gaa ttt agt agt act gct gat tca aca agt act ctg tca agc gaa 1824
Gly Glu Phe Ser Ser Thr Ala Asp Ser Thr Ser Thr Leu Ser Ser Glu
595 600 605
gca ttt caa ctt aga gaa tac tcc act acc ttc acg ttt cca aca aat 1872
Ala Phe Gln Leu Arg Glu Tyr Ser Thr Thr Phe Thr Phe Pro Thr Asn
610 615 620
atg aca aag ata aag gta tct tta ggt gct att gaa ggt gca gga gga 1920
Met Thr Lys Ile Lys Val Ser Leu Gly Ala Ile Glu Gly Ala Gly Gly
625 630 635 640
ttc tat tta gat aga att gaa ttc att cca gta gat gaa aat cac gat 1968
Phe Tyr Leu Asp Arg Ile Glu Phe Ile Pro Val Asp Glu Asn His Asp
645 650 655
aac aga gta aca cta gaa aaa gca cag aaa gcc gtg aat gcc ttg ttt 2016
Asn Arg Val Thr Leu Glu Lys Ala Gln Lys Ala Val Asn Ala Leu Phe
660 665 670
aca gcg gga aga aac gca cta caa aca gat gtg aca gat tac aaa gta 2064
Thr Ala Gly Arg Asn Ala Leu Gln Thr Asp Val Thr Asp Tyr Lys Val
675 680 685
gat cag gtt tcc att tta gtg gat tgt gta tca ggg gag tta tat cca 2112
Asp Gln Val Ser Ile Leu Val Asp Cys Val Ser Gly Glu Leu Tyr Pro
690 695 700
aat gag aaa cgc gaa cta ctc agt tta gtc aaa tac gca aaa cgt ttg 2160
Asn Glu Lys Arg Glu Leu Leu Ser Leu Val Lys Tyr Ala Lys Arg Leu
705 710 715 720
agc tat tct cgt aat tta ctc cta gat cca aca ttc gat tct att aat 2208
Ser Tyr Ser Arg Asn Leu Leu Leu Asp Pro Thr Phe Asp Ser Ile Asn
725 730 735
tcg tca gat gag aat ggc tgg tac gga agt aat ggt att gca att gga 2256
Ser Ser Asp Glu Asn Gly Trp Tyr Gly Ser Asn Gly Ile Ala Ile Gly
740 745 750
aat ggg aac ttt gta ttc aaa gga aac tat tta att ttc tca ggt acc 2304
Asn Gly Asn Phe Val Phe Lys Gly Asn Tyr Leu Ile Phe Ser Gly Thr
755 760 765
aat gat aca caa tac cca acg tat ctc tat caa aaa att gat gaa tcc 2352
Asn Asp Thr Gln Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser
770 775 780
aag ctc aaa gaa tat aca cgc tat aaa ctg aga gga ttt atc gaa aat 2400
Lys Leu Lys Glu Tyr Thr Arg Tyr Lys Leu Arg Gly Phe Ile Glu Asn
785 790 795 800
agt caa gat tta gaa gca tat gtg att cgc tat gat gca aaa cat gaa 2448
Ser Gln Asp Leu Glu Ala Tyr Val Ile Arg Tyr Asp Ala Lys His Glu
805 810 815
aca ttg gat gta tcc aat aat cta ttg ccg gat att tct cct gta aat 2496
Thr Leu Asp Val Ser Asn Asn Leu Leu Pro Asp Ile Ser Pro Val Asn
820 825 830
gca tgc gga gaa cca aat cgt tgt gct gca tta caa tac ctg gat gaa 2544
Ala Cys Gly Glu Pro Asn Arg Cys Ala Ala Leu Gln Tyr Leu Asp Glu
835 840 845
aat cca agg tta gaa tgt agt tcg ata caa gac ggt att tta tct gat 2592
Asn Pro Arg Leu Glu Cys Ser Ser Ile Gln Asp Gly Ile Leu Ser Asp
850 855 860
tcg cat tcg ttc tct ctc aat ata gat aca ggt tct att gat ttc aat 2640
Ser His Ser Phe Ser Leu Asn Ile Asp Thr Gly Ser Ile Asp Phe Asn
865 870 875 880
gag aac gta gga att tgg gtg ttg ttt aaa att tec aca ccg gaa ggg 2688
Glu Asn Val Gly Ile Trp Val Leu Phe Lys Ile Ser Thr Pro Glu Gly
885 890 895
tat gcg aaa ttt gga aac cta gaa gtg att gaa gat agc cca gtc att 2736
Tyr Ala Lys Phe Gly Asn Leu Glu Val Ile Glu Asp Ser Pro Val Ile
900 905 910
gga gaa gca tta gcc cgt gta aaa cgc caa gaa acg aag tgg cga aac 2784
Gly Glu Ala Leu Ala Arg Val Lys Arg Gln Glu Thr Lys Trp Arg Asn
915 920 925
aag ttg aca caa ctg cga acg gaa aca caa gcg att tat aca cga gca 2832
Lys Leu Thr Gln Leu Arg Thr Glu Thr Gln Ala Ile Tyr Thr Arg Ala
930 935 940
aaa caa gcc att gat aat gta ttc aca aat gca cag gac tct cac tta 2880
Lys Gln Ala Ile Asp Asn Val Phe Thr Asn Ala Gln Asp Ser His Leu
945 950 955 960
aaa ata ggt acg aca ttt gcg gca att gtg gct gcg cga aag att gtc 2928
Lys Ile Gly Thr Thr Phe Ala Ala Ile Val Ala Ala Arg Lys Ile Val
965 970 975
caa tcc ata cgc gaa gcg tat atg tca tgg tta tca atc gtt cca ggt 2976
Gln Ser Ile Arg Glu Ala Tyr Met Ser Trp Leu Ser Ile Val Pro Gly
980 985 990
gta aat tat cct att ttt aca gag ttg aat gag aga gta cag cga gca 3024
Val Asn Tyr Pro Ile Phe Thr Glu Leu Asn Glu Arg Val Gln Arg Ala
995 1000 1005
ttt caa tta tat gat gta cgg aat gtc gtg cgt aat ggc cga ttc 3069
Phe Gln Leu Tyr Asp Val Arg Asn Val Val Arg Asn Gly Arg Phe
1010 1015 1020
ctg aat gga gta tcg gat tgg att gtg aca tct gat gta aag gta 3114
Leu Asn Gly Val Ser Asp Trp Ile Val Thr Ser Asp Val Lys Val
1025 1030 1035
caa gaa gaa aat ggg aac aat gta tta gtt ctt tcc aat tgg gat 3159
Gln Glu Glu Asn Gly Asn Asn Val Leu Val Leu Ser Asn Trp Asp
1040 1045 1050
gcg caa gta tta caa tgt ctg aag ctc tat caa gat cgc gga tat 3204
Ala Gln Val Leu Gln Cys Leu Lys Leu Tyr Gln Asp Arg Gly Tyr
1055 1060 1065
atc ttg cgt gta acg gca cgt aaa gaa gga ttg gga gaa gga tat 3249
Ile Leu Arg Val Thr Ala Arg Lys Glu Gly Leu Gly Glu Gly Tyr
1070 1075 1080
att aca att acg gat gaa gaa ggg cat aca gat caa ttg aca ttt 3294
Ile Thr Ile Thr Asp Glu Glu Gly His Thr Asp Gln Leu Thr Phe
1085 1090 1095
ggc aca tgt gag gaa ata gat gca tct aac acg ttc gta acc aca 3339
Gly Thr Cys Glu Glu Ile Asp Ala Ser Asn Thr Phe Val Thr Thr
1100 1105 1110
ggt tat att aca aaa gaa cta gaa ttt ttc cca gat aca gag aaa 3384
Gly Tyr Ile Thr Lys Glu Leu Glu Phe Phe Pro Asp Thr Glu Lys
1115 1120 1125
gtg cgt ata gaa att ggg gaa aca gaa gga acc ttc cag gta gaa 3429
Val Arg Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Gln Val Glu
1130 1135 1140
agt ata gag tta ttt ttg atg gaa gat cta tgt taa 3465
Ser Ile Glu Leu Phe Leu Met Glu Asp Leu Cys
1145 1150
<210>2
<211>1154
<212>PRT
<213>苏云金芽胞杆菌(Bacillus thuringiensis)
<400>2
Met Asp Ile Arg Asn Gln Asn Lys Tyr Glu Val Val Tyr Pro Ala Ile
1 5 10 15
Asn Glu Thr Thr Ser Asn Thr Thr Ser Lys Tyr Pro Leu Ala Ser Asp
20 25 30
Pro Ile Lys Gln Tyr Gln Asn Met Asn Tyr Lys Asp Ser Leu Asn Ile
35 40 45
Ile Glu Gly Asn Asn Val Ile Thr Pro Val Ser Gly Thr Ala Val Leu
50 55 60
Ala Thr Ala Arg Lys Ile Gly Gly Lys Ile Val Lys Ala Ile Gly Glu
65 70 75 80
Gln Ile Leu Ser Lys Ile Leu Lys Glu Ile Leu Asp Tyr Leu Trp Pro
85 90 95
Ser Ser Ser Ser Ser Asn Ser Trp Glu Glu Met Met Lys Glu Val Glu
l00 105 110
Tyr Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Arg Asn Lys Ala Leu
115 120 125
Ala Val Leu Glu Gly Ile Gly Asn Ala Val Glu Ser Tyr Tyr Ser Ala
130 135 140
Leu Glu Ala Trp Glu Leu Glu Ser Ser Glu Arg Ser Leu Glu Leu Val
145 150 155 160
Leu Glu Arg Tyr Gln Phe Ala Val Gln Phe Ala Arg Ser Ser Met Pro
165 170 175
Ser Phe Ala Ile Ile Asn Tyr Glu Ile Pro Leu Leu Ala Thr Tyr Ala
180 185 190
Asn Ala Ala Asn Val His Leu Leu Leu Met Arg Asp Ile Gln Ile Tyr
195 200 205
Gly Asp Arg Trp Gly Ile Ser Gln Asn Asp Met Asn Leu Phe Leu Lys
210 215 220
Glu Gln Glu Ile Tyr Thr Ser Glu Tyr Ser Glu His Cys Val Lys Trp
225 230 235 240
Tyr Asn Glu Gly Leu Asn Gln Leu Lys Thr Lys Gly Gly Ala Ser Gly
245 250 255
Leu Val Trp Glu Asn Tyr Asn Ser Phe Arg Thr Glu Met Thr Ile Met
260 265 270
Val Leu Asp Leu Val Ala Ile Phe Pro Ala Tyr Asn Met Ser Lys Tyr
275 280 285
Pro Ile Glu Ser Thr Val Glu Leu Thr Arg Thr Ile Tyr Thr Asp Pro
290 295 300
Leu Gly Tyr Thr Gly Tyr Ser Asn Asp Glu His Pro Thr Tyr Tyr Ser
305 310 315 320
Ser Ala Lys Pro Phe Ser Ser Ile Glu Ser Arg Ala Val Leu Ala Pro
325 330 335
Ser Leu Phe Lys Trp Ile Thr Gln Leu Glu Val Tyr Thr Lys Lys Tyr
340 345 350
Ser Tyr Ser Ser Gln Tyr Thr Thr Leu Trp Thr Gly Leu Arg Val Ile
355 360 365
Ala Gln Pro Thr Lys Asp Phe Thr Asp Thr Val Tyr Asp Tyr Gly Ser
370 375 380
Ser Ser Gly Ser Glu Asn Lys Asp Val Phe Asp Leu Tyr Gly Asn Asp
385 390 395 400
Val Tyr Asp Thr Gln Ser Val Val Ser Ser Tyr Lys Pro Thr Gly Gly
405 410 415
Gly His Phe Gly Val Pro Gln Phe Arg Leu Phe Trp Ile Thr Lys Ser
420 425 430
Asn Gly Leu Arg Glu Gln Ile Phe Asn Tyr Ala Asn Asn Met Gly Ser
435 440 445
Tyr Ser Ala Tyr Arg Phe Ser Lys Asp Glu Leu Pro Ile Glu Leu Leu
450 455 460
Gln Pro Pro Leu Phe Gly Asp Ile Glu Glu Tyr Ser His Arg Leu Ser
465 470 475 480
His Val Ser Glu Val Ile Lys Asp Tyr Gly Glu Gly Ile Ile Pro Val
485 490 495
Leu Gly Trp Thr His Val Ser Val Thr Arg Asp Asn Arg Ile Tyr Pro
500 505 5l0
Asp Lys Ile Thr Gln Leu Pro Ala Val Lys Met Tyr Glu Leu Leu Ser
515 520 525
Ser Ala Val Val Val Lys Gly Pro Gly Phe Thr Gly Gly Asp Leu Val
530 535 540
Lys Arg Thr Gly Asn Gly Gly Ile Gly His Phe Asn Val Ser Val Glu
545 550 555 560
Ser Pro Gly Thr Gln Arg Tyr Arg Leu Arg Ile Arg Tyr Ser Ser Glu
565 570 575
Val Ser Gly Val Phe His Met Gln Ile Asn Asp Ile Glu Thr Ile Gln
580 585 590
Gly Glu Phe Ser Ser Thr Ala Asp Ser Thr Ser Thr Leu Ser Ser Glu
595 600 605
Ala Phe Gln Leu Arg Glu Tyr Ser Thr Thr Phe Thr Phe Pro Thr Asn
610 615 620
Met Thr Lys Ile Lys Val Ser Leu Gly Ala Ile Glu Gly Ala Gly Gly
625 630 635 640
Phe Tyr Leu Asp Arg Ile Glu Phe Ile Pro Val Asp Glu Asn HiS Asp
645 650 655
Asn Arg Val Thr Leu Glu Lys Ala Gln Lys Ala Val Asn Ala Leu Phe
660 665 670
Thr Ala Gly Arg Asn Ala Leu Gln Thr Asp Val Thr Asp Tyr Lys Val
675 680 685
Asp Gln Val Ser Ile Leu Val Asp Cys Val Ser Gly Glu Leu Tyr Pro
690 695 700
Asn Glu Lys Arg Glu Leu Leu Ser Leu Val Lys Tyr Ala Lys Arg Leu
705 710 715 720
Ser Tyr Ser Arg Asn Leu Leu Leu Asp Pro Thr Phe Asp Ser Ile Asn
725 730 735
Ser Ser Asp Glu Asn Gly Trp Tyr Gly Ser Asn Gly Ile Ala Ile Gly
740 745 750
Asn Gly Asn Phe Val Phe Lys Gly Asn Tyr Leu Ile Phe Ser Gly Thr
755 760 765
Asn Asp Thr Gln Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser
770 775 780
Lys Leu Lys Glu Tyr Thr Arg Tyr Lys Leu Arg Gly Phe Ile Glu Asn
785 790 795 800
Ser Gln Asp Leu Glu Ala Tyr Val Ile Arg Tyr Asp Ala Lys His Glu
805 810 815
Thr Leu Asp Val Ser Asn Asn Leu Leu Pro Asp Ile Ser Pro Val Asn
820 825 830
Ala Cys Gly Glu Pro Asn Arg Cys Ala Ala Leu Gln Tyr Leu Asp Glu
835 840 845
Asn Pro Arg Leu Glu Cys Ser Ser Ile Gln Asp Gly Ile Leu Ser Asp
850 855 860
Ser His Ser Phe Ser Leu Asn Ile Asp Thr Gly Ser Ile Asp Phe Asn
865 870 875 880
Glu Asn Val Gly Ile Trp Val Leu Phe Lys Ile Ser Thr Pro Glu Gly
885 890 895
Tyr Ala Lys Phe Gly Asn Leu Glu Val Ile Glu Asp Ser Pro Val Ile
900 905 910
Gly Glu Ala Leu Ala Arg Val Lys Arg Gln Glu Thr Lys Trp Arg Asn
915 920 925
Lys Leu Thr Gln Leu Arg Thr Glu Thr Gln Ala Ile Tyr Thr Arg Ala
930 935 940
Lys Gln Ala Ile Asp Asn Val Phe Thr Asn Ala Gln Asp Ser His Leu
945 950 955 960
Lys Ile Gly Thr Thr Phe Ala Ala Ile Val Ala Ala Arg Lys Ile Val
965 970 975
Gln Ser Ile Arg Glu Ala Tyr Met Ser Trp Leu Ser Ile Val Pro Gly
980 985 990
Val Asn Tyr Pro Ile Phe Thr Glu Leu Asn Glu Arg Val Gln Arg Ala
995 1000 1005
Phe Gln Leu Tyr Asp Val Arg Asn Val Val Arg Asn Gly Arg Phe
1010 1015 1020
Leu Asn Gly Val Ser Asp Trp Ile Val Thr Ser Asp Val Lys Val
1025 1030 1035
Gln Glu Glu Asn Gly Asn Asn Val Leu Val Leu Ser Asn Trp Asp
1040 1045 1050
Ala Gln Val Leu Gln Cys Leu Lys Leu Tyr Gln Asp Arg Gly Tyr
1055 1060 1065
Ile Leu Arg Val Thr Ala Arg Lys Glu Gly Leu Gly Glu Gly Tyr
1070 1075 1080
lle Thr Ile Thr Asp Glu Glu Gly His Thr Asp Gln Leu Thr Phe
1085 1090 1095
Gly Thr Cys Glu Glu Ile Asp Ala Ser Asn Thr Phe Val Thr Thr
1100 1105 1110
Gly Tyr Ile Thr Lys Glu Leu Glu Phe Phe Pro Asp Thr Glu Lys
1115 1120 1125
Val Arg Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe Gln Val Glu
1130 1135 1140
Ser Ile Glu Leu Phe Leu Met Glu Asp Leu Cys
1145 1150
Claims (4)
1、一个来自于苏云金芽胞杆菌杀虫晶体蛋白基因cry7Ba1,它的核苷酸序列如序列表SEQ IDNO:1所示。
2、权利要求1编码的苏云金芽胞杆菌杀虫晶体蛋白Cry7Ba1,它的氨基酸序列如序列表SEQID NO:1所示。
3、权利要求2所述的苏云金芽胞杆菌杀虫晶体蛋白Cry7Ba1,它与序列表SEQ ID NO:1所示的氨基酸序列具有80%以上同源性。
4、权利要求2所述的苏云金芽胞杆菌杀虫晶体蛋白Cry7Ba1,该蛋白的部分氨基酸序列与序列表SEQ ID NO:1所示的氨基酸序列中第1至658个氨基酸序列具有50%以上同源性。
Priority Applications (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNB2005100195960A CN100510081C (zh) | 2005-10-17 | 2005-10-17 | 苏云金芽胞杆菌的杀虫晶体蛋白基因cry7Bal |
JP2008535871A JP2009511075A (ja) | 2005-10-17 | 2006-10-17 | バチルス・チューリンゲンシスの殺虫性結晶タンパク質をコードする遺伝子cry7Ba1 |
PCT/CN2006/002724 WO2007045160A1 (en) | 2005-10-17 | 2006-10-17 | Gene cry7bal encoding an insecticidal crystal protein of bacillus thuringiensis |
CN2006800386848A CN101292035B (zh) | 2005-10-17 | 2006-10-17 | 编码苏云金芽胞杆菌的杀虫晶体蛋白的基因cry7Bal |
EP06791266A EP1937818A4 (en) | 2005-10-17 | 2006-10-17 | FOR AN INSECTICIDE CRYSTAL PROTEIN FROM BACILLUS THURINGIENSIS CODING GEN CRY7BAL |
AU2006303767A AU2006303767A1 (en) | 2005-10-17 | 2006-10-17 | Gene cry7Ba1 encoding an insecticidal crystal protein of Bacillus thuringiensis |
CA002624158A CA2624158A1 (en) | 2005-10-17 | 2006-10-17 | Gene cry7bal encoding an insecticidal crystal protein of bacillus thuringiensis |
BRPI0617496-5A BRPI0617496A2 (pt) | 2005-10-17 | 2006-10-17 | gene de proteÍna de cristal inseticida isolado de bacillus thuringiensis, constructo de dna recombinante, microorganismo e proteÍna de cristal inseticida de bacillus thuringiensis |
US12/089,992 US7838293B2 (en) | 2005-10-17 | 2006-10-17 | Gene Cry7ba1 encoding an insecticidal crystal protein of Bacillus thuringiensis |
MX2008004990A MX2008004990A (es) | 2005-10-17 | 2006-10-17 | Gen cry7bai que codifica una proteina cristalina insecticida de bacillus thuringiensis. |
ZA200802283A ZA200802283B (en) | 2005-10-17 | 2008-03-11 | Gene cry7Bal encoding an insecticidal crystal protein of Bacillus thuringiensis |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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CNB2005100195960A CN100510081C (zh) | 2005-10-17 | 2005-10-17 | 苏云金芽胞杆菌的杀虫晶体蛋白基因cry7Bal |
Publications (2)
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CN1952151A true CN1952151A (zh) | 2007-04-25 |
CN100510081C CN100510081C (zh) | 2009-07-08 |
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CNB2005100195960A Expired - Fee Related CN100510081C (zh) | 2005-10-17 | 2005-10-17 | 苏云金芽胞杆菌的杀虫晶体蛋白基因cry7Bal |
CN2006800386848A Expired - Fee Related CN101292035B (zh) | 2005-10-17 | 2006-10-17 | 编码苏云金芽胞杆菌的杀虫晶体蛋白的基因cry7Bal |
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CN2006800386848A Expired - Fee Related CN101292035B (zh) | 2005-10-17 | 2006-10-17 | 编码苏云金芽胞杆菌的杀虫晶体蛋白的基因cry7Bal |
Country Status (10)
Country | Link |
---|---|
US (1) | US7838293B2 (zh) |
EP (1) | EP1937818A4 (zh) |
JP (1) | JP2009511075A (zh) |
CN (2) | CN100510081C (zh) |
AU (1) | AU2006303767A1 (zh) |
BR (1) | BRPI0617496A2 (zh) |
CA (1) | CA2624158A1 (zh) |
MX (1) | MX2008004990A (zh) |
WO (1) | WO2007045160A1 (zh) |
ZA (1) | ZA200802283B (zh) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010078708A1 (zh) * | 2009-01-09 | 2010-07-15 | 华中农业大学 | 苏云金芽胞杆菌杀线虫晶体蛋白基因cry1518-35 |
CN102643773A (zh) * | 2011-02-18 | 2012-08-22 | 华中农业大学 | 过表达CodY蛋白的苏云金芽胞杆菌基因工程菌YBT-881-L1的筛选及鉴定 |
CN103421097A (zh) * | 2012-05-18 | 2013-12-04 | 华中农业大学 | 杀线虫晶体蛋白基因cry003-148及应用 |
CN103421098A (zh) * | 2012-05-18 | 2013-12-04 | 华中农业大学 | 杀线虫晶体蛋白基因cry003-131及应用 |
CN109929015A (zh) * | 2019-04-17 | 2019-06-25 | 东北农业大学 | 苏云金芽胞杆菌杀虫基因cry79Aa1、表达蛋白及其应用 |
US11447531B2 (en) | 2016-10-21 | 2022-09-20 | Vestaron Corporation | Cleavable peptides and insecticidal and nematicidal proteins comprising same |
US11472854B2 (en) | 2012-03-09 | 2022-10-18 | Vestaron Corporation | Insecticidal peptide production, peptide expression in plants and combinations of cysteine rich peptides |
US11692016B2 (en) | 2012-03-09 | 2023-07-04 | Vestaron Corporation | High gene expression yeast strain |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007147096A2 (en) | 2006-06-15 | 2007-12-21 | Athenix Corporation | A family of pesticidal proteins and methods for their use |
JP2012529910A (ja) * | 2009-06-16 | 2012-11-29 | ダウ アグロサイエンシィズ エルエルシー | Dig−5殺虫性cry毒素 |
CN102329760B (zh) * | 2011-10-19 | 2013-01-09 | 青岛农业大学 | 杀蛴螬类害虫的苏云金芽孢杆菌新菌株及其杀虫蛋白 |
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US5262324A (en) * | 1991-11-06 | 1993-11-16 | Mycogen Corporation | Coleopteran-active Bacillus thuringiensis isolates and genes encoding coleopteran-active toxins |
GB9318207D0 (en) | 1993-09-02 | 1993-10-20 | Sandoz Ltd | Improvements in or relating to organic compounds |
CN1160458C (zh) * | 1998-12-03 | 2004-08-04 | 中山大学 | 苏云金杆菌分子伴侣基因、含有该基因的载体及菌株 |
CN1111600C (zh) * | 1999-08-26 | 2003-06-18 | 中国科学院遗传研究所 | 杀虫蛋白基因及其应用 |
CN1152959C (zh) * | 2000-08-25 | 2004-06-09 | 上海市农业科学院生物技术研究中心 | 全合成苏云金杆菌杀虫晶体蛋白基因 |
NZ588825A (en) * | 2003-02-20 | 2011-06-30 | Athenix Corp | AXMI-014 delta-endotoxin |
WO2007147096A2 (en) * | 2006-06-15 | 2007-12-21 | Athenix Corporation | A family of pesticidal proteins and methods for their use |
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CN100510081C (zh) | 2009-07-08 |
US7838293B2 (en) | 2010-11-23 |
US20090130758A1 (en) | 2009-05-21 |
WO2007045160A1 (en) | 2007-04-26 |
ZA200802283B (en) | 2009-03-25 |
CA2624158A1 (en) | 2007-04-26 |
AU2006303767A1 (en) | 2007-04-26 |
MX2008004990A (es) | 2008-10-01 |
BRPI0617496A2 (pt) | 2011-07-26 |
CN101292035B (zh) | 2011-05-18 |
EP1937818A1 (en) | 2008-07-02 |
EP1937818A4 (en) | 2009-07-01 |
CN101292035A (zh) | 2008-10-22 |
JP2009511075A (ja) | 2009-03-19 |
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