苏云金芽孢杆菌S1478-1分离株的特性鉴定及cry1Ac同源基因克隆  

Identification of Bacillus thuringiensis Isolate S1478-1 and Cloning of cry1Ac Homolog

作　　者：张文飞[1,2] 姚淑霞[1] 谢柳[1,2] 杨明坤[3] 王锐萍[3] 叶大维[4] 方宣钧[2] 

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]海南海德热带农业资源研究所,三亚572025 [3]海南师范大学生命科学院,海口570200 [4]南开大学生命科学院,天津300071

出　　处：《基因组学与应用生物学》2009年第3期471-476,共6页Genomics and Applied Biology

基　　金：中国Bt资源收鉴与利用项目;国家863项目(2006AA022189)共同资助

摘　　要：本研究对从海南岛尖峰岭热带雨林自然保护区的土壤样品中分离出的Bt菌株S1478-1进行了特性鉴定,研究表明S1478-1分离株菌落形态和生长特征和Bt参照菌株HD73极其相似。16SrDNA序列分析表明,S1478-1分离株与其它B.thuringiensis、B.cereus和B.anthracis的16SrDNA序列相似性达到99%。分离株能产菱形伴胞晶体,SDS-PAGE蛋白电泳分析表明,菌株在生长后期,形成芽孢同时分泌130kD大小的晶体蛋白。生物测定表明S1478-1分离株对小菜蛾具有很高的毒杀活性,LC50值高达5.159×108cfu/mL。初步显示S1478-1分离株可作为防治鳞翅目害虫的生物农药菌株。利用PCR-RFLP方法鉴定S1478-1分离株含有cry1Ac同源基因,以PCR粘性端克隆方法扩增全长基因,序列测定表明该基因ORF为3537bp,编码1178个氨基酸,推定的编码蛋白分子量为133.3kD,与其它cry1Ac基因序列最高达到99%同源,因此,该基因可作为杀虫工程菌及培育转基因抗虫作物的候选基因。The identifications of a Bacillus thuringieusis isolate S1478-1, collected from the Jianfengling tropical rain forest reserves in Hainan Island, were carried out in the research. The results showed that the colonial morphology and growth curve of Bt isolate S1478-1 have quite similar with that of the standard strain kurstaki HD73. 16S rDNA conserved sequence of Bt isolate S 1478-1 was sequenced and its blast analysis of the sequence revealed that the Bt isolate S1478-1 should have high similarity compared with that of other B. thuringiensis ,B. cereus and B. onthracis. The isolate S1478-1 can produces the bipyramid acrystallin inclusion during the sporulation stage, which exhibits high specific toxicity to PluteUa xylostella larvae （LC50：5.159×10^8 cfu/mL）. Further research indicated that the isolate S1478-1 harbor crylAc-homolog gene that could express 130 kD insecticidal crystal protein base on the identification by the approaches of PCR-RFLP and SDS-PAGE. The full length sequence of crylAc-homolog was amplified by PCR and added it into pUC19 vector to make the construct named as pCRY1Ac1478. The sequencing result showed that the cloned crylAc-homolog has a ORF composed of 3 537 bp,which encode putative 133.3 kD protein with 1 178 amino acids. Homologous analysis demostrated that the cloned gene would be crylAc type gene with 99% similarity. In this studies we would like to recommend that the isolate S1478-1 can be as initiative for bio-pesticide production and either its crylAc-homolog as the candidate for biotechnological crop development.

关 键 词：苏云金芽孢杆菌 S1478—1分离株 cry1Ac同源基因 生物测定 基因登录号GQ227507 

分 类 号：S476[农业科学—农业昆虫与害虫防治]