苏云金芽孢杆菌cry基因PCR-RFLP鉴定体系的建立  

Establishment of PCRRFLP Identification System of cry Genes from Bacillus thuringiensis

作　　者：宋福平[1,2,3,4,5] 张杰[1,2,3,4,5] 谢天健 杨自文[1,2,3,4,5] 戴莲韵[1,2,3,4,5] 李国勋 

机构地区：[1]中国农业科学院植物保护研究所 [2]中国农业科学院生物技术研究中心 [3]湖北省农业科学院Bt研究开发中心 [4]中国林业科学院森林保护研究所 [5]东北农业大学昆虫科学系

出　　处：《中国农业科学》1998年第3期13-18,共6页Scientia Agricultura Sinica

基　　金：国家"863"计划资助

摘　　要：根据苏云金芽孢杆菌（Ｂｔ）ｃｒｙ２、ｃｒｙ３和ｃｒｙ４／ｃｒｙ１０型基因的保守区分别设计了３对通用引物Ｓ５ｕｎ２／Ｓ３ｕｎ２、Ｓ５ｕｎ３／Ｓ３ｕｎ３和Ｓ５ｕｎ４／Ｓ３ｕｎ４。ＰＣＲ扩增产生约１．２ｋｂ、１．４ｋｂ和１．５ｋｂ的产物，然后分别用ＨｉｎｃＩ／ＭｓｐＩ、ＢｇｌＩ／ＰｓｔＩ和ＢｓｔＥＩ／ＤｒａＩ酶切，并经ＲＦＬＰ分析鉴定Ｂｔ菌株的ｃｒｙ基因型。利用该方法对含已知ｃｒｙ２、ｃｒｙ３和ｃｒｙ４／ｃｒｙ１０型基因的Ｂｔ菌株和遗传工程菌进行基因分析，得到的结果与预测的ＲＦＬＰ相符，证明该方法可行。在此基础上对１４株Ｂｔ分离株的基因型进行了鉴定，其中有９株分离株含ｃｒｙ２型基因；仅菌株Ｂｔ２２含ｃｒｙ３型基因；所有被测菌株均不含ｃｒｙ４／ｃｒｙ１０型基因。According to the conserved regions of cry2, cry3 and cry4/cry10type genes from Bacillus thuringiensis (Bt) , three pairs of universal primers, S5un2/S3un2 for cry2type genes, S5un3/S3un3 for cry3type genes and S5un4/S3un4 for cry4/cry10type genes, were designed. The PCR amplified products with above three pairs of primers,1.2kb, 1.4kb and 1.5kb fragments were digested with HincII/MspI, BglII/PstI or BstEII/DraI respectively. The crytype genes of Bt strains can be determined by analysis of the RFLP. By using this method, the Bt strains and genetically engineered bacteria containing known cry2, cry3, cry4/cry10type genes were identified, with the results matching the predicted restriction patterns. It can be concluded that this method is feasible. Furthermore, the cry genes in 14 Bt isolates were determined. 9 isolates contain cry2type genes. The isolate Bt22 contains cry3type genes. No isolate contains cry4/cry10type genes. 

关 键 词：苏云金芽孢杆菌 CRY基因 PCR-RFLP 鉴定体系 

分 类 号：Q939.103[生物学—微生物学]