Bt菌株QCL-1中cry2Ac10基因的克隆、表达和活性研究  

Study on Cloning,Expression and Insecticidal Activity of A Novel Gene cry2Ac10 from Bacillus thuringiensis Strain QCL-1

作　　者：白雪峰[1] 李长友[1] 程林友[1] 郑桂玲[1] 周洪旭[1] 李国勋[1] 

机构地区：[1]莱阳农学院植物保护学院,山东青岛266109

出　　处：《生物技术》2007年第4期10-13,共4页Biotechnology

基　　金：国家973计划项目(2003CB114201)资助

摘　　要：目的：从高毒力Bt菌株中克隆cry2Ac10基因,并研究其表达和杀虫活性。方法：以Bt菌株QCL-1质粒为模板,利用cry2特异性引物FY2A5和FY2A3进行PCR扩增,将目的片段克隆到表达载体pET-21b（＋）,构建T7启动子控制的大肠杆菌重组表达质粒pET21b-cry2Ac。经IPTG诱导后,SDS-PAGE检测基因表达情况,然后对表达产物进行生物活性测定。结果：从菌株QCL-1中克隆出目的基因,该基因的编码框由1 872个碱基组成,编码的蛋白质由623个氨基酸组成,与已报道的Cry2Ac氨基酸同源性为97.4%-99.7%。该基因（GenBank accession EF405952）已被国际Bt基因命名委员会正式命名为cry2Ac10。该基因在大肠杆菌BL21（DE3）中能够正常表达70kDa的蛋白,表达产物对棉铃虫、粘虫和粉纹夜蛾幼虫具有高毒力,同时对甜菜夜蛾幼虫生长有抑制作用,其中对棉铃虫和粘虫初孵幼虫的LC50分别为30.0μg/g和16.7μg/g。结论：成功克隆和表达了cry2Ac10基因,并明确了cry2Ac10蛋白的活性,为该基因的研究和应用奠定基础。Objective： To screen pesticidal protein with high toxicity from B. thuringiensis, a novel gene, cry2Ac10, was cloned from the Bt strain QCL - 1, and the expression and insecticidal activity of the gene were tested. Methods： The cry2A gene was amplified by designing a pair of special primers, FY2A5 and FY2A3, based on the plasmid of Bt strain QCL- 1. The recombinant vector, pET21b - cry2Ac was constructed in E. coli BL21 （DE3） by controlled of T7 promotor. Cry2A protein induced by IPTG was determined by bioassay. Results： The cry2Ac10 gene was cloned and identified as GcnBank accession number No. EF405952. Allotment results showed that the cry2Ac was composed of 1872 base pairs, encoding 623 amino acids, which were homolog of 97.4-99.7% compared with pubhshed Cry2Ac. The cry2Ac gene inserted into pET- 21b （ ＋ ） and transformed into E. coli BL21（DE3） was expressed as 70 kDa proteins to a high level after being induced by IPTG. The insecticidal protein form transformant possessed high toxicity to the larvae of Helioeverpa armigera, Mythimna separata. LC50 of the protein was 30.0μg/g to H. armigera and 16.7μg/g to M. separata respectively. Conclusion： cry2Ac10 was cloned and expressed successfully and it had strong insecticidal activity.

关 键 词：苏云金芽孢杆菌 cry2Ac10基因 基因克隆 蛋白表达 生物活性 

分 类 号：Q939.96[生物学—微生物学]