对蛴螬有活性的苏云金芽胞杆菌菌株的筛选与基因鉴定

Screening and Identification of cry Gene from Bacillus thuringiensis Strains Toxic to the Larvae of Cockchafers

为了寻找对地下害虫蛴螬有杀虫活性的基因资源,以实验室分离的500株苏云金芽胞杆菌(Bacillus thuringiensis,Bt)菌株为材料,通过杀虫活性测定筛选到43株对蛴螬有活性的菌株,其中菌株C9对3种供试昆虫均有活性。光学及扫描电镜检测发现这些菌株均释放球形晶体。SDS-PAGE分析显示这些菌株均表达约130kD的蛋白。采用3种方法分析43株菌株的杀虫晶体蛋白基因类型,结果显示:其中21株只含cry8Ca;13株同时含cry8Ca和cry8Ea基因;QC4等8株菌株只能用引物HRM8F/HRM8R获得高度保守区域约110bp的扩增产物,说明这些基因的5’端序列与已知基因差异较大。测序结果经BLAST分析显示,产物序列同cry8基因的相似性均在98%以上,但其全长基因类型还需要其它方法确定。菌株QCM的PCR-RFLF图谱不同于已发现的cry8基因,推测其中含有新基因。

To identify gene resources toxic to cockchafers,500 strains of Bacillus thuringiensis isolated in our laboratory were investigated.Forty-three strains had insecticidal activity against cockchafers,and C9 was entomocidal to all the three species of cockchafers tested.Results of optical and scanning electronic microscope observation indicated that all of these Bt strains contained crystals in spherical shape.SDS-PAGE analysis indicated that all these strains could express protein with 130 kD molecular mass.Three methods were used to study the cry gene-types of these 43 strains.In 21 strains,there was only cry8Ca type gene,In 13 strains,cry8Ca and cry8Ea type genes co-existed.In 8 strains,PCR products of high-conserved region（about 110 bp） could be obtained only with primers HRM8F/HRM8R,indicating that it is quite different between the 5＇ flanking sequences of these genes with known cry8 genes.Results of sequencing and BLAST analysis indicated that the sequences similarity between the products and cry8 gene is more than 98%.However,the exact gene types need to be determined by further studies.Strain QCM may contain a novel cry8 gene for its unique PCR-RFLP patterns.