苏云金芽孢杆菌新疆分离株cry3基因的克隆和序列分析

Cloning and Sequence Analysis on Bacillus thuringiensis cry 3 Gene

从新疆天山北坡土壤及昆虫上分离到的苏云金芽孢杆菌（Bacillus thuringiensis,Bt）菌株对鞘翅目害虫具有高毒力,其总DNA约为15 kb,以此为模板,并设计特异引物,扩增出800 bp和550 bp DNA片段,将回收的片段与pBS-T质粒连接,转化大肠杆菌（Escherichia coli）,用蓝白斑法筛选,将阳性克隆子（1号-5,700 bp左右、G4号-3,500 bp左右、G4号-4,720 bp左右）测序,与GenBank中已知的序列进行比对,结果表明1号-5与cry3基因家族的杀虫蛋白基因序列同源性可达84%~100%。

These Bacillus thuringiensis（Bt） strains,used in this experiment and separated from soil and insects of north slope of Tianshan Mountains in Xinjiang,showed high virulence to Coleoptera pests.They contained total DNA about 15 kb as PCR template,specific primers were designed,800 bp and 550 bp DNA were amplified.Then these fragments were recovered and connected with vector pBS-T,and transformed the product into E.coli DH5α.The positive clone was obtained with the blue-white blot screening method,the sequensing was conducted on positive clone（1-5 about 700 bp,G4-3 about 500 bp,G4-4 about 720 bp）,then the known genes were compared with the sequences registered on the GenBank.The result indicated that the strains 1-5 had a higher sequence homology of 84%-100% with insecticidal toxin protein gene of the cry 3 gene family.