松叶面抗虫共生工程菌的构建

Construction of A Pine Needle Symbiotic Engineered Bacterium Exhibiting the Insecticidal Activity to Dendrolimus punctatus

将来自Bacillusthuringiensissubsp.kurstaki的杀虫基因cryIAc通过综合质粒载体pEG601,整合到松树共生细菌B.cereus(Bc752)的染色体上,得到的工程菌对马尾松毛虫幼虫有明显的杀虫活性。此综合质粒含有能在营养期表达BtcryIAc基因的强启动子、cryIAc杀虫基因、四环素抗性标记基因tetr、8.0kb的EcoRI-NcoⅠB.cereus(O147)染色体片段。将综合质粒通过电击导入Bc752中,综合质粒与Bc752染色体发生同源重组,将BtcryIAc基因整合到Bc752的染色体上。通过对转化子的DNA酶切分析、PCR扩增、SDS-PAGE凝胶电泳检测、Westernblot、电镜观察、毒力测定,结果表明BtcryIAc基因已经整合到松树共生细菌Bc752的染色体上,并可高效表达。

Bt crylAc gene from Bacillus thuringiensis subsp, kurstaki was introduced into the chromosome of pine symbiotic bacterium Bacillus cereus （Bc752） by an integrative vector pEG601. The vector contains a strong vegetative promoter, the crylAc gene （identified as a 7.4 kb SphI - NruI fragment of DNA from B. thuringiensis HD - 73）, the tetracycline resistance gene （ tetr ） and an 8.0 kb EcoRI - NcoI fragment of chromosomal DNA from Bc752 to allow for homologous recombinant between the vector and the bacterial chromosome. Transformation of Bc752 with plasmid pEG601 by electroporation resulted in the engineered symbiotic bacteria. Insertion of the vector DNA into the chromosome was demonstrated by analysis of DNA restriction enzymes and PCR amplification. Recombination strains containing Bt crylAc gene produced the 133ku protoxin which could be detected by SDS- PAGE, ELISA, electronmicroscope observation and insect bioassays.