嗜线虫致病杆菌抗生素基因簇克隆及遗传体系建立

Directly cloning the gene cluster of potential nucleoside antibiotics and establishing the genetic manipulation system of Xenorhabdus szentirmaii DSM 16338

【目的】嗜线虫致病杆菌(Xenorhabdus)是昆虫病原线虫的共生菌,研究此共生菌分泌的抗菌活性的次级代谢产物基因蔟信息。【方法】通过直接PCR将目的基因簇分为数段扩增,利用天然酵母重组系统实现体外重组;同时采用果聚糖蔗糖转移酶基因sacB负筛选系统,通过两次同源臂重组构建基因敲除突变株,建立嗜线虫致病杆菌DSM 16338的遗传操作系统。【结果】成功获得推测目的基因簇;缺失所推测基因簇的3个相连基因,筛选到大片段缺失突变株GW2及调控基因敲除突变株GW4。【结论】利用高效的直接克隆方法获得了基因簇,成功对DSM 16338推测基因簇完成了基因中断,建立了该菌的遗传操作系统,为阐述此类细菌天然产物的生物化学多样性奠定了基础。

【Objective】Cloning the potential nucleoside antibiotic gene cluster from Xenorhabdus szentirmaii DSM 16338 and establishment the genetic manipulation system for X.szentirmaii DSM 16338.【Method】Cloning of the target gene cluster was done by direct PCR amplification of different segments that were joined together,and the in vitro homologous recombination of different segments was fulfilled by using a native yeast recombination system.Meanwhile,the sacB gene of fructan-sucrose transferase was employed as a negative selection marker to construct gene knockout mutant strains through double homologous arm recombination for establishing genetic manipulation system for X.szentirmaii DSM 16338.【Result】The predicted target gene cluster was successfully obtained.Large fragment deletion mutant strain designated as GW2 was constructed by knocking out three consecutive genes of this predicted gene cluster.In order to avoid the regulatory gene inhibiting the transcription expression of the entire predicted gene cluster in the negative regulatory process,the regulatory gene knockout mutant strain GW4 was selected.【Conclusion】A target gene cluster in X.szentirmaii DSM 16338 was obtained by using an efficient direct cloning method.The gene cluster was also interrupted and the genetic manipulation system of Xenorhabdus szentirmaii DSM 16338 was constructed successfully.This study will provide a foundation for further studies on the biological and chemical diversity of natural products produced by bacteria of the genus,Xenorhabdus.