嗜线虫致病杆菌抗生素基因簇克隆及遗传体系建立

doi:10.3969/j.issn.1000-2006.201602023

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南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (5): 175-180.doi: 10.3969/j.issn.1000-2006.201602023

嗜线虫致病杆菌抗生素基因簇克隆及遗传体系建立

吴蓉¹(), 汪桂¹, 邓子新², 陈文青²^,^*(), 苏二正¹^,^*()

1.南京林业大学轻工与食品学院, 江苏南京 210037
2.武汉大学药学院, 湖北武汉 430072

收稿日期:2016-02-24 修回日期:2018-06-16 出版日期:2019-10-08 发布日期:2019-10-08
通讯作者: 陈文青,苏二正
基金资助:
国家自然科学基金面上项目(31270100)

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

WU Rong¹(), WANG Gui¹, DENG Zixin², CHEN Wenqing²^,^*(), SU Erzheng¹^,^*()

1. College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037,China
2. School of Pharmaceutical Science, Wuhan University, Wuhan 430072, China

Received:2016-02-24 Revised:2018-06-16 Online:2019-10-08 Published:2019-10-08
Contact: CHEN Wenqing,SU Erzheng

摘要/Abstract

摘要：

【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。

关键词: 嗜线虫致病杆菌, 克隆, 遗传体系, 突变株筛选

Abstract:

【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 forX. 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.

Key words: Xenorhabdus szentirmaii, clone, genetic system, mutant screening

中图分类号:

吴蓉,汪桂,邓子新,等. 嗜线虫致病杆菌抗生素基因簇克隆及遗传体系建立[J]. 南京林业大学学报（自然科学版）, 2019, 43(5): 175-180.

WU Rong, WANG Gui, DENG Zixin, CHEN Wenqing, SU Erzheng. Directly cloning the gene cluster of potential nucleoside antibiotics and establishing the genetic manipulation system of Xenorhabdus szentirmaii DSM 16338[J].Journal of Nanjing Forestry University (Natural Science Edition), 2019, 43(5): 175-180.DOI: 10.3969/j.issn.1000-2006.201602023.

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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

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