[1]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(05):175-180.[doi:10.3969/j.issn.1000-2006.201602023]
 WU Rong,WANG Gui,DENG Zixin,et al.Directly cloning the gene cluster of potential nucleoside antibiotics and establishingthe genetic manipulation system of Xenorhabdus szentirmaii DSM 16338[J].Journal of Nanjing Forestry University(Natural Science Edition),2019,43(05):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
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《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
43
期数:
2019年05期
页码:
175-180
栏目:
研究简报
出版日期:
2019-09-20

文章信息/Info

Title:
Directly cloning the gene cluster of potential nucleoside antibiotics and establishing the genetic manipulation system of Xenorhabdus szentirmaii DSM 16338
文章编号:
1000-2006(2019)05-0175-06
作者:
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)
Author(s):
WU Rong1WANG Gui1DENG Zixin2CHEN 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)
关键词:
嗜线虫致病杆菌 克隆 遗传体系 突变株筛选
Keywords:
Xenorhabdus szentirmaii clone genetic system mutant screening
分类号:
Q74
DOI:
10.3969/j.issn.1000-2006.201602023
文献标志码:
A
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。
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 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.

参考文献/References:

[1] GLAZER I, SALAME L, DVASH L, et al. Effects of tannin-rich host plants on the infection and establishment of the entomopathogenic nematode Heterorhabditis bacteriophora [J]. Journal of Invertebrate Pathology, 2015, 128: 31-36. DOI: 10.1016/j.jip.2015.02.002. [2] FERREIRA T, VAN RCEENEN C A, TAILLIEZ P, et al. First report of the symbiotic bacterium Xenorhabdus indica associated with the entomopathogenic nematode Steinernema yirgalemense [J]. Journal of Helminthology, 2016, 90(1):108-112. DOI: 10.1017/S0022149X14000583. [3] RAZIA M, RAJA R K, PADMANABAN K, et al. 16S rDNA-based phylogeny of non-symbiotic bacteria of entomopathogenic nematodes from infected insect cadavers[J]. Genomics, Proteomics & Bioinformatics, 2011, 9(3): 104-112. DOI: 10.1016/S1672-0229(11)60013-2. [4] 王立婷, 丘雪红, 吴春艳, 等. 昆虫病原线虫和共生细菌定殖关系的研究进展[J]. 环境昆虫学报, 2013, 35(2): 232-241. DOI: 10.3969/j.issn.1674-0858.2013.02.15. WANG L T, QIU X H, WU C Y, et al. Advances in the species-specific colonization of entomopathogenic nematodes by their symbiotic bacteria [J]. Journal of Environmental Entomology, 2013, 35(2): 232-241. [5] LENGYEL K, LANG E, FODOR A, et al. Description of four novel species of Xenorhabdus, family Enterobacteriaceae: Xenorhabdus budapestensis sp. nov., Xenorhabdus ehlersii sp. nov., Xenorhabdus innexi sp. nov., and Xenorhabdus szentirmaii sp. nov [J]. Systematic and Applied Microbiology, 2005, 28(2): 115-122. DOI: 10.1016/j.syapm.2004.10.004. [6] OHLENDORF B, SIMON S, WIESE J, et al. Szentiamide, an N-formylated cyclic depsipeptide from Xenorhabdus szentirmaii DSM 16338 [J]. Natural Product Communications, 2011, 6(9): 1247-1250. DOI: 10.1007/s12571-011-0142-3. [7] GUALTIERI M, OGIER J C, PAGES S, et al. Draft genome sequence and annotation of the entomopathogenic bacterium Xenorhabdus szentirmaii strain DSM16338 [J]. Genome Announcements, 2014, 2(2):e00190. DOI: 10.1128/genomeA.00190-14. [8] 汪桂. Septacidin及潜在核苷类抗生素基因簇的研究 [D]. 南京:南京林业大学, 2014. WANG G. Research on gene clusters of septacidin and the potential nucleoside antibiotic [D].Nanjing:Nanjing Forestry University, 2014. [9] CHEN W, HUANG T, HE X, et al. Characterization of the polyoxin biosynthetic gene cluster from Streptomyces cacaoi and engineered production of polyoxin H [J]. Journal of Biological Chemistry, 2009, 284(16):10627-10638. DOI: 10.1074/jbc.M807534200. [10] CHEN W, YAN L, JIE L, et al. An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis [J]. Protein & Cell, 2016, 7(9): 673-683. DOI: 10.1007/s13238-016-0289-y. [11] KOUPRINA N, LARIONOV V. Transformation-associated recombination(TAR)cloning for genomics studies and synthetic biology[J]. Chromosoma, 2016, 125(4): 621-632. DOI: 10.1007/s00412-016-0588-3. [12] YAMANAKA K, REYNOLDS K A, KERSTEN R D, et al. Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A [J]. Proceedings of the National Academy of Sciences, 2014, 111(5): 1957-1962. DOI: 10.1073/pnas.1319584111. [13] LALIOTI M D, HEATH J K. A new method for generating point mutations in bacterial artificial chromosomes by homologous recombination in Escherichia coli [J]. Nucleic Acids Research, 2001, 29(3): e14. DOI: 10.1093/nar/29.3.e14. [14] KIESER T, BIBB M J, BUTTNER M J, et al. Practical streptomyces genetics: a laboratory manual 2000 [M]. Norwich: The John Innes Foundation, 1991. [15] SAMBROOK J, ERITSCH E F, MANIATIS T. Molecular cloning: a laboratory manual [M]. 2nd ed. NewYork: Cold Spring Harbor Laboratory Press, 1989. [16] FORST S A, TABATABAI N. Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus [J]. Applied and Environmental Microbiology, 1997, 63(3): 962-968. DOI: 10.1016/S0027-5107(96)00251-5.

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备注/Memo

备注/Memo:
收稿日期:2016-02-24 修回日期:2018-06-16 基金项目:国家自然科学基金面上项目(31270100)。 第一作者:吴蓉(rongwu@njfu.edu.cn)。*通信作者:陈文青(wqchen@whu.edu.cn),副教授,负责关键技术的实施,ORCID(0000-0003-2557-8960); 苏二正(ezhsu@njfu.edu.cn),教授,负责总体实验的设计,ORCID(0000-0002-3868-567X)。 引文格式: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.
更新日期/Last Update: 2019-10-08