防御假单胞菌双突变体H78ΔrsmA/E中hmgA基因对藤黄绿菌素生物合成的抑制  

Inhibition of the hmgA gene on pyoluteorin biosynthesis in rsmA/E dual mutant of Pseudomonas protegens H78

作　　者：关业俊 王正 向涛 张雪洪[1] 黄显清[1] GUAN Ye-Jun;WANG Zheng;XIANG Tao;ZHANG Xue-Hong;HUANG Xian-Qing(State Key Laboratory of Microbial Metabolism,College of Life Science and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China)

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点试验室,上海20024

出　　处：《微生物学通报》2020年第8期2331-2337,共7页Microbiology China

基　　金：上海市自然科学基金(19ZR1427600);国家自然科学基金(31270083,31470196)。

摘　　要：【背景】防御假单胞菌(Pseudomonas protegens) H78是分离于油菜根际的一株生防菌,其能合成藤黄绿菌素(pyoluteorin,Plt)等多种广谱抗生素,H78的rsmA/E双突变体中Plt合成被完全抑制。【目的】通过转座子诱变技术,筛选H78ΔrsmA/E双突变体中重新激活Plt合成的下游调控因子。【方法】通过同源重组的方法在pltL基因下游插入红色荧光蛋白(redfluorescentprotein,RFP)基因来指示Plt操纵子表达的激活情况;利用转座子随机插入突变、半随机PCR技术筛选并定位目标基因;通过基因回补等方法进一步验证基因功能。【结果】从约2万株H78ΔrsmA/E的转座子突变体中筛选到一株高产Plt和某种黑色素的菌株,并确定其插入位点为hmgA基因,hmgA基因回补能重新抑制H78ΔrsmA/E的Plt合成。【结论】假单胞菌双突变体H78ΔrsmA/E中hmgA基因对Plt的合成存在强烈抑制作用,是潜在的RsmA/E下游调控基因。本研究为进一步阐明Plt合成的调控机制与网络及通过基因工程提高Plt产量奠定了基础。[Background] Pseudomonas protegens H78, a biocontrol strain isolated from the rape rhizosphere, can produce multiple broad-spectrum antibiotics such as pyoluteorin(Plt). Plt biosynthesis is completely inhibited by the rsmA/E dual mutation in Pseudomonas protegens H78. [Objective] The aim of this study is to screen the downstream regulatory factors for reactivating Plt biosynthesis in the H78ΔrsmA/E dual mutant by transposon mutagenesis. [Methods] The red fluorescent protein(RFP) gene was inserted downstream of the Plt biosynthetic gene pltL by homologous recombination to indicate the activation of Plt operon expression. The target gene was screened and located by the transposon-based random insertion mutation and the semi-random PCR. The gene function was further confirmed by the gene complement method. [Results] A mutant with high yield of Plt was screened from about 20 000 transposon insertion mutants of H78ΔrsmA/E, and its mutation site was determined to be within the hmgA gene. In turn, the hmgA complementation can inhibit Plt biosynthesis in the H78ΔrsmA/E strain. [Conclusion] In the H78ΔrsmA/E dual mutant of P. protegens, the hmgA gene shows strong inhibitory effect on Plt biosynthesis. The hmgA gene is a potential downstream regulatory gene of RsmA/E. This study lays a foundation for further elucidating the regulatory mechanism and network of Plt biosynthesis and improving the yield of Plt through genetic engineering.

关 键 词：防御假单胞菌 H78ΔrsmA/E双突变体 转座子诱变 hmgA基因 藤黄绿菌素 

分 类 号：S476[农业科学—农业昆虫与害虫防治]