gacS基因插入失活对绿针假单胞菌G-05的两种胞外次生代谢物合成的影响

Effects of insertional inactivation of gacS gene on two secondary metabolites in Psedomonas chlororaphis G-05

一株来自大棚温室甜椒根际的绿针假单胞菌Pseudomonas chlororaphis G-05,可分泌抗生物质吩嗪-1-羧酸,并具有抑制辣椒疫霉的生物防治功效。【目的】为了系统研究该菌株的生物防治功能及抗生物质合成与分泌机制。【方法】首先通过生化法和16S rDNA同源比对法对该菌株进行系统分类的初步鉴定,再根据基因的同源性从G-05基因组DNA中克隆长1.4 kb的gacS基因的部分保守区段,采用抗庆大霉素基因(gentamycin resistance cassette,aacC1)插入失活的策略构建了该基因突变株G-05S。【结果】在King's B(KMB)或PPM培养基中,突变株G-05S合成吩嗪-1-羧酸的能力受到明显抑制。然而,突变株G-05S分泌的吲哚乙酸与野生株相比无显著差异。互补实验表明,gacS基因的表达可以使突变株G-05S的吩嗪-1-羧酸的合成恢复到野生株水平。【结论】由此推测,GacS(Global activator sensor)对不同次生代谢物的调控具有特异性。

Phenazine-1-carboxylic acid biosynthesized and secreted by Pseudomonas chlororaphis G-05 isolated from the rhizosphere of pepper in greenhouse （Huaian, China）, contributes to its biological suppression of R. solani growth. [Objective] Our aim is to elucidate its biocontrol function and regulation mechanism. [Methods] We first identified the strain with biochemical method and homology comparison of 16S rDNA. A conservative DNA fragment of gacS gene was then obtained from the genomic DNA of the wild type strain G-05 by polymerase chain reaction （PCR）. According to homologous recombination technology, a mutant G-05S was then created with insertional inactivation of gentamycin resistance cassette （aacC1）. [Results] In comparison with the wild type strain G-05, the gacS-deficient mutant G-05S produced trace amount of phenazine-1-carboxylic acid in King＇s B （ KMB ） or Pigment Production Medium（PPM） medium, respectively. However, it produced indole-3-acetic acid （IAA） in the same way as the wild type strain. When the gacS gene was introduced with the shuttle vector pME6032, the mutant G-05S produced the same phenazine-1-carboxylic acid as the wild type strain. [Conclusion] The regulation mediated by gacS gene on secondary metabolites is specific and differential in some biocontrol agents.