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

摘要:

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。

Abstract:

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.