不同碳源对生防荧光假单胞菌2P24产抗生素2,4-二乙酰基间苯三酚的影响

Effect of carbon sources on production of 2,4-diacetylphoroglucinol in Pseudomonas fluorescens 2P24

【目的】包括碳源代谢等不同环境因子可调控生防菌株生防相关因子表达,进而影响其防病效果。荧光假单胞菌2P24可防治多种植物病原真菌、细菌引起的土传病害,抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphoroglucinol,2,4-DAPG)是其主要生防因子之一。本文利用平板对峙法及遗传学方法研究不同碳源对菌株2P24产生2,4-DAPG的影响及相关的调控途径。【方法】利用平板对峙法检测了菌株2P24在添加葡萄糖、果糖和蔗糖等碳源的土豆浸液培养基中对棉花立枯丝核菌(Rhizoctonia solani)的拮抗能力及菌株2P24中影响2,4-DAPG产生的相关基因的表达。另外,利用Tn5转座子对含有2,4-DAPG合成基因phl A报告质粒p970Gm-phl Ap的野生型菌株2P24进行随机突变,在果糖土豆浸液培养基中筛选提高phl A基因表达的突变菌株。【结果】平板对峙实验表明,菌株2P24以葡萄糖为碳源时其抑菌活性最强,蔗糖次之,而以果糖等为碳源时菌株2P24无抑菌活性;转录融合实验进一步表明葡萄糖可促进phl A基因的表达,果糖则不影响phl A基因的表达。在果糖土豆浸液培养基中,转座子随机突变实验获得了5株可明显提高phl A基因表达的突变菌株。Tn5插入位点和序列分析显示其中一个突变体是Tn5破坏了che B基因。转录检测表明与野生菌株相比,che B突变体中phl A基因的表达和2,4-DAPG的前体物质间苯三酚(phloroglucinol,PG)产量都显著提高。游动性实验发现突变che B基因可显著降低该菌株的游动性。【结论】上述结果表明菌株2P24中不同碳源在转录水平上可影响phl A基因的表达,进而影响2,4-DAPG产生。遗传学结果也显示,che B基因参与调控2,4-DAPG生物合成过程。

[Objective] Many environmental factors,such as carbon sources,regulate the biosynthesis of antimicrobial compounds and influence the biocontrol capacity of Pseudomonas fluorescens.P.fluorescens 2 P24 protects various crop plants against root diseases caused by plant pathogens.Among a range of antimicrobial compounds secreted by 2 P24,2,4-diacetylphloroglucinol（2,4-DAPG） is the major determinant of its biocontrol potential.This study investigated the impact of exposing strain 2 P24 to selected carbon sources on the production of 2,4-DAPG.[Methods] Antifungal activity of strain 2 P24 was tested on potato infusion agar with different carbon sources against Rhizoctonia solani.The reporter strain 2 P24（p970 Gm-phl Ap） was subjected to a random mini-Tn5 insertion mutagenesis.The collection of Tn5 insertion mutants was then screened for improved phl A expression on potato infusion agar with fructose.[Results] Strain 2 P24 cultured on potato infusion with glucose strongly inhibited the growth of R.solani,whereas no inhibition was observed on potato infusion agar with or without fructose.Five mutants with significantly increased phl A expression were identified and the interrupted locus in one of them was identified as the che B gene.Genetic analysis showed that the expression of phl A and the production of phloroglucinol（PG） were strongly increased in the che B mutant as compared with the parental strain.[Conclusion]Carbon resources influenced the expression of phl A and 2,4-DAPG production and some genetic factors involved in carbon sources to regulate the production of 2,4-DAPG.