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

摘要:

[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phlA报告质粒p970Gm-phlAp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phlA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phlA基因的表达，果糖则不影响phlA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phlA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cheB基因。转录检测表明与野生菌株相比，cheB突变体中phlA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cheB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phlA基因的表达，进而影响2，4-DAPG产生。遗传学结果也显示，cheB基因参与调控2，4-DAPG生物合成过程。

Abstract:

[Objective] Many environmental factors, such as carbon sources, regulate the biosynthesis of antimicrobial compounds and influence the biocontrol capacity of Pseudomonas fluorescens. P. fluorescens 2P24 protects various crop plants against root diseases caused by plant pathogens. Among a range of antimicrobial compounds secreted by 2P24, 2,4-diacetylphloroglucinol (2,4-DAPG) is the major determinant of its biocontrol potential. This study investigated the impact of exposing strain 2P24 to selected carbon sources on the production of 2,4-DAPG. [Methods] Antifungal activity of strain 2P24 was tested on potato infusion agar with different carbon sources against Rhizoctonia solani. The reporter strain 2P24 (p970Gm-phlAp) was subjected to a random mini-Tn5 insertion mutagenesis. The collection of Tn5 insertion mutants was then screened for improved phlA expression on potato infusion agar with fructose. [Results] Strain 2P24 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 phlA expression were identified and the interrupted locus in one of them was identified as the cheB gene. Genetic analysis showed that the expression of phlA and the production of phloroglucinol (PG) were strongly increased in the cheB mutant as compared with the parental strain.[Conclusion] Carbon resources influenced the expression of phlA and 2,4-DAPG production and some genetic factors involved in carbon sources to regulate the production of 2,4-DAPG.