辣椒溶杆菌X2-3 LC_Clp转录因子功能分析

Function analysis of LC_Clp transcription factor in Lysobacter capsici X2-3

摘要:

辣椒溶杆菌(Lysobacter capsici) X2-3是从小麦根际分离的一株对多种病原真菌和卵菌有抑菌活性的菌株，目前对该菌株产生的抗菌物质及其产生调控机制尚不明确。【目的】明确转录因子LC_Clp对该菌株抗菌物质产生的调节作用，为深入了解L. capsici X2-3的生防机制提供依据。【方法】从转座子EZ-Tn5随机插入突变体库中筛选获得X2-3的LC_clp基因突变体M356，通过恢复性克隆获得功能互补菌株，分析LC_clp基因在拮抗活性、胞外酶分泌以及调控基因表达方面的差异。【结果】与X2-3相比，M356对测试病原真菌、卵菌的抑菌活性和产生体外抗菌物质的能力完全丧失，蛋白酶和纤维素酶产生量明显减少，几乎不产几丁质酶；所检测的转录调节因子、次生代谢及胞外酶等相关基因的表达量均显著低于野生株X2-3，而互补菌株MCS28和X2-3水平相当。【结论】LC_Clp不仅与菌株的抗菌物质合成及抑菌活性有关，还影响胞外酶的产生，并调控多种基因的表达，具有广泛的调节作用。

Abstract:

Lysobacter capsici X2-3, a plant growth promoting rhizobacteria (PGPR) strain isolated from the wheat rhizosphere, shows strong antimicrobial activity against many plant pathogenic fungi and oomycetes. However, little is known about the antibiotics produced by X2-3 as well as their regulation mechanism. [Objective] Our study aimed to clarify the regulation effect of LC_Clp transcription factor on the antibiotic production of L. capsici X2-3 and to provide reference for deep understanding of its biocontrol mechanism. [Methods] We selected a LC_clp mutant M356 from the mutant bank that was generated by the random insertion of the EZ-Tn5 transposon and certified by plasmid rescue. Additionally, we analyzed the differences of LC_clp gene in antimicrobial activity, secretion of extracellular enzymes, and regulation of gene expression among X2-3, mutant M356 and the complementary strain MCS28. [Results] Antimicrobial activity of mutant M356 against pathogenic fungi and oomycetes was completely lost as compared with that of X2-3 and the antibiotics produced by M356 was undetected by HPLC method. Extracellular enzymes such as protease, cellulose and chitinase that was secreted by mutant M356 decreased significantly. Expression of genes relating transcriptional regulators, secondary metabolism and extracellular enzymes were lowered dramatically in M356 as compared with that of wild-type strain X2-3. However, all the items tested in complementary strain MCS28 were similar with those of wild-type strain X2-3. [Conclusion] As a global regulator, LC_Clp regulates the synthesis of antibiotics, the production of extracellular enzymes and the expression of multiple genes in L. capsici X2-3.