层出镰刀菌乌头酸酶家族的功能研究

Functions of the aconitase family in Fusarium proliferatum

【目的】层出镰刀菌是引起苜蓿根腐病的主要病原菌之一,探究层出镰刀菌中乌头酸酶家族蛋白的功能特性,为深入认识层出镰刀菌基础生理代谢的分子机制提供依据。【方法】利用hmmsearch工具,对真菌中含有乌头酸酶结构域的蛋白进行检索,并进行系统进化分析;通过实时荧光定量PCR及SWISS MODEL建模技术分别分析FpACO基因的表达模式与蛋白结构;利用同源重组双交换方法构建层出镰刀菌乌头酸酶基因敲除突变体;分析ΔFpACO3、ΔFpACO4-1、ΔFpACO4-2敲除突变体的生长、产孢、孢子形态、环境胁迫响应及致病力等表型变化;进一步测定敲除突变体中线粒体代谢相关生理生化指标的变化情况。【结果】FpACO4-1与FpACO4-2在产孢及孢子形态发生中发挥作用;FpACO3、FpACO4-1、FpACO4-2参与调控层出镰刀菌对细胞壁胁迫及金属离子胁迫的敏感性;FpACO3、FpACO4-1、FpACO4-2影响线粒体代谢,包括总乌头酸酶活性、ATP含量、过氧化氢含量及三羧酸循环关键基因表达等。【结论】乌头酸酶家族参与调控层出镰刀菌产孢、孢子形态分化、细胞壁胁迫及金属离子胁迫响应和线粒体代谢等过程。

[Objective]Fusarium proliferatum is one of the major pathogens causing root rot of alfalfa.This study aims to investigate the function of the aconitase family in F.proliferatum and give insights into the molecular mechanisms underlying the physiological metabolism of this pathogen.[Methods]We employed the hmmsearch tool to identify the proteins containing the aconitase domain in F.proliferatum and then carried out the phylogenetic analysis.real-time PCR and SWISS-MODEL were employed to analyze the expression profiles of FpACO genes and the protein structures,respectively.The homologous recombination method was used to construct the FpACO-deleted mutants of F.proliferatum.Furthermore,we explored the growth,sporulation,spore morphology,stress responses,and pathogenicity ofΔFpACO3,ΔFpACO4-1,andΔFpACO4-2 and measured the mitochondrial metabolism indicators of the mutants.[Results]FpACO4-1 and FpACO4-2 were involved in sporulation and spore morphogenesis.FpACO3,FpACO4-1,and FpACO4-2 were responsible for regulating the sensitivity of F.proliferatum to cell wall stress and metal ion stress.Moreover,FpACO3,FpACO4-1,and FpACO4-2 affected the mitochondrial metabolism indicators,including the total aconitase activity,the ATP level,the hydrogen peroxide level,and the expression of key genes in the tricarboxylic acid cycle.[Conclusion]The aconitase family members are involved in the regulation of the processes such as sporulation,spore morphogenesis,response to cell wall stress and metal ion stress,and mitochondrial metabolism in F.proliferatum.