梨黑斑病菌CHD基因克隆及其在代谢梨果皮酚酸类物质中的作用解析

CHD in Alternaria alternata: gene cloning and degradation performance against phenolic acids in pear fruit peel

摘要:

【背景】 植物病原真菌必须有效解毒寄主组织中酚类物质等具有抗菌活性的次生代谢产物才能成功侵染和定殖，目前已有研究表明部分病原真菌可通过合成邻苯二酚1,2-双加氧酶(catechol 1,2-dioxygenase, CHD)等降解寄主中的酚类物质，但梨黑斑病菌互隔交链孢(Alternaria alternata)对果皮酚类物质是否具有降解作用及作用机制尚不清楚。【目的】 对梨黑斑病菌中CHD进行克隆和生物信息学分析，并研究其对梨果皮酚酸类物质的降解作用，以期为进一步揭示A. alternata CHD在代谢梨果皮酚类物质过程中的分子机制提供支撑。【方法】 采用同源克隆法从A. alternata中克隆CHD；通过Smart、SOPMA等软件对其进行生物信息学分析；利用四元超快速液相色谱仪检测A. alternata代谢后酚酸含量的变化，并通过RT-qPCR分析CHD在代谢酚酸类过程中的表达情况。【结果】 从A. alternata克隆得到2个CHD基因，分别编号为AaCHD1和AaCHD2，其蛋白均含有双加氧酶的C末端和N末端保守结构域；系统发育分析表明，其分别与番茄匍柄霉(Stemphylium lycopersici)及异旋孢腔菌(Cochliobolus heterostrophus)中的CHD氨基酸序列具有高度同源性。进一步分析发现A. alternata能够以酚酸类物质为唯一碳源生长，但对梨果皮主要酚酸类物质的代谢作用存在差异，对阿魏酸、绿原酸、咖啡酸的代谢强于对香豆酸；同时外源酚酸促进了A. alternata中AaCHD1和AaCHD2显著上调表达，其中外源咖啡酸的诱导作用最强，其次是阿魏酸和对香豆酸。【结论】 A. alternata的AaCHD对梨果皮酚酸类物质具有降解作用且降解效果因底物种类而异，为全面揭示AaCHD在A. alternata克服梨果皮酚类物质的防御作用进而侵染寄主的分子机制提供一定的理论依据。

Abstract:

[Background] To successfully infect and colonize the host tissue, phytopathogenic fungi need to detoxify the antifungal secondary metabolites such as phenolic compounds in the host. Studies have shown some pathogenic fungi can degrade phenolic compounds in the host by synthesizing catechol 1,2-dioxygenase (CHD). However, whether the Alternaria alternata can degrade phenols in pear fruit peel and the mechanism remain unclear. [Objective] To clone CHD from A. alternata, conduct bioinformatics analysis of CHD, and study the degradation performance of phenolic acids in pear fruit peel by CHD, thus providing support for further clarifying the molecular mechanism of this enzyme. [Methods] CHD was cloned from A. alternata by homologous cloning. Smart and SOPMA were used for bioinformatics analysis. The changes in phenolic acid content after metabolism of A. alternata were determined by quaternary ultra-fast liquid chromatography, and the expression level of CHD during the metabolism of phenolic acids was analyzed by RT-qPCR. [Results] Two CHD genes were cloned from A. alternata and designated as AaCHD1 and AaCHD2. The proteins encoded by the two genes both contained C-terminal and N-terminal conserved domains of dioxygenases. The phylogenetic analysis showed that AaCHD1 and AaCHD2 were highly homologous to the CHD amino acid sequences in Stemphylium lycopersici and Cochliobolus heterostrophus, respectively. Furthermore, A. alternata can grow with phenolic acids as the sole carbon source and differentially metabolize phenolic acids in pear fruit peel. Specifically, it showed stronger degradation effects on ferulic acid, chlorogenic acid, and caffeic acid than on p-coumaric acid. At the same time, exogenous phenolic acids up-regulated the expression of AaCHD1 and AaCHD2, and exogenous caffeic acid had the strongest up-regulatory effect, followed by ferulic acid and p-coumaric acid. [Conclusion] AaCHD in A. alternata played a role in degrading phenolic acids in pear fruit peel, and the degradation effects vary with different phenolic acids, which provides a theoretical basis for comprehensively revealing the molecular mechanism of AaCHD in overcoming the defense effect of phenolic acids in pear peel.