苹果树腐烂病菌Argonaute基因鉴定及功能初步分析

Preliminary function analysis of Argonaute genes fromValsa mali

Argonaute(AGO)蛋白作为沉默复合体RISC的重要组成部分,直接影响小RNA分子的调控功能。为了明确苹果树腐烂病菌(Valsa mali)AGO蛋白的生物学功能,以揭示苹果树腐烂病菌小RNA分子作用机制奠定基础。本研究从苹果树腐烂病菌基因组中比对出2个AGO蛋白的编码基因VMAGO1和VMAGO3,序列分析发现其均具有Argonaute的标志性功能结构域PAZ和PIWI,并与粗糙脉孢菌和构巢曲霉的AGO蛋白具有高度的同源性。基于同源重组原理成功获得了VMAGO1和VMAGO3的基因单缺失突变体,其菌落、菌丝形态及菌落生长速率与野生型03-8相比没有明显变化。此外,VMAGO1和VMAGO3的缺失不影响病菌对盐离子胁迫的响应,而VMAGO3能够参与病菌对pH、H2O2胁迫的响应,且VMAGO3的缺失能够影响病菌的致病力,其具体调控机理还需进一步验证。

As the main component of RISC,Argonaute（AGO）protein mediates a variety of life activities through regulating the gene silencing pathway.Exploration of the function of AGO proteins of Valsa mali will lay the foundation for uncovering the mechanism function of small RNAs in V.mali.In this study,two proteins were obtained based on the genome information of V.mali by homologous alignment,and both of them contain the typical protein domains of AGO,PAZ and PIWI domain.Meanwhile,they were highly homologous to the AGO proteins of Neurospora crassaand Aspergillus nidulans.Thus,the two genes encoded by AGO protein were named as VMAGO1 and VMAGO3.The gene deletion mutants of VMAGO1 and VMAGO3were obtained by PEG-mediated transformation and confirmed by PCR detection and Southern blot analysis.The morphology of colony,hyphae and growth rate of gene deletion mutants（ΔVMAGO1 andΔVMAGO3）showed no significant difference with the wild type 03-8.TheΔVMAGO1andΔVMAGO3also showed no significant change when they were cultured on PDA plates containing different concentration of salt ions.When they were cultured on the mediumof different pH and the medium containing 0.05% H_2O_2,ΔVMAGO3could grew slowly in the culture of pH=11and showed enhanced tolerance to stress of H2O2.Moreover,infection analysis showed that the pathogenicity ofΔVMAGO3strain enhanced significantly.