金龟子绿僵菌致病相关小RNA对寄主蝗虫靶基因调控的研究

Metarhizium is an insect pathogenic fungi widely used as a biocontrol agent, the anti-immunity of host insects is one of an important factor for the instability of control efficacy.Small RNAs,as regulators,play an important role in host-microbial interaction. Especially，it have recently been reported that small RNAs of plant pathogenic fungi can regulate immunity-related target genes of plants for a successful infection.However, little is known about the roles of small RNAs in the regulation of pathogenesis from entomopathogenic fungi. In our preliminary studies, the existence of small RNAs and small RNAs machinery in Metarhizium anisopliae and its host locust was confirmed. Based on this work, in-depth study of the critical issue for the cross-kingdom regulation of host target genes by pathogenicity-related small RNAs in Metarhizium will be carried out. Firstly, pathogenicity-related small RNAs in Metarhizium and its expression profiles will be analyzed and identified by high-throughput sequencing approach.Secondly,host target genes of these Metarhizium small RNAs will be analyzed by bioinformatics analysis. Finally, experimental methods,such as Real-time PCR, immunoprecipitation,gene knock-out and small RNA overexpression,will be employed to confirm the regulation of host target genes by Metarhizium small RNAs.The findings should help to understand the regulatory mechanism of pathogenicity of small RNAs in entomopathogenic fungi,also provide a reliable basis for the genetic alteration of the production strain of Metarhizium.

绿僵菌是一类广泛应用于生物防治的昆虫病原真菌，而影响其防治效果不稳定的一个重要因素是寄主昆虫的免疫抵抗作用。小RNA在寄主与病原菌相互作用中发挥重要调控作用。特别是近期Science杂志报道植物病原真菌小RNA可调控植物的免疫相关靶基因以获得成功侵染。然而，迄今小RNA在昆虫病原真菌对寄主致病过程中的作用未见报道。前期工作中，我们确认金龟子绿僵菌及其寄主蝗虫均存小RNA和小RNA沉默机制。本项目在此基础上，围绕该菌致病相关的小RNA跨物种调控寄主蝗虫靶基因进行研究。采用高通量测序分析和筛选，获得绿僵菌致病相关的小RNA及表达特征；再通过生物信息学方法分析其寄主蝗虫的靶基因；并进一步结合定量PCR、免疫共沉淀、基因敲除与过表和致病力测定等技术手段深入分析绿僵菌小RNA对寄主蝗虫靶基因的调控作用。研究结果将有助于阐明小RNA在昆虫病原真菌致病过程中的作用机理，为菌株改良提供可靠的理论依据。

绿僵菌是一类广泛应用于生物防治的昆虫病原真菌，而影响其防治效果不稳定的一个重要因素是寄主昆虫的免疫抵抗作用。小RNA在寄主与病原菌相互作用中发挥重要调控作用。然而，迄今小RNA在昆虫病原真菌对寄主致病过程中的作用未见报道。前期工作中，我们发现绿僵菌及其寄主蝗虫均存小RNA和小RNA沉默机制。本研究在此基础上，围绕罗伯茨绿僵菌致病相关的小RNA跨物种调控寄主蝗虫靶基因的关键问题，分别从以下4个方面进行了深入研究：（1）绿僵菌致病过程中的miRNA组成及表达分析，（2）绿僵菌致病过程中上调表达miRNA的寄主蝗虫靶基因分析，（3）绿僵菌关键miRNA在寄主体内与寄主靶基因结合的免疫共沉淀分析，（4）绿僵菌关键miRNA对寄主致病力的影响分析。具体研究结果如下：（1）针对绿僵菌侵染蝗虫后混合样品中绿僵菌miRNA的分析，结果发现：在绿僵菌侵染蝗虫后0h，12h，24h，36h样品中，分别检测到已知的绿僵菌miRNA分别有0、1、2和3个。（2）针对绿僵菌致病过程中上调表达miRNA的寄主蝗虫靶基因分析，结果发现3个上调表达的绿僵菌miRNA作用于数百种寄主蝗虫的靶基因，这充分说明绿僵菌miRNA在其侵染致病过程中发挥重要作用。（3）针对绿僵菌关键miRNA在寄主体内与寄主靶基因结合的免疫共沉淀分析：一方面，免疫共沉淀样品中确实发现有7个绿僵菌特有的miRNA及其所调控的315个寄主蝗虫靶基因；这些寄主蝗虫靶基因主要富集于mRNA surveillance pathway，Antigen processing and presentation等通路。另一方面，在免疫共沉淀样品中鉴定到5个已知的蝗虫miRNA和20个新的miRNA。（4）通过STTM (Small Tandem Target Mimic)技术，特异性地针对绿僵菌miR-1的生物学功能进行了分析；结果发现：相比较于野生型菌株，STTM-miR-1转基因突变菌株的毒力明显增强，且虫子尸体表面产孢能力也明显增强。其中部分工作已经撰写成文并投至杂志Applied and Environmental Microbiology；目前已经收到小修意见。总之，这些结果的获得拓展了人们对于小RNA在绿僵菌等昆虫病原真菌致病过程中作用机制的理解，也为绿僵菌生产菌株的的遗传改造提供重要的理论基础。