金龟子绿僵菌产孢调控中微小RNA及其靶基因的鉴定与功能研究

Metarhizium is an entomogenous fungi widely used as a biocontrol agent to control insect pests. The level of Metarhizium commercialization depends critically on the efficient production of spores. MilRNAs (microRNA-like RNAs),which exist in almost all higher eukaryotes, play an important role in the regulate of the growth and development of many organisms. However, little is known about the roles of milRNA in the regulation of sporulation from fungi. In our preliminary studies, the existence of milRNA transcript invovled in the sporulation process from Metarhizium anisopliae was confirmed..Based on this work, in-depth study of the critical issue of the milRNA and function in the sporulation process from Metarhizium was carried out. Firstly, milRNAs and its target genes involved in the regulation sporulation were systematically identified by high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation(HITS-CLIP).At the same time, expression profiles of milRNAs and its target genes before and after conidiation were analyzed by microarray and Real-time PCR. Secondly,the regulatory role of miRNAs and its target genes were further confirmed by bioinformatics network analysis. Finally, experimental methods,such as small RNA overexpression, silencing and reporter gene systems, were employed to confirm the biological function of the milRNAs and its target genes.The findings should help to clarify the mechanism of milRNAs regulation in the fungal sporulation process, also provide an important basis for the genetic alteration of the production strain of Metarhizium.

绿僵菌已被广泛运用于农林害虫的生物防治，其商品化程度关键在于孢子高效生产。微小RNA广泛存在于多种真核生物，在调控生物生长发育中发挥重要作用。然而，有关微小RNA在真菌产孢调控中的作用尚未见报道。我们前期研究确认金龟子绿僵菌在产孢中确存微小RNA转录。本项目在此基础上，围绕该菌产孢过程中的微小RNA及功能关键问题进行研究。通过紫外交联免疫共沉淀Argonaute -小RNA-mRNA复合体和高通量测序的方法，系统地鉴定其产孢中小RNA及其靶基因；同时，采用基因芯片和定量PCR技术分析它的小RNA及其靶基因在产孢前后的表达差异；再通过生物信息学网络分析，进一步确认起调控作用的小RNA及其靶基因；最后利用微小RNA过表达、抑制和报告基因系统等分子手段对确认的微小RNA及其靶基因进行生物学功能验证。研究结果将有助于阐明真菌产孢中的小RNA调控机制，也为绿僵菌生产菌株的的遗传改造提供重要理论依据。

绿僵菌已被广泛运用于农林害虫的生物防治，其商品化程度高低关键在于孢子高效生产。微小RNA广泛存在于多种真核生物，在调控生物生长发育中发挥重要作用。然而，有关微小RNA在真菌产孢调控中的作用尚未见报道。本研究以罗伯茨绿僵菌为研究对象，分别从以下4个方面进行了深入研究：（1）绿僵菌小RNA通路关键功能基因的分析，（2）绿僵菌产孢前后的miRNA高通量测序分析，（3）利用HITS-CLIP技术分析绿僵菌miRNA及其靶基因（位点），（4）利用STTM技术分析绿僵菌关键miRNA生物学作用。研究结果如下：（1）绿僵菌小RNA通路中关键功能基因Dicer和AGO基因各有两个，通过基因敲除，发现各突变株的产孢量均不同程度的低于野生型菌株，特别是△dcl2和△ago1，下降尤为明显；此外，对Dicer突变株的miRNA表达量进行分析，发现△dcl2和△dcl1△dcl2中miRNA表达量显著下调，这说明在绿僵菌中Mrdcl2可能主要参与miRNA的生物发生；这部分工作已被杂志Applied and Environmental Microbiology正式接受待发表。（2）高通量Solexa测序技术完成了绿僵菌miRNA的系统鉴定，发现了数个在产孢前后具有显著差异表达的miRNA。进一步的靶基因预测发现，miRNA作用于各种的靶基因，其中主要为转录调控因子；这充分说明miRNA在绿僵菌的产孢前后发挥重要作用，这部分工作已在Fungal biology正式发表。（3）通过紫外交联免疫共沉淀法（CLIP）成功分离纯化到miRNA-mRNA-AGO三聚复合物中的核酸以及相关的miRNA；通过高通量测序分析发现：数十个符合标准的miRNA以及数千个非重复靶位点。（4）通过STTM技术，对miR-7的生物学功能进行了分析，发现STTM-miR-7突变株的产孢量比野生型菌株下降一个数量级；以在12小时的孢子萌发率为指标，发现STTM-miR-7突变株的孢子萌发率高于野生型的菌株。这些结果的获得拓展了人们对小RNA调控绿僵菌产孢的理解，且将有助于最终阐明真菌产孢中的小RNA调控机制，也为绿僵菌生产菌株的的遗传改造提供重要的理论基础。