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

项目名称： 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

项目编号： No.31272096

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 黄勃

作者单位： 安徽农业大学

项目金额： 75万元

中文摘要： 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。

中文关键词： 绿僵菌；小RNA；产孢；紫外交联免疫共沉淀；STTM

英文摘要： 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 confir

英文关键词： Metarhizium robertsii；Small RNA；Sporulation；HITS-CLIP；Small Tandem Target Mimic