微小RNA(miR-7)靶向调控罗伯茨绿僵菌产孢的分子机理研究

Metarhizium has been successfully used to control a variety of agricultural and forestry pests, however, the low sporulation rate is an restrictive factor for its application and spreading. MicroRNA is an important regulatory factor for gene expression in eukaryotes, which is involved in the regulation of different developmental stages and stress responses. In our preliminary studies, the sporulation and the conidiophore branches of Metarhizium robertsii significantly reduced after the expression of miR-7 inhibited by STTM (short tandem target mimics) technology.. Therefore, firstly, phenotype analysis of the miR-7 inhibited or overexpressed mutant strains were carried out. Next, the specific target genes and the sporulation regulation pathways of them were analyzed by UV-crosslinking immunoprecipitation and bioinformatics analysis, then the 2-3 key target genes were further confirmed by sensor method applying in vivo and by the identification of 3’-UTR in mutant strains. Finally, experimental methods, such as the gene knockout/complementary mutant were employed to verified the biological function of the 2-3 key target genes, proving their influences in sporulation process, then confirmed the key genes targeted by miR-7 and the sporulation regulation pathways they belong to, and providing a theoretical basis and method for elucidating the sporulation regulation mechanism of miR-7 in Metarhizium and the genetic improvement of strains。

绿僵菌成功用于多种农林害虫的生物防治；但产孢低成为限制其应用推广的一个重要因素。MicroRNA是真核生物重要的基因表达调控因子，参与不同发育时期和逆境响应的调控。前期发现：利用STTM（short tandem target mimics）技术抑制miR-7的表达后，罗伯茨绿僵菌的产孢量和分生孢子梗分支数均显著减少。因此，项目拟开展miR-7调控其产孢分子机制的研究。首先分析miR-7被抑制和过表达后转基因菌株的产孢表型变化；其次通过免疫共沉淀和生物信息学分析其特异的靶基因及所在产孢调控通路，并进一步通过结合体内Sensor及回复突变3’-UTR确认2-3个miR-7的关键靶基因；最后采用基因敲除/回复突变技术，对2-3个关键靶基因的生物学功能分析以验证其对产孢影响，进而明确miR-7调控产孢的关键基因及所在产孢调控通路；为阐明绿僵菌miR-7调控产孢机制及菌株遗传改良提供重要理论依据。

绿僵菌是一类应用广泛的生物防治昆虫病原真菌，而绿僵菌致死昆虫的首要因素是需要有足够的分生孢子附着到昆虫体表，进而穿透体壁，体内定殖，致死昆虫。此前研究发现绿僵菌小RNA产生通路的关键因子MrAgo1基因的缺失可以显著降低分生孢子的产量，因此我们推测小RNA（包括milRNA）参与调控绿僵菌的产孢。基于前期研究，本项目针对绿僵菌2个milRNA（milR-7和milR-9）的功能及作用机制开展了研究，分别从以下5个方面展开深入研究：（1）罗伯茨绿僵菌milR-7的功能分析及靶基因预测；（2）罗伯茨绿僵菌产孢期特异的milRNA表达分析及3×Flag-Ago1免疫共沉淀分析；（3）罗伯茨绿僵菌milR-9的功能分析；（4）罗伯茨绿僵菌milR-9的靶基因预测分析；（5）罗伯茨绿僵菌milR-9的靶基因功能分析。研究结果具体如下：（1）分析milR-7的被抑制菌株（STTM-milR-7）的产孢量，结果显示：与野生型菌株（WT）产孢量相比，STTM-milR-7产孢量降低了90%。（2）针对罗伯茨绿僵菌产孢期特异的milRNA分析及3× Flag-Ago1免疫共沉淀分析，结果发现1个milRNA（milR-9）在产孢过程中表达量显著高于其他milRNA。（3）针对milR-9的功能分析，结果发现：与WT相比，Pre-milR-9缺失突变株（△milR-9）的产孢量下降了58%，而Pre-milR-9过表达菌株（OVER-milR-9）的产孢量则上升了70%。（4）针对罗伯茨绿僵菌milR-9的靶基因预测分析，结合3× Flag-Ago1免疫共沉淀数据分析、罗伯茨绿僵菌milRNA和mRNA的表达分析，结果发现前期预测的80个候选靶基因中有6个候选靶基因符合转录水平调节规律（也即可能是milR-9真正的靶基因）。（5）针对罗伯茨绿僵菌milR-9的靶基因功能分析，结果发现：与WT相比，6个候选靶基因中有1个基因 (MAA_09111， NUDIX hydrolase domain protein)的基因缺失突变株产孢量上升了60%（推测该基因可能是产孢负调控因子）。总之，这些结果的获得揭示了绿僵菌miRNA靶向调控产孢的分子机制，拓展了人们对绿僵菌产孢调控机制的理解，也为绿僵菌的遗传改良提供了理论基础。