基于小菜蛾抗菌肽表达调控的MicroRNAs及其功能研究

MicroRNAs(miRNAs)是生物体内一类新型保守的非编码单链小分子RNA，通过抑制或降解功能基因的mRNA,参与细胞生长、发育等生命活动的调控过程。为探索miRNAs 对昆虫抗菌肽表达的调控，本项目拟以小菜蛾为研究对象：构建miRNA 文库及深度测序，鉴定小菜蛾体内miRNAs的种类和数量；基因芯片筛查调控小菜蛾抗菌肽表达的miRNAs（AMP-miRs），利用活体过量表达或RNAi干扰AMP-miRs在小菜蛾体内的表达，运用定量RT-PCR、Northern blot检测小菜蛾抗菌肽及相关免疫基因的表达差异，阐明AMP-miRs在小菜蛾抗菌肽产生过程中的功能；利用荧光素酶载体系统和RNAi技术明确AMP-miRs调控的靶标基因。通过上述研究，阐明AMP-miRs调控小菜蛾抗菌肽表达的机理。研究结果不仅有助于深入理解害虫的免疫调控机制，也为通过调控害虫的免疫系统来控制害虫奠定基础。

MicroRNAs (miRNAs) are small endogenous non-coding RNAs that post-transcriptionally regulate gene expression in eukaryotes. They are known to play diverse roles in physiological processes such as homeostasis and development.In this project, in order to elucidate the regulation of expression of antibacterial peptids of Plutella xylostella by miRNAs, the miRNAs library will be constructed and the diversity and quantity of miRNAs will be identified by deep-sequencing. Microarray will be used to screen the miRNAs which ragulated the transcripion of antibacterial peptides(named AMP-miRs),and then over-expression and RNA interference (RNAi) will be used to over-express or knock down the expression of the AMP-miRs, either completely silencing or impairing the signal transduction pathway that regulates the immune response in the P.xylostella. Silencing of AMP-miRs may result in easily infection of P. xylostella by pathogenic microorganisms and increase the mortality. Quantitative RT-PCR and Northern blot will be used to verify the spatial and temporal expression profiles of antibacterial peptides genes and immune-related genes after AMP-miRs are silenced by dsRNA to elucidate the functions of AMP-miRs in the immune system. The software miRanda will be used to predict antibacterial peptides target of immune-related miRNAs, and then confirmed by luciferase system and RNAi. The results of this project will not only provide information to understand the regulation of the expression of antibacterial peptides on the RNA level,but also lay a basis for biological control of insect pests based on the regulation of insect immune system.