基于小菜蛾抗菌肽表达调控的MicroRNAs及其功能研究

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.

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。

为阐明microRNA对抗菌肽表达的调控机理，本项目利用RNA-Seq测定了玫烟色棒束孢Isaria fumosorosea，绿僵菌素Destruxin, Bacillus thuringiensis (Bt)处理小菜蛾后不同时间的转录组和小RNA文库，筛选了小菜蛾免疫外界微生物的免疫相关microRNA和mRNA，并对microRNA和mRNA的关联性进行了动态分析，预测了其靶标相关性，初步确定了免疫相关microRNA的靶标及其在免疫系统中的功能；并利用RNAi技术明确了部分microRNA靶标的功能。具体结果如下：.1、.筛选了miR-2, miR-9a, miR-745, miR-7b,和miR-2767等主要调控Toll信号通路产生的抗菌肽及酚氧化酶级联反应来抵御I. fumosorosea的侵染的microRNA，qRT-PCR明确了其对I. fumosorosea侵染表达时效性。.2、.筛选并确定了miR-276主要调节ßGBP和Relish的表达，从而调控酚氧化酶级联反应和Imd信号通路产生的抗菌肽来免疫Destruxin的毒性，qRT-PCR明确了其对Destruxin免疫表达时效性。.3、.确定了mir-317直接参与调控小菜蛾抗菌肽Gloverin1（Px_105389810）基因的表达，miR-1调控Lysozyme，miR-263参与lectin的表达来调控小菜蛾免疫Bt的侵染。荧光素酶系统明确了其相互靶标关系。.4、.RNAi明确了抗菌肽cecropin和gloverin主要受Toll通路调控，cecropin和gloverin分别对绿僵菌和Bt具有杀菌活性；lectin可被细菌诱导且蛋白对Bt侵染具有防御作用。.通过本项目研究发表7篇SCI文章（IF≥4,3篇），2篇中文核心期刊，申请专利2个，培养硕士研究生11名，博士3名，博士后1名。