二化螟microRNA调控蜕皮激素合成的分子机制

miRNA广泛地参与昆虫的发育调控，但其对蜕皮激素合成的调控机制尚不明确，本项目拟在前期工作的基础上，以二化螟为研究对象，研究miRNA调控蜕皮激素合成的分子机制：1) 构建二化螟蜕皮激素合成通路，扩增通路中所有基因的全长。利用已发现的miRNA进行靶标预测, 找到能够靶向蜕皮激素合成途径基因的miRNA；2)利用双荧光素酶载体对miRNA基因进行靶标验证；3)对验证成功的miRNA及其靶标, 开展不同龄期和不同组织的表达谱和相关性分析；4)分别注射miRNA类似物或抑制物，过表达或干扰miRNA，检测靶标基因的mRNA和蛋白水平的变化，测定蜕皮激素滴度，观察miRNA干扰后对二化螟发育的影响；5)对其中关键miRNA基因开展害虫基因沉默控制的应用研究。预期研究成果将明确miRNA在蜕皮激素合成中的功能，进一步揭示miRNA对昆虫发育调控的分子机制，为害虫基因沉默控制寻找合适的作用靶标。

In order to find important functional genes for developing new techniques in RNAi pest control, this project intends to study the regulation of ecdysone biosynthesis by microRNA in rice stem borer. 1) Constructing ecdysone biosynthesis pathway of rice stem borer, amplifying the full length transcripts with RACE strategy, predicting the miRNA targets with miRanda to determine the miRNA target genes in the biosynthesis pathway of rice stem borer. 2) Validating the miRNA targets with luciferase report system to confirm the the regulation roles of miRNA. 3) For those miRNAs and its target genes in the ecdysone biosynthesis pathway, studying the expression profile in different developmental stage or different tissues. 4)Injecting the miRNA mimics to increase the miRNA expression and siRNA to downregulate the miRNA expression, then confirming the RNAi efficiency by qPCR, estimating the concentration change of ecdysone after RNAi, observing the phenotype change. This project will uncover the regulation roles of miRNA in the ecdysone biosynthesis and provide alternative targets for pest control.