miR-14和miR-284介导桔小实蝇变态发育的分子调控机制

The research focuses on the molecular regulation mechanism of miR-14 and miR-284 in metamorphic development of the oriental fruit fly, Bactrocera dorsalis, one of the most economically important fruit fly pests devastating fruit and vegetable industry. On the basis of previous study about identification of miRNAs and corresponding targets prediction, a series of molecular techniques including RNAi, dual luciferase reporter system, RNA binding protein immunoprecipitation and fluorescence in situ hybridization are utilized comprehensively to identify the target regulatory sequence of miR-14 and miR-284; to study the regulation mode of miR-14 and miR-284; to examine the effects of miR-14 and miR-284 on metamorphic development of B. dorsalis; to analyze target regulation of miRNAs; and finally to clarify the molecular regulation mechanism of miR-14 and miR-284 mediated in metamorphic development of the fruit fly. The results will enrich the knowledge about regulation mechanism of insect metamorphic development, promote to seek novel small RNAs as insecticide targets and provide new control strategy on the fruit flies. Therefore, it has important theoretical significance and application prospects.

本项目以果蔬危险性害虫桔小实蝇Bactrocera dorsalis为研究对象，紧密围绕miR-14和miR-284介导桔小实蝇变态发育的分子调控机制这一关键科学问题，综合运用RNAi、双荧光素酶报告系统、RNA结合蛋白免疫共沉淀、荧光原位杂交等技术和手段，在前期miRNAs挖掘及其靶基因预测的基础上，鉴定桔小实蝇miR-14和miR-284对Kr-h1的靶向调控区域，系统分析miR-14和miR-284的表达调控模式，阐明miR-14和miR-284对桔小实蝇变态发育的影响，进而明确桔小实蝇miR-14和miR-284对靶基因的转录调控，最终揭示miR-14和miR-284介导桔小实蝇变态发育的分子调控机制。研究结果不仅有助于解析昆虫变态发育的调控机理，还有利于寻获新的小分子RNA杀虫靶标，为实蝇类害虫防控提供新的思路，因此具有重要的理论意义和应用前景。

本项目以果蔬危险性害虫桔小实蝇为研究对象，综合运用双荧光素酶报告系统、RNA结合蛋白免疫沉淀、原位杂交、RNAi和CRISPR/Cas9等技术和方法，分别对miR-14、miR-284与预测靶基因Kr-h1，miR-309a和预测靶基因pnr、tret体外验证发现，仅miR-309a与预测靶基因pnr结合，后续围绕miR-309a调控桔小实蝇发育的分子机制展开，在明确miR-309a调控卵巢发育及靶基因预测的基础上，通过揭示miR-309a对pnr、pnr对Vgs的靶向调控关系，构建miR-309a-pnr调控通路模型，阐明miR-309a靶向pnr调控桔小实蝇卵巢发育的分子机制。研究结果不仅在理论上揭示miRNA调控双翅目昆虫卵巢发育的新机制，丰富对昆虫miRNA生理功能的认识；同时在实践上有利于寻获新的小分子RNA杀虫靶标，为研发桔小实蝇生殖力调控的新型防控技术提供新思路。因此，具有重要的理论意义和应用前景。