蚊特异高效miRNA转导系统的构建及amiRNAi的杀虫效果研究

miRNAs regulate a large number of genes, many of which are involved in key developmental processes of mosquito and interactions between the pathogens and mosquito vectors. Investigating the potential roles of miRNAs will contribute to development of novel approaches to control vector-borne diseases. However, an excellent in vivo transducing system still be expected for further investigation. Mosquito densoviruses (MDV) are mosquito-specific parvoviruses and infect susceptible mosquito cells persistently. We previously showed that MDV can be as valuable vector for gene expression in vivo. These attributes make them potential in development of mosquito-specific transducing system. Unfortunately, there is a major limitation of this transducing system, the the strict size limit of the recombinant genome imposed by the icosahedral geometry of the viral particl and powerless for secondary transmission in vivo for defective genome. So, an intronic pre-miRNA expression strategy is engaged to offer a new method to overcome limitations for the efficient use of MDV, and then, transducing system is used to overexpress and knock down specific miRNA in vitro and in vivo, and transduction efficiency and expression levels are estimated. Furthermore, the key genes linked to the development of mosquito are selected as targets to design artificial miRNA(amiRNA), the recombinant virus transducing system is used as vecter to silence target gene by amiRNA interference(amiRNAi) in vivo, the bioinsecticidal activity and potential of MDV as a biopesticide are evaluated.

miRNA是基因表达的调控分子，对蚊的生长发育具有重要影响，但目前尚缺乏有效的转导系统。MDV是蚊虫特有病毒，申请者在前一个已完成的国家基金中证实其作为载体在蚊体内可高效表达外源基因，具有发展为蚊特异转导系统和生物杀虫剂的潜能，但缺陷型的重组病毒成为其应用的障碍。根据以上科学问题和研究基础，本项目拟：⑴以内含子内pre-miRNA的表达策略，构建表达miRNA的非缺陷型重组MDV转导系统；⑵利用该系统在蚊虫细胞和幼虫中overexpress和knock down特异性miRNA，测试其转导效率、表达效率以及对蚊虫生长发育的影响；⑶利用该系统介导的amiRNAi技术，以蚊虫生长发育关键基因为靶标，实现对目标基因的沉默，并测试其对蚊虫的杀伤效果。总体目标是：构建蚊特异高效的MDV转导系统，测试amiRNAi的杀虫效果，为蚊虫miRNA的功能研究和生物杀虫剂的研发提供应技术方法和应用基础。

蚊虫不但骚扰人类的生活，而且传播许多重要的疾病，生物防制是控制蚊媒的主要发展方向之一。蚊浓核病毒（MDV）是蚊类特有病毒，具有发展为生物杀虫剂的潜在价值，但野生病毒杀虫活性低，作用速度慢。目前，基因沉默技术已经在害虫防制上初获成功，但应用于蚊虫却缺乏有效的转导系统。申请者在国家基金（非连续1年期小额）项目的资助下完成了项目研究的基础工作，为今后研究的深入奠定了基础。1.成功以重组埃及伊蚊浓核病毒AeDNV为载体证实了人造内含子在蚊体内外可有效的剪切；2.在内含子剪切后，病毒的外显子可以有效拼接并顺利表达了报告基因；3.利用内含子内表达miRNA的策略，成功在C6/36细胞内表达了白纹伊蚊aal-let-7与aal-mir-210两种重要的miRNA，证实了该策略。4.对白纹伊蚊生长过程中起重要作用的性别决定基因Transformer2 与doublesex进行了研究，证实了蚊体内存在多种transformer2异构体，及其对doublesex的重要调控作用；5.对transformer2基因的抑制可以影响doublesex的性别特异性拼接进程，从而影响蚊虫的性别分化进程，以及血餐后卵巢的发育，证实tranformer2基因可以作为浓核病毒控蚊的重要靶基因之一。以上研究均为构建重组蚊浓核病毒介导的蚊特异性基因沉默奠定了技术，实验室内测试其介导的基因沉默对蚊虫的杀伤效果，为蚊虫特异高效生物杀虫剂的研发奠定基础。