重组蚊浓核病毒介导的蚊特异性基因沉默及其杀虫效果研究

Mosquito-borne diseases are major international public health problems that continue to pose a public health threat. Biological control has become an important component of environmentally friendly mosquito-borne disease management. Mosquito densoviruses (MDV) are mosquito-specific parvoviruses, have previously been shown as potential biopestiside. However, the wild-type virus has a relatively slow speed of action and low effective. At present, gene silencing technique already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. But for mosquito, an excellent in vivo transducing system is still expected. We previously showed that MDV can be as valuable vector for gene expression in vivo, which shown the potential of MDV as a 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 particle and powerless for secondary transmission in vivo for defective genome. So, an intronic small RNA 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 knock down specific gene 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) or siRNA, the recombinant virus transducing system is used as vecter to silence target gene by amiRNA interference(amiRNAi) or RNAi in vivo, the bioinsecticidal activity and potential of MDV as a biopesticide are evaluated.

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。申请者前一期国家基金的研究表明，MDV可作为载体在蚊体内高效表达外源基因并有效提高其毒力，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以内含子内小RNA的克隆策略，构建表达siRNA和artificial miRNA的非缺陷型重组MDV转导系统；⑵以蚊虫生长繁育关键基因为靶标，利用该系统实现蚊体内目的基因的沉默，实验室内测试其对蚊虫的杀伤效果。总体目标是：构建重组蚊浓核病毒介导的蚊特异性基因沉默转导系统，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。

一、目的背景：蚊媒传染病仍然是世界性的公共卫生问题，生物防制是控制蚊媒传染病的发展方向之一。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，但在应用于蚊虫却缺乏有效的转导系统。总体目标是：构建重组蚊浓核病毒介导的蚊特异性基因沉默转导系统，实验室内测试其介导的基因沉默对蚊虫的杀伤效果，为蚊类特异高效生物杀虫剂的研发奠定基础。.二、研究内容 ⑴以内含子内小RNA的表达策略，构建表达siRNA和artificial miRNA的非缺陷型重组MDV转导系统；⑵以蚊虫生长繁育关键基因为靶标，利用该系统实现蚊体内目的基因的沉默，实验室内测试其对蚊虫的杀伤效果。.三、重要结果、关键数据及其科学意义.在国自然资助下主要研究结果包括：1.成功构建了非缺陷型的可自我复制与包装的重组蚊浓核病毒；2.通过内含子内小RNA表达策略，成功利用重组病毒介导的在蚊细胞系C6/36细胞内与白纹伊蚊幼虫阶段过表达与抑制内源性蚊虫miRNA与人造artifical miRNA；3.系统测试了重组病毒在蚊体内外基因稳定性。4.利用白纹伊蚊与埃及伊蚊幼虫为载体，初步的测试了病毒大规模生产的新型方法。5选择蚊虫生长发育的关键基因之一作为靶基因，有效的降低了该种靶基因的表达水平，并增强了病毒对蚊虫的病原性，包括增加增加了病毒的死亡率，增强了亚致死效应，包括蚊虫的幼虫期、化蛹时间、羽化时间等。该蚊虫特异的小RNA重组病毒载体的成功构建，为蚊虫基因功能研究提供有力的武器，为蚊虫防制提供了新的策略。

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