miRNAs介导的水通道蛋白(AsAQPs)表达调控在中华按蚊成蚊越冬抗寒中的作用研究

Anopheles sinensis, as the major vector of malaria in China, survives in winter as adult mosquitoes and the number of the population reduces significantly at the time, thus the transmission ability of the diseases reduced as well. Little was known about the molecular mechanism about the An. sinensis overwintering. During freezing environment, whether an organism survives often depends on the ability to manage its body water and cryoprotectants as glycerol. Aquaporins (AQPs), which rapidly redistribute water and certain small solutes across the cell membrane, are tightly associated with physiological response and survival of osmotic challenge risk caused such as cold. MicroRNA are small non-coding RNAs capable of regulating AQPs genes expression. We have proved that there are two AQPs expressed in An. sinensis, they have function of either transport water or glycerol and expressed significantly different during cold. Based on fact that two AsAQPs were discovered, we hypothesis that the unique functions of AsAQPs may related the low temperature resistance ability of mosquitoes during winter and miRNAs could selectively regulate the activity of AsAQPs. The project is to characterization the function of AsAQPs during low temperature, to confirm that AsAQPs are key point for mosquitoes to survive during cold stress，and to explore the possibility of identifying miRNAs that could selectively modulate their expression, which will provide the information on the molecular mechanism of mosquitoes overwintering and produce data on the strategy-design of mosquitoes control.

中华按蚊是我国及东南亚地区最重要的媒介昆虫之一，该蚊以成蚊方式越冬，按蚊越冬的特征及其调控机理可为媒介控制提供科学依据。研究发现miRNAs在昆虫抗冻中发挥重要调节作用，但靶标尚不明确；AQP对细胞内外水分调节及甘油等转运是昆虫抗寒的重要方式，而哺乳动物AQPs的转录受到miRNAs调节。本课题组在前期研究中成功克隆到中华按蚊的两种AsAQPs，它们在低温下中华按蚊中的表达水平有明显变化。由此推测，中华按蚊这两种AsAQPs可能在miRNAs的调控下，在其越冬抗低温过程中发挥重要作用。本项目拟分别通过体外培养细胞系研究中华按蚊miRNAs对AsAQPs蛋白表达的调控；采用基因沉默技术确认AsAQP1和AsAQP2在抗寒越冬过程中作用；并通对不同状态下中华按蚊miRNAs和AsAQPs表达水平分析miRNAs介导的AQPs表达调控在其成蚊越冬抗寒中的作用，从而为按蚊越冬抗寒提供分子解释

中华按蚊是我国及东南亚地区最重要的媒介昆虫之一，是传播疟疾的主要媒介，以成蚊越冬。本研究基于前期从中华按蚊中克隆到2个水通道蛋白（AsAQP1和AsAQP2）基因的基础上，对其参与中华按蚊越冬的机理及其调控进行研究，初步阐述中华按蚊越冬的特征及其调控机制，为媒介控制措施提供研究基础。本研究对这两个谁通道蛋白的基因分析，证实这两个基因具有通道蛋白的典型特征。并通过体外功能验证AsAQP1具有转运甘油和尿素等小分子的功能，AsAQP2具有转运水的功能。通过对这两个水通道蛋白的研究发现，这两种蛋白在雄性按蚊中的表达水平要高于雌性按蚊。在雌性按蚊中，AsAQP1在按蚊马氏管和脂肪体中高表达，而AsAQP2则在马氏管和中肠中高表达。在雌性按蚊吸食血餐后，AsAQP1在4-12小时高表达，随后表达水平下降，而AsAQP2则在血餐后48-72小时高表达。此外，在实验室对中华按蚊成蚊越冬环境进行模拟，结果表明中华按蚊在8℃至10℃实验室条件下可以存活3个月且吸血后的按蚊存活时间比未吸血的长，具备了越冬条件。进一步通过低温（18℃，12小时光照/12小时黑暗）诱导中华按蚊滞育模型，通过对中肠及卵巢的发育情况判定中华按蚊可实验室诱导滞育。进一步研究低温条件下AsAQPs 的表达水平，结果表明，两种水通道蛋白在低温（8℃）下的表达水平均比常温下（26℃）低，可能与低温下按蚊体内代谢水平下降有关。在此基础上对常温和低温下中华按蚊进行了转录组测序，结果表明低温下高表达的基因有230个，常温下高表达的基因有170个，选取差异表达显著的15个基因进行验证，其中有6个基因能成功扩增，荧光定量结果与转录组测序结果一致，且这6个基因均在卵巢、头部及脂肪体高表达，可能与抗寒有关。