球形芽孢杆菌下调卵黄蛋白原基因表达在其抑制按蚊产卵中的作用和机制研究

Vector control with Bacillus shpaericus(Bs) is an effective way to prevent the transmission of mosquito-borne diseases. However, some mosquitoes can survive from treatment with sublethal dose of Bs（SdBs）because of nonstandard using, which would change their biological traits. Thus, current researches are focus on exploring the interaction mechanism between SdBs and mosquitoes. We previously found that fecundity of the surviving Anopheles dirus from SdBs was significantly decreased, but the mechanism is still unclear. Our further study found that: Bs was detected in the fat body of mosquitoes survive from SdBs, and the treatment with SdBs suppressed the expression of vitellogenin (Vg) gene, which was regulated by TOR signal pathway. As it is known to all, Bs killed the mosquito larva by secreting Bin toxin proteins. Thus, we assumed that Bs may affect the fat cells by secreting toxin proteins, then inhibit the TOR signaling pathway to downregulate Vg expression and finally control An. dirus’s fecundity. As a result, we plan to investigate the role and mechanism of Bin and TOR signal pathway in the effect of Bs inhibit An. dirus’s fecundity, with the technology of micro-injection, RNA interference, and so on. This study will be a theoretical basis for widespread use of Bs in the future, and will also provide a new control target to reduce the mosquito population density.

目前使用生物杀虫剂球形芽孢杆菌(Bs)控制蚊媒数量是阻断蚊传疾病传播的有效措施，然而亚致死剂量Bs的使用将会导致部分蚊虫存活，并改变其生物学性状。探究亚致死剂量Bs与蚊媒之间的相互作用机制是当前研究的焦点。课题组前期研究发现：亚致死剂量Bs处理后存活大劣按蚊(Ad)的产卵能力显著下降，但机制尚不清楚。进一步研究发现：存活按蚊脂肪体细胞中检测到Bs存在，且Bs处理显著下调脂肪体细胞中TOR信号通路调控的卵黄蛋白原（Vg）基因表达水平。研究证实Bs是通过分泌毒素蛋白Bin发挥杀蚊幼虫的作用，因此我们推测Bs进入脂肪体组织后，可能通过毒素蛋白Bin抑制脂肪体细胞TOR信号通路，从而下调Vg表达并抑制产卵。鉴此，本研究拟采用微量注射、RNAi等技术，探讨Bs毒素蛋白和TOR信号通路在Bs抑制Ad产卵中的作用和机制，这将为Bs的推广应用奠定更扎实的理论基础，也为降低致病蚊虫种群密度提供新的作用靶点。

亚致死剂量球形芽孢杆菌的使用可改变存活大劣按蚊的产卵能力。本课题通过微量注射、RNAi、RACE PCR、转录组测序等技术，对球形芽孢杆菌下调卵黄蛋白原基因的表达抑制大劣按蚊产卵的作用机制进行了初步研究。首先通过RACE PCR技术扩增了大劣按蚊卵黄蛋白原基因，并对AdVg主要功能域蛋白进而了表达和纯化，通过qPCR明确了Bs通过下调Vg抑制大劣按蚊产卵。在机制探讨方面，初步证实Bs菌体在存活的大劣按蚊成蚊体内通过抑制TOR信号通路下调Vg表达抑制大劣按蚊产卵；为了全面分析Bs抑制大劣按蚊产卵的分子机制，进行了转录组测序分析，筛选出了4个候选基因，推测这些基因可能也参与Bs抑制大劣按蚊产卵，相关的验证工作正在进行。另外，我们还对Bs抑制大劣按蚊产卵能力的现象是否存在普遍性问题进行了研究，发现亚致死剂量Bs处理增强了斯氏按蚊的产卵能力，并对相应的分子机制进行了初步的探讨。综合上述结果，本研究为探讨亚致死剂量Bs抑制大劣按蚊产卵的分子机制奠定了坚实的工作基础。同时对于探索亚致死剂量Bs与不同种属按蚊繁殖能力之间的关系提供了重要的启示性线索。项目资助发表科研论文7篇，已接收科研论文1篇，已投稿SCI论文1篇。培养硕士生2名，均已取得硕士学位。项目投入经费20万元，支出16.5661万元，各项支出基本与预算相符。剩余经费3.4339万元，剩余经费计划用于本项目研究后续支出。