马铃薯甲虫蜕皮激素合成、分解及蜕皮时的信号转导途径研究

In the Colorado potato beetle, L. decemlineata, the moulting hormone (JH) orchestrates ecdysis and metamorphosis of larvae, adult reproduction, and overwintering diapause of the adults. cDNA fragments from 12 genes putatively associated with ecdysteriod hormone biosynthesis, metabolism and signaling transductions have been previously identified. Among them were putative MH biosynthesis enzymes encoded by Halloween genes such as cyp306a1, cyp307a1, cyp302a1,cyp314a1 and cyp315a1，MH catabolism related gene cyp18a1, signaling pathway involving genes such as SRC and BR，and the genes resposible to MH signaling pathway such as E75,HR3,HR4 and FTZ-F1. A dsRNA-produced vector was constructed and transferred to Escherichia coli HT115. The engineered bacterium was cultured, and a great amount of dsRNA derived from cyp18a1,cyp306a1,cyp307a1,cyp302a1,cyp314a1 and cyp315a1 was obtained. The bacteria liquid contained each of these dsRNAs caused larval mortality, reduced larval weight, decreased pupation ratio and affected development when dietaryly introduced to 2nd-instar larvae. The purpose of the present scientific plan is to clone the 12 genes encoding CYP18A1,CYP306A1,CYP307A1,CYP302A1,CYP314A1,CYP315A1,SRC, BR, E75,HR3,HR4 and FTZ-F1 by RACE. The physiological functions are confirmed in vivo by RNAi and rescue experiments with MH, MH analogs halofenozide, and MH antagonist. Moreover, we will obtain pure enzymes of CYP18A1,CYP306A1,CYP307A1,CYP302A1,CYP314A1 and CYP315A1 by expressed their genes in Sf9 cell line, cultured the cells and obtained the proteins, and will perform in vitro bioassay to test the activities, substrate specifications and optimal reactive conditions of these enzymes. In addition, prothoracic gland are isolated, cultured in vitro and treated with different dsRNAs, or different enzyme inhibitors. The biosynthetic activities for MH of these prothoracic glands are compared in order to further define the physiological function of the enzyme CYP306A1,CYP307A1,CYP302A1,and CYP315A1.

马铃薯甲虫是新疆重要的马铃薯害虫，持续扩散能力强，入侵其它马铃薯产区的风险大，严重威胁我国马铃薯生产。蜕皮激素(MH)调控马铃薯甲虫蜕皮与发育、生殖系统成熟和滞育3种生理过程，决定马铃薯甲虫生活史、后代数量和空间分布。我们已鉴定了12种MH合成、代谢和信号转导相关的基因片段；开发了发酵法生产dsRNA的技术；发现喂食MH合成与分解相关基因的dsRNA后，严重影响马铃薯甲虫的存活率、存活个体体重和化蛹率。本项目拟克隆这12个基因的cDNA全长；通过RNA干扰、活体与离体分析、真核表达技术验证这些蛋白的功能；明确MH调控幼虫蜕皮的信号转导途径。研究结果可补充、完善和丰富MH信号产生和转导的机理，开拓广阔的后续研究领域；也可揭示马铃薯甲虫生活史的薄弱环节，制订出针对性强、效果佳的综合配套治理措施；还可作为农药开发新靶标和高通量快速筛选生长调节剂农药的检测分子。具有重要理论意义和潜在应用价值

马铃薯甲虫是新疆重要的马铃薯害虫，持续扩散能力强，入侵其它马铃薯产区的风险大，严重威胁我国马铃薯生产。蜕皮激素(MH)调控马铃薯甲虫幼虫发育、生殖系统发育与成熟、滞育和飞行肌的解离和再生4种生理过程，决定马铃薯甲虫生活史、后代数量和空间分布。我们已克隆24种MH合成、代谢和信号转导相关蛋白的可能编码基因片段；开发了发酵法生产dsRNA的技术；发现喂食2种MH降解相关基因的dsRNA后，幼虫生长缓慢，蜕皮受阻。在此基础上，本项目拟深入研究12个关键基因。克隆它们的全长；验证其编码蛋白的生理学功能；明确这些蛋白相互作用调控幼虫蜕皮的分子机理。研究结果可补充和完善MH信号产生和转导机理，开拓广阔的后续研究领域；也有助于揭示幼虫发育的敏感时期，制订出针对性强、效果佳的综合配套治理措施；还可作为农药开发新靶标和高通量快速筛选生长调节剂农药的检测分子。具有重要理论意义和潜在应用价值。