RNAi介导的舞毒蛾JHEH基因功能及代谢调控机制研究

The gypsy moth, Lymantria dispar, is a key lepidopteran species that has been estimated to colonize at least 300 different forest species worldwide. Juvenile hormone (JH) as a group of sesquiterpenoids biosynthesized by the corpora allata is an important insect hormone in the haemolymph that regulates insect larval development, reproduction, and metamorphosis. Juvenile hormone epoxide hydrolase (JHEH) is an important enzyme in the degradation pathways of juvenile hormone (JH) in insects. JHEH titers regulate the entire process of insect development. Now, we had cloned a JHEH gene of gypsy moth. The transcriptional profiles of JHEH were detected by qRT-PCR and found that it was expressed throughout all stages. JHEH gene was successfully knocked down by dsRNA in the instar larvae. In order to known the function of JHEH in the JH degradation and JH signaling pathway. In this project, we will conduct the following studies to explore the functions of JHEH gene in gypsy moth including (1) the specific expression of JHEH in gypsy moth; (2) the effects of silencing JHEH gene on the development of gypsy moth; (3) The synergy of JHEH, JHE and JHDK in JH degradation; (4) characterization of JH signal transduction of JHEH in gypsy moth; (5) the contents of juvenile hormone and molting hormone are determinated by HPLC. These results will demonstrate physiological functions of JHEH gene in JH degradation, JH signal transduction and wing form of gypsy moth. In addition, it will provide a new way for formulate new pesticide based on finding key genes participated in important physiological function.

保幼激素(juvenile hormone,JH)是一种倍半萜类衍生物。保幼激素环氧化物水解酶（JHEH）作为保幼激素降解的的酶类，在昆虫的整个生长发育过程中起重要作用。前期我们克隆获得舞毒蛾JHEH基因，其在舞毒蛾不同虫态均有表达差异，通过喂食JHEH的dsRNA证实能够显著降低JHEH基因的表达量。围绕JHEH如何参与保幼激素的代谢与信号传导这一科学问题，通过分子生物学相关技术，将开展如下研究内容:（1）舞毒蛾JHEH基因特异性表达（2）JHEH对舞毒蛾生长发育影响（3）在保幼激素代谢中JHEH、JHE和JHDK三者间的协同作用（4）JH信号转导通路分析（5）保幼激素及蜕皮激素含量测定。通过以上研究阐明舞毒蛾JHEH基因重要生理功能，说明JHEH基因在舞毒蛾幼虫体内保幼激素代谢、信号转导及成虫翅型分化中参与的激素代谢调节机制，为新杀虫剂的创制研究提供了一个潜在的有价值的新靶标。

昆虫体内保幼激素（JH）对昆虫的生长发育具有重要的调控作用，而Jheh为JH代谢途径中的代谢基因，探索Jheh基因功能和与相关基因的关系对明确昆虫生理与寻找杀虫作用靶标具有重要意义。.本研究基于舞毒蛾转录组数据库，从舞毒蛾体内克隆获得三条JH降解酶基因Jhe、Jheh与Jhdk，并对其进行了相关的注释。使用qPCR检测了Jhe、Jheh与Jhdk在舞毒蛾不同发育阶段的时间表达模式，并使用ELISA法检测了舞毒蛾在不同发育阶段中JHIII滴度的变化；最后通过注射法对舞毒蛾六龄幼虫进行Jheh基因的RNAi实验，并验证其对Jhe、Jhdk及JH信号通路基因Kr-h1基因的影响，分析JHIII滴度对昆虫化蛹及其生理变化的影响。.主要研究结果如下：. 1.舞毒蛾JH降解酶基因的克隆与分析.基于舞毒蛾转录组数据库，运用RT-PCR技术克隆获得JH代谢途径中三个降解酶基因Jhe、Jheh与Jhdk的ORF,推导出其编码的氨基酸序列，进而使用ClustalW2、ProtParam、ProtScale以及TMHMM等生信软件分析了三个JH降解酶基因的蛋白质保守序列、核心结构域、跨膜结构等潜在生理功能。. 2.JH降解酶基因与JHIII在不同发育阶段的表达模式.Jhe在舞毒蛾三龄至五龄幼虫中表达量呈先下调后上调的趋势，在每龄幼虫的初期与末期表达量都较高，这与JHIII滴度的变化趋势相一致，但时间上JHIII的表达呈现明显的滞后性，说明在舞毒蛾幼虫中Jhe对JHIII具有一定的调控作用。Jhdk与Jheh基因在每龄期中均呈现先上调后下调的表达趋势，而JHIII滴度在三龄至五龄幼虫中先降后升，二者的变化趋势相反，JH信号对Jheh与Jhdk的表达产生负向调控。蛹期JHIII滴度水平较低，与Jheh与Jhdk低表达保持一致。成虫期JHIII滴度较低可能与Jhe高表达有关。. 3.Jheh基因沉默对舞毒蛾六龄幼虫发育的影响.RNAi介导的Jheh基因沉默显著降低了Jheh基因在转录水平上的表达量，导致了Jhe基因在24 h时出现上调，jhdk基因在36 h时出现显著下调，JH含量指示基因Kr-h1表现为转录水平上的上调，JH滴度增加。