飞蝗几丁质酶5两个亚型的基因编辑及功能研究

Chitinases play important roles in degrading chitin in development, molting and peritrophic membrane of Locusta migratoria and the Chitinase 5 of the L. migratoria is encoded by two genes, LmCht5-1 and LmCht5-2. The dsRNA of LmCht5-1 injection in 5th nymph of L. migratoria led to severe molting defects and lethality, while no obvious effects were detected with RNAi of LmCht5-2. Although the two Cht5 genes have the complementary expression pattern through the egg development, the functional differentiation of LmCht5-1 and LmCht5-2 in whole development remains not to be elucidated. In this study, we will employ the recent developed genome editing technology CRISPR/Cas9 system in the functional analysis of Cht5 genes. We will first optimize the Cas9 protein according to the codon bias of L. migratoria and construct CRISPR/Cas9 system for gene editing; then, the specific and high efficient sgRNA for LmCht5-1 and LmCh5-2 will be designed and screened in embryonic cells using surrogator reporter system. Later, the CRISPR/Cas9 gene editing system will be tested and optimized in vivo, and then the selected sgRNA for LmCht5-1 and LmCh5-2 will be injected into the egg of L. migratoria using the optimized system coupled with LmCas9 mRNA, respectively. Finally, we will obtain the LmCht5 knockout L. migratoria to reveal the functional differentiation and compensating effect of LmCht5 by using systematical examination the phenotypes and chitin variation of the LmCht5 knockout L. migratoria. The platform of L. migratoria genome edting technology will facilitate the gene function analysis and lay the basis for applification of chitinases as the target of pest-control.

几丁质酶在昆虫生长发育过程中具有重要作用。飞蝗几丁质酶5有2个亚型（LmCht5-1和LmCht5-2），前期研究表明RNA干扰LmCht5-1可阻断飞蝗5龄若虫的蜕皮发育，而RNA干扰LmCht5-2无明显表型。LmCht5-1和LmCht5-2在飞蝗卵期发育过程中表达模式存在显著差异，它们在飞蝗卵、若虫发育及成虫期的功能尚未系统阐明。本项目拟构建飞蝗密码子优化的CRISPR/Cas9系统，在飞蝗胚胎细胞中筛选靶基因高效的sgRNA，建立飞蝗活体CRISPR/Cas9基因编辑系统；将靶基因sgRNA与LmCas9 mRNA共注射飞蝗受精卵，获得LmCh5-1和/或LmCht5-2敲除的阳性个体；研究LmCht5两个亚型分别在飞蝗卵、若虫发育及成虫期的生理功能及基因补偿效应。在建立飞蝗基因编辑技术的基础上，研究结果将为飞蝗防治的几丁质酶分子靶标提供重要理论依据。

飞蝗是重要的农业害虫，也是研究渐变态昆虫的模式物种之一。飞蝗一生经历卵、若虫和成虫3个发育阶段。飞蝗表皮的周期性蜕皮是研究表皮发育代谢模式及设计生物防治靶标的重要生物学行为依据。飞蝗胚胎发育过程中有4次表皮生成和3次表皮降解过程，分别为浆膜表皮、胚胎第一层表皮、预若虫表皮和1龄若虫表皮。飞蝗几丁质酶5包含LmCht5-1和LmCht5-2两个基因。LmCht5-1在飞蝗预若虫表皮生成和降解时期高表达，定位于表皮细胞、预若虫表皮和部分若虫1龄表皮中，参与预若虫表皮蜕皮时原表皮中几丁质的降解。LmCht5-2在飞蝗浆膜表皮和预若虫表皮降解时均高表达，将其干扰后，引起浆膜表皮和预若虫降解困难而导致孵化失败。LmCht5-1和LmCht5-2早期害虫防治提供新型分子靶标。本课题依据蝗虫密码子偏好性构建高效LmCas9，建立昆虫细胞水平双荧光报告载体靶位点筛选系统，获得dsRNase2基因编辑飞蝗，建立飞蝗CRISPR/Cas9基因编辑技术平台，为飞蝗基因功能研究提供研究平台和实验动物模型。