飞蝗翅特异表皮蛋白基因LmACP19和LmACP7差异表达调控机制

Wings are important accessory structure of insects, and cuticular proteins play an important role in the formation of insect wings. According to our previous studies, LmACP19 and LmACP7 are the cuticular protein genes expressed specifically in wings that are involved in the formation of endocuticle and exocuticle in Locusta migratoria, respectively. Silencing them would be result in abnormal development of wings. Among them, LmACP19 has a higher expression level at post-ecdysial, whereas LmACP7 at pre-ecdysial, which is relate to ecdysone (20E) signal. However, the mechanism of such a differential expression is still unclear. This project will focus on the regulatory mechanism of the two wing specific protein genes, LmACP19 and LmACP7, during the formation of the endocuticle and exocuticle of L. migratoria. Our research will mainly focus on: 1) Based on the screened the key transcription factor LmBTB that induced by 20E, to explore its function in the formation of wing cuticle; 2) identifying its interacting protein and analyzing differential expression mechanisms of LmACP19 and LmACP7 regulated by LmBTB and its interacting protein from individuals and cells level by using dual-luciferase reporter system, electrophoretic mobility shift assay, ChIP-qPCR and RNAi assays; 3) revealing the regulatory of differential expression of LmACP19 and LmACP7 as mediated by 20E signal by using LmBTB. Our study is expected to contribute to better understanding of the regulatory mechanisms in the formation of endocuticle and exocuticle as mediated by ecdysone in insects wing, and provides theoretical basis for the effective control of migrant pests through regulating wings formation.

翅是昆虫重要的附属结构，表皮蛋白在翅形成中具有重要作用。项目前期研究表明，飞蝗翅特异表皮蛋白基因LmACP19和LmACP7分别参与翅内、外表皮形成，沉默后均导致翅发育异常。LmACP19在蜕皮后高表达，LmACP7在蜕皮前高表达，其表达模式与蜕皮激素信号（20E）相关，但其差异表达调控机制尚不清楚。本项目拟围绕这一科学问题，开展如下研究工作：1）根据已获得的受20E诱导的转录因子LmBTB，利用RNAi结合透射电镜技术探讨其在飞蝗翅表皮形成中的功能；2）鉴定LmBTB相互作用蛋白，利用双荧光素酶报告系统、凝胶阻滞、染色质免疫沉淀、RNAi等方法在个体和细胞水平解析其对LmACP19和LmACP7差异表达调控机制；3）明确LmBTB介导的20E信号对LmACP19和LmACP7差异表达调控作用。研究结果可揭示昆虫翅内外表皮蛋白的差异表达调控机制，为基于调控翅形成进行害虫防控提供科学依据。

翅是昆虫重要的附属结构，表皮蛋白在翅的形成和发育中具有重要作用。项目前期研究发现，飞蝗翅特异表皮蛋白基因LmACP19和LmACP7分别参与翅内、外表皮形成，沉默后均导致翅发育异常。LmACP19在蜕皮后高表达，LmACP7在蜕皮前高表达，其表达模式与蜕皮激素信号（20E）相关，但，但其具体作用机制及其差异表达调控机制尚不清楚。本项目围绕这一科学问题，开展了如下研究内容：1）根据已获得的受20E诱导的转录因子LmBTB，利用RNAi结合透射电镜技术探讨其在飞蝗翅表皮形成中的功能；2）解析了LmACP19和LmACP7在飞蝗翅发育中的功能及作用机制；3）明确LmBTB介导的20E信号对LmACP19和LmACP7差异表达调控作用。. 主要取得如下结果：1）基于课题组转录组数据库和基因组数据库，鉴定了BTB家族因子并将其命名为LmBTBD6。表达分析发现该基因主要在飞蝗翅芽中高表达并响应20E信号通路调控。生物学功能研究发现，LmBTBD6主要参与若虫到成虫转变过程中翅的发育，LmBTBD6的表达缺失导致成虫翅发育异常，超微结构观察发现翅表皮结构变薄。2）为了探讨翅表皮蛋白的功能，首先从显微和超微结构观测了若虫到成虫翅发育中的结构变化和内外表皮形成过程，为研究翅特异表皮蛋白基因LmACP19和LmACP7的生物学功能奠定了基础。然后采用免疫组化和免疫胶体金方法发现LmACP7主要定位于翅的外表皮，而LmACP19的定位不同于LmACP7，主要在细胞层中。LmACP19和LmACP7的表达缺失均能导致细胞凋亡，且翅内表皮变薄引起翅发育畸形。3）沉默20E受体EcR和核受体因子LmHR39后，LmBTBD6表达显著下调，表明LmBTBD6响应LmHR39介导的20E诱导表达。进一步研究发现，沉默LmBTBD6后，翅特异表皮蛋白基因LmACP7表达量降低，而翅表皮蛋白基因LmACP19表达升高，表明LmBTBD6调控翅表皮蛋白基因LmACP7和LmACP19差异表达，且LmHR39-LmBTBD6能够介导蜕皮激素信号对飞蝗翅发育的调控。研究结果揭示了渐变态昆虫--飞蝗翅内外表皮蛋白基因在翅形成中作用及其差异表达调控机制，为基于调控翅形成进行害虫防控提供了科学依据。