家蚕丝腺中BR-C转录因子互作蛋白的鉴定及功能研究

In insects, transcription factors Broad-Complex (BR-C), containing two conserved domains of protein-protein interaction domain BTB and C2H2 zinc-finger domain, contribute to the cross-talk between 20-hydroxyecdysone (20E) and juvenile hormone (JH), two hormones that coordinately orchestrate insect growth and development. Previous studies about insect BR-C mainly focused on its response to 20E and JH as well as roles on the transcriptional regulation of downstream targets. But, its interacting proteins have not identified yet. In silkworm, several recognized motifs for BR-C were predicted from the upstream genomic region of transcriptional initiation site of genes encoding silk proteins and regulators related to silk proteins biosynthesis, suggesting that BR-C may involve in regulating the biosynthesis of silk proteins in silkworm silk gland. But, no direct experimental evidence is found to support this hypothesis...In this application, we propose to identify the proteins that interact with transcription factor BR-C in silkworm silk gland and further characterize its roles. First, we will take advantage of the powerful tools of yeast two-hybrid system screens, BiFC and Co-IP to identify the interacting proteins of BR-C in silk gland. Second aim is to determine how BR-C and its interacting proteins coordinately respond to 20E and/or JH. Finally, it is necessary to decipher the roles of BR-C and its interacting proteins in the development of silk glands and transcription of silk protein genes based on the methods of transient RNAi, transgenic RNAi, transgenic overexpression, luciferase reporter gene assay, EMSA, and so on. ..We expect that the results from these studies will not only provide new insights into the pivotal role of the interaction between BR-C and its interacting proteins in orchestrating 20E and/or JH signaling in silkworm and other insects, but also help us to understand the roles of BR-C mediated 20E and/or JH signaling on the silk gland development and silk protein biosynthesis in silkworm as well as to get new cues for genetically improving the yields and qualities of silk production.

昆虫BR-C是含BTB蛋白结合结构域和锌指结构域的转录因子，其主要介导了蜕皮激素（20E）和保幼激素（JH）信号通路间的相互作用（Cross-talk）。以往对昆虫BR-C的研究集中于BR-C如何调控下游基因转录，而有关其互作蛋白的鉴定仍是空白。而在家蚕中，BR-C还可能参与了对家蚕丝蛋白合成的调节，但也没有直接的实验证据。鉴于此，本项目拟以家蚕BR-C为靶标，利用酵母双杂交文库扫描、BiFC和Co-IP等技术鉴定家蚕丝腺中与BR-C蛋白互作的蛋白，并应用瞬时RNAi、转基因RNAi及过表达、荧光素酶报告基因分析、EMSA等技术，系统分析BR-C及其互作蛋白如何协同应答20E和JH信号并调控丝腺发育及丝蛋白基因的表达。相关研究结果不仅可以完善家蚕及昆20E和JH的信号传导机制，而且还有助于揭示家蚕丝腺生长发育及丝蛋白合成的激素调节机理，从而为家蚕茧丝经济性状的分子改良提供新的线索。

昆虫BR-C转录因子包含2个负责转录调控的锌指结构域和1个负责蛋白互作的BTB结构域，其主要参与蜕皮激素（20E）和保幼激素（JH）通路的信号传导。以往对昆虫BR-C的研究集中于BR-C如何调控下游基因转录，而有关其互作蛋白的鉴定及翻译后修饰仍是空白。本项目旨在鉴定BR-C的相互作用蛋白并解析其在BR-C介导激素信号传导中的作用。围绕项目研究计划，我们首先从家蚕基因组中鉴定了56个含BTB结构域的基因，而BTB-BACK-PHR、BTB-BACK-Kelch和BTB-FLYWCH等亚家族BTB基因在昆虫进化过程中发生了复制扩张。其次，我们以家蚕BR-C的BTB结构域为诱饵，通过酵母双杂交筛选发现蛋白激酶C（PKC）信号通路的锚定蛋白RACK1能与BR-C发生蛋白互作，一系列生化实验证实两者的互作介导了BR-C的PKC磷酸化并促使其入核。另外，通过液相色谱-串联质谱（LC-MS/MS）分析发现，家蚕BR-C的S186残基是PKA的磷酸化修饰位点；后续的实验证实PKA对BR-C的磷酸化抑制了BR-C对丝胶蛋白基因Ser1及表皮蛋白基因WCP10等下游靶基因的转录调控作用，并发现20E能通过抑制cAMP/PKA信号通路来抑制BR-C的磷酸化。此外，我们不仅发现20E能促进家蚕丝腺DNA复制相关基因的转录，并可引起丝腺细胞发生凋亡，而且证实有丝分裂关键调控因子AuroraB激酶在家蚕细胞周期中的作用。这些结果不仅为完善昆虫内分泌激素信号转导通路奠定了基础，也为阐明内分泌激素在调控家蚕丝腺发育及丝蛋白基因转录中的作用机制提供了新的理论支撑。