Yorkie促进家蚕丝腺和翅芽生长的功能及机理研究

The Hippo-Warts-Yorkie signaling pathway coordinately regulates cell proliferation and apoptosis and is now appreciated as a major growth control pathway in vertebrates as well as arthropods, emerging as a hotspot in modern biomedical research. As a key component in this pathway, Yorkie promotes cell proliferation and growth, and inhibits cell death, resulting in growth of organs and organisms in Drosophila and mammals. However, in the silkworm, Bombyx mori, no studies about the Hippo-Warts-Yorkie pathway and Yorkie have been reported yet. In this proposal, we first aim to identify all key genes in the Hippo-Warts-Yorkie pathway in Bombyx by using bioinformatics, and to investigate their developmental profiles and tissue distributions using quantitative real-time PCR and Western blotting. Next, we are going to unravel the physiological function and molecular mechanism about how Yorkie promotes growth and development of silk gland and wing disc in Bombyx, using the loss-of-function (systemic RNAi and knock-out) and gain-of-function (baculovirus-mediated overexpression and the GAL4/UAS transgenic system) methods. Using the GAL4/UAS transgenic system, we have previously shown that Ras1CA overexpression specifically in the Bombyx posterior silk gland results in improved fibroin synthesis and silk yield. Finally, in this project, we will simultaneously overexpress YorkieCA and Ras1CA specifically in the Bombyx posterior silk gland. We expect that co-overexpression of YorkieCA and Ras1CA will further improve fibroin synthesis and silk yield in comparison with Ras1CA overexpression.

Hippo-Warts-Yorkie信号通路通过调控细胞分裂和细胞凋亡进而调控器官和个体的生长，是现代生物医学的研究热点之一。Yorkie作为Hippo-Warts-Yorkie通路中的关键分子，对果蝇和哺乳动物器官和个体的生长具有显著促进作用；但在家蚕中，到目前为止，尚无该通路以及Yorkie的相关研究报导。本项目将对家蚕Hippo-Warts-Yorkie通路的关键基因进行生物信息学分析，并在mRNA水平和蛋白水平上确定它们的时空表达谱；采用系统RNAi和基因敲除等"丧失功能"方法、杆状病毒超表达和GAL4/UAS超表达等"获得功能"方法研究Yorkie促进家蚕丝腺和翅芽生长的生理功能和分子机制；利用GAL4/UAS转基因系统，在家蚕后部丝腺中同时超表达YorkieCA和Ras1CA，期望能在单独超表达Ras1CA的基础上进一步提高蚕丝产量。

Hippo-Warts-Yorkie 信号通路通过调控细胞分裂和细胞凋亡进而调控器官和个体的生长。通过研究我们发现该信号通路基因在家蚕中非常保守，基因芯片和定量PCR分析表明，该通路的基因在进行有丝分裂的组织，像生殖腺，翅芽表达量非常丰富，除了Yorkie，该通路基因在同一种组织中表达模式基本一致，但是在不同的组织中，尤其是翅芽和后部丝腺中，基因的表达模式几乎相反。我们用系统RNAi和杆状病毒超表达的方法研究了Yorkie的功能，研究结果表明当用RNAi的方法干扰该基因的表达，家蚕的发育推迟，产卵量减少；该基因超表达时，家蚕的发育会加快，后部丝腺增大。该研究表明Yorkie对家蚕个体的生长发育有重要的促进作用。利用PiggyBac转基因技术在家蚕的后部丝腺特异性过表达Yorkie的持续激活形式YorkieCA，得到丝腺变大，蚕丝产量提高的可稳定遗传的转基因家蚕品系,进一步提高了蚕丝产量；利用GAL4/UAS转基因系统，在家蚕后部丝腺中同时超表达YorkieCA和Ras1CA，双阳性转基因家蚕的蚕丝产量与野生型相比没有明显的区别，Ras与Yorkie相互抑制使过表达失去作用。