激素和胰岛素信号调控家蚕幼虫丝腺细胞核内有丝分裂的机制研究

Silkworm is an important economic insect, and the main economic value of silkworm is the silk production. The basis of silk protein synthesis is endomitosis of silk gland cells in silkworm. Because the mechanism of endomitosis in silk gland cells is unclear, the high yield breeding process of silkworm were hampered. In preliminary studies we found that the endomitotic DNA synthesis of silk gland cells was increased during the vigorous feeding phase and gradually inhibited in the next molting phase, suggested nutrition and hormones may be related to the regulation of endomitosis in silk gland cells. We will use BrdU labeled technology and in vitro culture technology to identify the regulation of nutrition and hormones on endomitosis in silk gland cells; we will use western blot method to clarify the regulation of insulin signal pathway in the silk gland; Using the iTraq technology, we will screen the difference protein in the silk glands that the silk gland cells DNA replication level is different, which verify whether there are changes of major genes of insulin signal pathway in the development and screen the key genes of regulation of endomitosis in silk gland cells; using transgenic technology, we will overexpress and interfere with the key gene and elucidate the molecular mechanism of it. This project may identify whether endomitosis in silk gland cells is regulated by nutrition and hormones; and lay the foundation for the further investigation of the mechanism underlying efficient endomitosis in silk gland cells and for cultivation of high yield varieties of silkworm.

家蚕是重要的经济昆虫，它的主要的经济价值是产丝。家蚕丝蛋白合成的基础是幼虫丝腺细胞高效核内有丝分裂，由于其机制尚不清楚，阻碍了高产丝家蚕新品种的培育进程。在前期研究中发现丝腺细胞DNA复制在幼虫蜕皮期被抑制，在蜕皮间期被大量激发，暗示丝腺细胞核内有丝分裂可能与营养和激素有关。本项目拟采用BrdU标记和离体培养技术，鉴定营养和激素是否调控丝腺细胞核内有丝分裂；通过免疫印迹，阐明胰岛素信号在丝腺组织中参与调控的路径；采用定量蛋白组学iTRaq技术，鉴定丝腺细胞DNA复制程度不同时期丝腺组织的差异蛋白，分析筛选调控丝腺细胞核内有丝分裂的关键基因；通过转基因技术，在丝腺中特异过表达或干涉关键基因，阐明该基因调控丝腺细胞核内有丝分裂的分子机制。通过本项目研究，可明确营养、激素和胰岛素信号是否调控家蚕丝腺细胞核内有丝分裂，可为解析家蚕丝腺细胞高效核内有丝分裂调控机制、加快高产丝家蚕新品种培育奠定基础。

家蚕是重要的泌丝经济昆虫，丝腺是泌丝器官，丝腺细胞高效的核内有丝分裂是家蚕丝蛋白生物合成的前提，解析丝腺细胞高效核内有丝分裂的机制可为家蚕高产丝新品种培育提供依据，具有极大的理论意义和应用价值。本项目围绕“激素和胰岛素信号调控家蚕幼虫丝腺细胞核内有丝分裂的机制研究”这一科学问题，将家蚕丝腺组织作为研究对象，取得了突破性的实验进展。主要包括：明确了家蚕在生长发育过程中幼虫丝腺细胞DNA复制情况，表明在家蚕幼虫期丝腺细胞DNA复制呈波动趋势，在刚蜕皮起蚕时DNA复制有自激发现象，之后DNA复制的激发不断增强，到盛食期达到最高，之后随着眠期的临近DNA复制程度逐渐减少，到家蚕幼虫眠期DNA复制程度达到最低，到五龄三天时丝腺细胞DNA复制大量被激发，而且在五龄时丝腺细胞DNA含量明显增多且细胞体积显著增大；说明了营养对丝腺细胞DNA复制至关重要；阐明了胰岛素能够激发丝腺细胞的DNA复制，蜕皮激素抑制丝腺细胞的DNA复制，而保幼激素对丝腺细胞的DNA复制没有显著影响；揭示了胰岛素信号通路参与调控丝腺细胞核内有丝分裂的路径，PI3K信号通路，TOR信号通路和ERK信号通路都参与了家蚕幼虫丝腺细胞DNA复制的调控；解析了胰岛素和蜕皮激素调控细胞周期的作用机制，胰岛素使得细胞周期短暂阻滞到G0/G1期并且能够促进DNA的复制，而蜕皮激素使得细胞周期阻滞到G2/M期，并且抑制DNA的复制；筛选出调控家蚕丝腺细胞核内有丝分裂的关键基因BmFoxO并进行了克隆鉴定及功能解析，过表达BmFoxO使得细胞周期阻滞到G0/G1期，并且抑制DNA复制，敲除BmFoxO使得细胞周期阻滞到G2/M期，并且促进DNA复制；解析BmFoxO调控细胞周期的分子机制，过表达BmFoxOCA能够诱导内源BmCyclinB的入核，并且过表达BmFoxOCA能够显著上调BmCyclinB的转录水平和蛋白水平。揭示了BmFoxO的共同调控因子BmCyclinB、BmCDK1、BmCDK10；以上研究结果不仅侧面验证了蜕皮激素、饥饿和胰岛素信号通路对家蚕丝腺细胞核内DNA复制的调控，也为进一步研究丝腺细胞高效核内有丝分裂调控机制奠定基础。