蛋白泛素化修饰异常与家蚕裸蛹的关系研究

Silk yield is one of the most concerned scientific issues in sericulture production. The clarifying of the molecular mechanism underlying the silk production will provide the theoretical guidance in breeding of the high production strains, development of silkworm gland bioreactor and improving the economic benefits of sericulture production..The expression and regulation of silk gene in mutated strain with nude pupa (Nd) is specific and it will be an excellent experimental material to investigate the regulation mechanism underlying the silk yield..Years of investigation results from proteome and transcriptome data in our lab showed that the proteasome pathway was significantly enhanced in silkworm with nude pupa comparing with the normal strain. We drew a hypothesis based on these data: the mutated fibroin heavy chain gene of nude pupa can promote the enhancing of the proteasome pathway in the posterior silk gland and thus triggering the abnormality of ubiquitination modification of the whole posterior silk gland which led to the nude pupa. .The project intends to use the gene editing technology of TALEN and CRISPR-Cas to knock out the endogenous fibroin heavy chain gene and obtain the mutated strains. And then the comparative proteomics research of ubiquitination modification will be performed in posterior silk gland of mutated strains, nude pupa, Qiufeng, and nude pupa × Qiufeng hybrids to explain the causes of dramatically increased proteasome pathway and clarify the molecular mechanism underlying the nude pupa formed.

蚕丝产量是蚕桑生产最关注的科学问题之一，阐明蚕丝产量分子调控机理，可以为培育蚕丝高产品种、开发高效家蚕丝腺生物反应器、提高蚕桑生产经济效益提供理论指导。.蚕丝突变品种裸蛹（Nd）的蚕丝蛋白基因表达调控具有独特性，是研究蚕丝产量分子调控机理的绝佳材料。.我们多年的家蚕蛋白质组和转录组的研究结果表明，具有裸蛹性状特性的家蚕后部丝腺细胞的蛋白酶体通路极显著高于正常品种。据此我们得出一个假设：裸蛹突变的丝素重链基因会促进家蚕后部丝腺蛋白酶体通路代谢水平极显著地提高，从而引发家蚕后部丝腺整体性的蛋白泛素化修饰异常，最终导致裸蛹。.本项目拟采用TALEN、CRISPR/Cas9基因编辑技术，突变家蚕内源性丝素重链基因，获得突变品种。采用比较蛋白质组学技术分析突变品种、裸蛹、秋丰、及裸蛹×秋丰杂交一代家蚕品种后部丝腺的蛋白泛素化修饰差异，揭示蛋白酶体通路代谢水平提高的原因，阐明裸蛹品种形成的分子机理。

蚕丝产量是蚕桑生产最关注的科学问题之一，阐明蚕丝产量分子调控机理，不尽有利于培育蚕丝高产品种，更有利于开发高效家蚕丝腺生物反应器。.蚕丝突变品种裸蛹（Nd）的蚕丝蛋白基因表达调控具有独特性，是研究蚕丝产量分子调控机理的绝佳材料。.我们从多年的家蚕蛋白质组和转录组的研究结果中发现裸蛹性状特性的家蚕后部丝腺细胞的蛋白酶体通路极显著高于正常品种，据此我们提出一个假设：裸蛹突变的丝素重链基因会促进家蚕后部丝腺蛋白酶体通路代谢水平极显著地提高，从而引发家蚕后部丝腺整体性的蛋白泛素化修饰异常，最终导致裸蛹。.本项目采用CRISPR/Cas9基因编辑技术，突变了家蚕内源性丝素重链、轻链、P25基因、以及丝胶基因，采用转录组学、比较蛋白质组学技术分析了突变品种、裸蛹、秋丰、及裸蛹×秋丰杂交一代家蚕品种后部丝腺的蛋白泛素化修饰差异，基因转录水平差异、蛋白质组差异、lncRNA和miRNA组差异，初步证明了裸蛹（Nd）蚕后部丝腺中大量突变的丝素重链蛋白诱发了蛋白酶体通路代谢水平异常的提高，造成了蛋白酶体通路代谢对丝腺内生长发育必须的正常蛋白质的非特异性降解，破坏了丝腺的正常生长，造成丝腺短小，最终成为裸蛹品种。.通过对裸蛹形成机理的研究，了解了蚕丝产量的基因表达调控机理，了解了丝素重链基因、丝素轻链基因纯合突变体容易致死，揭示了要开发高效家蚕丝腺生物反应器，可以以丝素P25基因、丝胶1 基因纯合突变体作为转基因实验受体，或者先获得外源转基因家蚕，再基因编辑内源的丝素重链基因、丝素轻链基因。为利用家蚕丝腺生物反应器高效表达外源基因奠定了理论基础。.项目已经获得了表达蜘蛛梨状丝、聚状丝、葡萄状丝、多聚牵引丝、藤壶粘丝等近10种外源基因的家蚕丝腺生物反应器，计划进一步结合研究获得的内源丝蛋白基因突变家蚕品种，培养高效表达丝蛋白纤维的转基因家蚕品种，创制特殊性能仿生丝纤维材料，为国防安全建设服务。