凡纳滨对虾TOR信号通路及其重要成员的作用机制研究

The signaling network anchored by the protein kinase TOR (target of rapamycin signaling) in eukaryotes impacts most major cellular functions, giving it an outsized role in regulating basic cell behaviors such as growth (mass accumulation) and proliferation. TOR signaling pathways in model organisms have been well studied, but the work concerning the TOR network in crustaceans has not yet commenced. In this work, the TOR signaling pathway of Pacific white shrimp (Litopenaeus vannamei), the most widely cultured shrimp in China will be studied. Most corresponding protein factors which has been found in TOR signaling pathway of model organisms or new components will be identified and analyzed in L. vannamei. Some important proteins will be selected and conduct further studies for their functional roles in pathway. . First, an overall analysis of all components of the TOR signaling pathway will be performed by using transcriptome sequencing technology. Then, the digital gene expression tag profiling (DGE) will be conducted following an injection of rapamycin to find out genes whose expression responds to TOR inhibition. Both fine characterized genes and new genes will be identified in this section. The phosphorylation of the proteins in the pathway will also be analyzed by western-blotting. Proteins responding to rapamycin injection by changing protein structure and activity will be found out. Based on the overall understanding of the TOR signaling pathway, some important genes will be selected to perform the RNAi experiments. The roles of the gene in regulating protein phosphorylation, gene expression and cell growth will be studied after the injection of dsRNA. Because many proteins involved in the TOR pathway form complexes or interact with other factor to exercise their functions, the yeast two-hybrid experiments will be carried out to verify the interactions between proteins. . In summary, the primary goal of the present study is to draw a comprehensive picture of the TOR signaling pathway of Pacific white shrimp, and give deeper comprehension of the roles of major components in this pathway. Results of this work will contribute much to our understanding of the pathway in growth control of crustacean. Genes involved in controlling growth, metabolism, and reproduction can be indicators for measuring and guiding feed design or breeding. Furthermore, combined with the application of genetic engineering technology to aquatic animals, manipulation of animal growth and body size can be achieved by controlling certain signaling pathways.

TOR信号途径（target of rapamycin signaling）是普遍存在于真核生物中的信号传导通路，主要参与细胞生长与分化以及营养代谢的调控。本研究以凡纳滨对虾为研究对象，首先采用转录组测序总体分析对虾中参与TOR信号通路的各种蛋白因子，然后根据纳巴霉素（rapamycin）刺激下，肌肉组织基因表达谱的差异和主要蛋白因子的磷酸化水平差异，分析已知基因的作用方式，并发掘参与TOR信号转导的新基因。在对TOR信号通路有整体认识的基础上，选择关键基因克隆其cDNA全长，并结合采用RNA干扰和酵母双杂交技术对基因的功能进行深入分析。通过本研究的开展，将对凡纳滨对虾的TOR信号通路进行全面系统的阐述，并对该通路中部分基因和编码蛋白的功能进行深入解析，对于进一步深入阐明对虾生长代谢调控机制奠定坚实的理论基础。

mTOR信号通路（target of rapamycin signaling）是普遍存在于真核生物中的信号传导通路，主要参与细胞生长与分化以及营养代谢的调控。. 本研究通过对凡纳滨对虾进行转录组测序，并结合模式生物中mTOR信号通路的已有研究结果，分析获得了33个可能参与凡纳滨对虾mTOR信号通路调控作用的基因片段。在此基础上，克隆了mTOR信号通路中4种基因的cDNA全长序列：raptor，eif4e2，eif4e1a和Rheb，并采用荧光定量PCR和Western杂交技术研究了其在对虾应答营养素刺激和生长抑制剂雷帕霉素刺激中的作用机制。通过RNA干扰技术研究了raptor基因抑制对mTOR信号通路的影响。结果表明凡纳滨对虾mTOR信号通路对雷帕霉素敏感，且可以接收氨基酸信号调控细胞生长。. 采用转录组测序技术分析了凡纳滨对虾肌肉组织中对雷帕霉素刺激做出反应的差异表达基因，发现与细胞自噬作用相关的大量基因对mTOR信号通路抑制反应灵敏。为进一步确定这些基因在细胞自噬中的作用机理，采用Western杂交技术研究了自噬过程相关蛋白含量和磷酸化水平的变化。结果表明对虾mTOR信号通路可通过调控细胞自噬作用调控细胞的生长代谢平衡。. 模式生物中大量的研究结果表明机体内存在复杂的信号调控通路参与细胞生长和增殖的调控，但是这方面的调控机制在甲壳动物中所知甚少。本项目的研究成果不仅为mTOR信号通路的系统发生研究提供了参考，从分子水平了解对虾的生长和代谢调控机制，对于对虾良种培育以及饲料配方的优化具有重要的指导意义。