海参自溶动态基因表达调控网络的构建

Sea cucumber (Stichopus japonicus) possesses strong ability of autolysis, which leads to severe deterioration in its quality during preservation, storage, transport and handling. Our previous studies supported by National Natural Science Foundation of China have demonstrated the regulation mode of cathepsin L and the biological characteristics of its target proteins in the autolysis of sea cucumber from gene expression levels. Furthermore, our previous studies have also indicated that the changes of various kinds of gene expressions are involved in autolysis of sea cucumber. This has provided a new clue to understand the mechanism of sea cucumber, but it also brought new scientific problems, including what kinds of key functional genes do exist in the autolysis of sea cucumber? What are the connections among the key functional genes? Therefore, concerning these two problems, the object of present study is to investigate the process of sea cucumber autolysis in transcriptional levels. Firstly, the dynamic transcriptomes of sea cucumber autolysis are constructed by technology of the molecular biology. The candidate functional genes are obtained according to the functional annotation, clustering and enrichment analysis of differentially expressed genes by Roche 454 high-throughput sequencing and bioinformatics software. Secondly, the key functional genes are obtained candidate functional genes after similarity and homology analysis, verificafion and screening of gene expression. Finally, the dynamic gene expression regulation network is constructed for autolysis of sea cucumber after analysis of enrichment significance and interaction among signaling pathways for key functional genes. Consequently, the studies of this project will provide foundational information for elucidation of the mechanism involved in the autolysis of sea cucumber.

海参具有极强的自溶能力，这给海参的保鲜、贮藏、运输和加工带来诸多不便。前期的青年科学基金项目从基因表达水平出发，明确了组织蛋白酶L对海参自溶的调控方式及靶点蛋白的生物信息。此外，研究还发现，海参自溶过程涉及多种基因表达的改变，这为研究海参自溶机理提供了新的线索，又带来了新的科学问题——海参自溶关键功能基因有哪些？各关键功能基因间有何联系？因此，针对这两个问题，本项目拟从转录水平上研究海参自溶过程。首先，利用分子生物学手段构建海参自溶动态转录组文库，应用Roche454测序技术，结合生物信息学分析软件，对差异表达基因进行功能注释、聚类及富集分析，获得候选功能基因；其次，对候选基因进行相似性、同源性分析，并进行表达验证与筛选，获得关键功能基因。最后，利用KEGG数据库，对关键功能基因进行信号通路富集显著性及相互作用分析，构建海参自溶动态基因表达调控网络，为揭示海参自溶机理奠定研究基础。

海参具有极强的自溶能力，这给海参的保鲜、贮藏、运输和加工带来诸多不便。本项目从转录水平上研究了紫外线诱导的海参自溶过程。首先，以紫外线（58 µW/cm2，照射30 min）为诱导因子，建立海参自溶模型。通过Illumina平台测序，构建海参自溶动态转录组文库。利用生物信息学分析软件，对基因序列进行功能注释、聚类及富集分析，海参基因GO注释结果显示，结合（binding）和细胞过程（cellular process）具有很高的富集显著性。其次，以未经紫外线照射的海参组（I_Sj_BW）为对照，与经紫外线照射的海参组（U_Sj_BW）的表达量进行比较，统计获得表达上调的基因456条，表达下调的基因536条，从而明确了海参体壁自溶中候选功能基因。结合溶酶体和吞噬体相关信号分子，筛选海参自溶关键功能基因21条。针对海参自溶中Cystatin和Actin关键功能基因，研究了海参自溶过程中肌肉层蛋白的降解特性，并探讨了半胱氨酸类蛋白酶的参与机理。针对海参自溶中Collagen关键功能基因，研究了海参胶原蛋白特性，并明确了海参肠内源酶对体壁胶原蛋白的降解作用。最后，利用KEGG数据库，对关键功能基因进行信号通路富集显著性及相互作用分析，构建了以溶酶体通路和吞噬体通路为基础的海参自溶基因表达调控网络。以上研究成果发表SCI/EI收录论文7篇、中文核心期刊论文3篇；国际会议口头报告5次、墙报展示1次，获得中国授权发明专利1件、申报中国发明专利3件，培养硕士研究生8名，为揭示海参自溶机理奠定了研究基础。

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