坛紫菜高温胁迫应答的定量差异蛋白质组学研究

Porphyra haitanensis is an economically important marine crop in China. A major problem in Porphyra farming is thallus decay in response to high temperature stress. Molecular studies of temperature stress can help to resolve the issue of thallus decay. In this study, combined with multi-dimensional liquid chromatography and tandem mass spectrometry, the technology of isobaric tag for relative and absolute quantitation (Itraq) of proteomics will be used to analyze the expression patterns of proteins in a high-temperature tolerant line of P. haitanensis when cultured under both high-temperature stress and normal temperatures. Based on the data of this quantitatively comparative proteomic study, we will construct expression profiles of the entire set of proteins, select the specific proteins related to high temperature stress, and construct an interaction network of all the differentially expressed proteins. We will also clone the full-length genes of key high temperature stress-specific proteins and analyze their dynamic expression under high-temperature stress over time by quantitative PCR. All the proteomics data will be collated with the transcriptome data to analyze the molecular mechanisms and regulatory networks of P. haitanensis in response to high-temperature stress. The transcriptome data have been obtained from the same line of P. haitanensis, cultured under both high-temperature stress and normal temperatures. We hope to show the principles of high-temperature tolerance in P. haitanensis, and present new genes which have important functions in this regard. These results will create a foundation for a breeding profile for P. haitanensis in relation to stress tolerance.

阐明坛紫菜高温胁迫应答的分子机制是借助分子生物学手段解决坛紫菜"高温烂菜"问题的基础。本研究拟以坛紫菜耐高温品系为材料，通过iTRAQ技术，结合多维液相色谱和串联质谱分析，构建坛紫菜应答高温胁迫不同时间水平的差异蛋白表达谱，筛选与坛紫菜高温胁迫应答相关的蛋白质，构建蛋白互作网络图；同时克隆高温胁迫应答关键蛋白的编码基因，并对各编码基因在高温胁迫不同时间水平下的动态表达规律进行实时荧光定量PCR分析；最后综合以上蛋白质组数据同坛紫菜应答高温胁迫的转录组数据进行关联分析，从转录组和蛋白质组两个水平共同解析坛紫菜应答高温胁迫的分子机制和调控网络。以期初步揭示坛紫菜的高温胁迫抗性机理，获得一批对坛紫菜耐高温性状表达有重要功能或调控作用、并有重要应用前景的新基因，为今后应用基因工程技术进行海藻抗逆育种提供理论指导和有自主知识产权的重要功能基因。

坛紫菜是我国南方沿海重要的水产养殖种类，产量占全国紫菜总产量的75%以上。水温是影响坛紫菜产量和质量的重要环境因子。近年来，随着全球气候变暖，高水温严重影响了坛紫菜生产的正常开展，“高温烂菜”频繁发生。为阐明坛紫菜高温胁迫应答的分子机制，以解决坛紫菜的“高温烂菜”。本研究首次构建了坛紫菜基因表达的序列数据库，并以坛紫菜耐高温品系Z-61为材料，采用RNA-seq技术和iTRAQ技术，构建了坛紫菜应答高温胁迫不同时间水平的基因和蛋白差异表达谱，筛选出了高温胁迫应答的关键蛋白；克隆获得了39条关键蛋白的编码基因，并摸清了这些基因在高温胁迫下表达特征；最后，综合以上蛋白质组学和转录组学的分析结果，解析了坛紫菜应答高温胁迫的分子调控网络，初步摸清了坛紫菜对高温胁迫应答的分子机制，获得了一批对坛紫菜耐高温性状表达有重要功能或调控作用、并有重要应用前景的新基因，为今后应用基因工程技术进行海藻抗逆育种提供了理论指导和有自主知识产权的重要功能基因。发表论文17篇（其中SCI收录9篇），申报发明专利4项（其中1项已获授权），获国家级水产新品种证书1项，培养研究生7名。