香港牡蛎Nrf2基因的克隆及其在高温抗逆中的功能研究

Hong Kong oyster, Crassostrea hongkongesis, is one of the most economically important shellfish in South China Sea. As sessible animals living in subtropical estuaries and intertidal zones, the Hong Kong oysters face a highly dynamic and stressful environment with long periods of high temperature, low salinity and hypoxia. They have adapted to their environment by evolving a complete and effective stress tolerance capacity and molecular mechanisms. However, these molecular mechanisms are poorly understood. By employing second-generation high-throughput sequencing technologies, we have screened out an extensive set of genes responding to enviromental stress from Crassostrea hongkongesis. Nrf2, a key antioxidant transcription factor, shows a 4 fold increase in expression during heat stress. Based on these studies, this proposal aims to: (1) clone the full-length of Nrf2 from Crassostrea hongkongesis by PCR,(2)indentify its protein structure and evolutional relationship by bioinformatic analysis,(3) study its spatial-temporal expression patterns by means of fluorescence quantitative PCR, Western blot analysis, mRNA and protein in situ hybridization,(4) study its transcriptional regulation in heat stress tolerance by reporter genes and chromatin co-immunoprecipitation,(5)explore some single nucleotide polymorphism (SNP) sites associated with heat stress tolerance in this gene region. Hopefully, our results would not only elucidate molecular mechanisms under which estuarine mollusk adapts to the highly stressful environments, but also provides a target gene and molecular basis for the breeding of heat stress tolerance oysters and other marine aquaculture shellfish.

香港牡蛎生活在环境复杂多变的亚热带河口潮间带地区，长期经受高温、低盐度和缺氧等锻炼，已经演化出完善而有效的逆境耐受能力和分子机制；但我们对这一机制知之甚少。本项目试图在对香港牡蛎进行转录组测序和基因表达谱分析，并筛选得到大量香港牡蛎抗逆相关功能基因的基础上，对香港牡蛎抗氧化应激关键转录因子nrf2基因进行深入研究。通过基因克隆和生物信息学分析，获得目标基因，明确蛋白结构和物种进化关系；综合运用荧光定量PCR、Western blot、mRNA和蛋白质原位杂交技术研究其时空表达模式；通过构建报告基因载体、染色质免疫共沉淀等方法研究其对高温抗逆相关基因的表达调控；同时鉴定一些与高温抗逆性状相关的单核苷酸多态性（SNP）位点。本项目的研究成果将有助于人们进一步了解贝类抗逆的分子机理，为牡蛎及其它海水养殖贝类的高温抗逆育种提供靶基因和分子依据。

香港牡蛎是解析亚热带河口无脊椎动物对高温抗逆分子机制的理想材料。Nrf2是调控细胞对抗外来异物和氧化损伤的关键转录因子，在参与细胞抗氧化应激和外源性有毒物质诱导的主要防御机制中发挥着重要的作用。到目前为止，在其它物种中已证实经Nrf2调节的内源性保护基因已经超过200个。本项目通过基因克隆获得了香港牡蛎Nrf2基因（chnrf2）序列。chnrf2基因全长3096bp，编码846个氨基酸。序列分析结果显示，chnrf2的Neh1、Neh2和Neh3结构域与脊椎动物Nrf2基因相对应的区域保守性较高。用荧光实时定量PCR方法对chnrf2进行表达特征分析，发现chnrf2在不同发育时期胚胎和成体不同组织中均有表达，在成体腮组织中表达水平最高。chnrf2基因在高温胁迫条件下表达量出现明显上升，并且与hsp70、bcl2、catalase等保护性基因呈现出一定的协同性。用Genome walking的方法对香港牡蛎hsp70、bcl2、catalase基因的启动子序列进行扩增，转录因子预测结果显示香港牡蛎hsp70、bcl2、catalase基因的启动子区域有多个Nrf2的潜在结合位点。这些结果暗示在高温胁迫条件下，chnrf2可能通过调节hsp70、bcl2、catalase基因的表达来发挥其在高温防御中的作用。通过将chnrf2序列与香港牡蛎454转录组测序序列比对，我们在chnrf2编码区内发现了5个SNP位点。我们的研究结果将有助于人们进一步了解贝类高温抗逆的分子基础，为牡蛎及其它海水养殖贝类的高温抗逆育种提供靶基因和分子依据。