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引用本文:王斌,武敏敏,胡利腾,张建设.太平洋真宽水蚤(Eurytemora pacifica) CRH-BP基因克隆及表达分析.海洋与湖沼,2019,(1):210-219.
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太平洋真宽水蚤(Eurytemora pacifica) CRH-BP基因克隆及表达分析
王斌, 武敏敏, 胡利腾, 张建设
浙江海洋大学海洋科学与技术学院 国家海洋设施养殖工程技术研究中心 舟山 316022
摘要:
促肾上腺皮质激素释放激素结合蛋白(CRH-BP)是一种糖基化蛋白,与促肾上腺皮质激素释放激素(CRH)具有很高的亲和力,对CRH诱导的脑垂体促肾上腺皮质激素(ACTH)的释放起到抑制作用。本研究采用RT-PCR与RACE基因克隆方法首次获得太平洋真宽水蚤(Eurytemora pacifica)CRH-BP基因全长cDNA序列(GenBank登录号:MF289345)1950bp,其中ORF区1245bp编码415个氨基酸,5'非编码区841bp,3'非编码区294bp,理论分子量/等电点为45.816/5.87。生物学序列分析结果表明,太平洋真宽水蚤CRH-BP氨基酸序列与桡足类中近亲真宽水蚤同源性最高;具有6个蛋白修饰位点及蛋白家族标签序列,具有明显的跨膜结构域及信号肽。实时荧光定量PCR分析结果表明,太平洋真宽水蚤CRH-BP基因在盐度、温度及pH环境胁迫中的mRNA表达量均具有一定的时间梯度表达性与浓度梯度表达性特征。本文通过对太平洋真宽水蚤CRH-BP基因结构及不同环境因子刺激下的表达特点的研究为探究桡足类在环境应激、生殖生理及免疫调控等方面奠定了理论基础。
关键词:  太平洋真宽水蚤  促肾上腺激素释放激素结合蛋白  基因克隆  环境胁迫  实时荧光定量PCR
DOI:10.11693/hyhz20180500139
分类号:Q789;Q955
基金项目:国家海洋局公益性行业科研专项,201505025号;舟山市海洋与渔业局2018年现代渔业发展市级专项。
cDNA CLONING AND EXPRESSION OF CRH-BP GENE OF COPEPOD EURYTEMORA PACIFICA
WANG Bin, WU Min-Min, HU Li-Teng, ZHANG Jian-She
Marine Science College of Zhejiang Ocean University, National Engineering Research Center of Marine Facilities Aquaculture, Zhoushan 316022, China
Abstract:
CRH-BP (Corticotropin releasing hormone binding protein) is a glycosylated protein in a high affinity with corticotropin releasing hormone (CRH). CRH induces the release of adrenocorticotropic hormone (ACTH), and CRH-BP can inhibit the release of ACTH by combining CRH. In this study, Eurytemora pacifica was selected as the research subject from which the full-length cDNA sequence of CRH-BP gene was obtained by RT-PCR and RACE methods. The full-sequence of CRH-BP (GenBank accession number:MF289345) was 1950bp, having a complete open reading frame of 1245bp encoding 415 amino acids, 5' and 3' non-encoding ones for 841bp and 296bp, respectively, the molecular weight of 45.816kDa, and isoelectric point of 5.89. Sequence comparison indicated high homology in amino acid sequence of CRH-BP cDNA to E. affinis, sharing 6 protein post-translational modification and protein family sequence tagged sites, an obvious signal peptide, and transmembrane domain. CRH-BP mRNA expression analysis of E. pacifica showed that Ep.CRH-BP gene expression could be up-regulated under salinity, temperature, or pH stress, depending on temporal and concentration conditions. Therefore, these results shall be helpful to further explore the structure and function of the copepod CRH-BP gene, and to provide a basis for studying the molecular mechanism of stress response from the copepod.
Key words:  CRH-BP  Eurytemora pacifica  gene cloning  environmental factors  RT-PCR
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