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中国明对虾(Fenneropenaeus chinensis) coat-ε基因全长cDNA克隆及组织分布
王修芳1,2, 刘庆慧2,3, 吴 垠1, 黄 倢2,3
1.大连海洋大学 大连 116023;2.农业部海洋渔业可持续发展重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;3.青岛海洋科学与技术国家实验室 海洋渔业科学与食物产出过程功能实验室 青岛 266071
摘要:
coat-ε基因表达的蛋白是组成COPⅠ的coatomer复合体的一个亚基,为获得中国明对虾(Fenneropenaeus chinensis) coat-ε基因全长序列,采用cDNA末端快速扩增(Rapid amplification of cDNA end, RACE)技术,扩增出coat-ε基因的3¢端和5¢端,测序结果经DNAMAN比对拼接得出coat-ε基因全长,基因全长1402 bp,5¢非编码区(UTR) 84 bp,3¢非编码区(UTR) 310 bp,开放阅读框1008 bp,预测编码335个氨基酸,其中,第230–300的氨基酸属于TPR超家族,SignalP 3.0 Server预测氨基酸序列没有信号肽,TMHMM Server v. 2.0分析此氨基酸不存在跨膜结构,PSORTⅡ Prediction预测该蛋白位于线粒体、细胞质、内质网中,属胞内蛋白。系统进化树显示,中国明对虾的coat-ε基因与节肢动物门的动物亲缘关系相近。采用实时荧光定量方法分析该基因在鳃、上皮、胃、肌肉、肝胰腺等不同组织中的相对表达,结果显示,coat-ε在肌肉中的相对转录表达量最高,在鳃和附肢的表达次之。本研究获得的中国明对虾coat-ε全长序列,可为该基因功能研究提供基础。
关键词:  中国明对虾  coat-ε  基因克隆  组织分布
DOI:10.11758/yykxjz.20150617002
分类号:
基金项目:国家重点基础研究发展计划(2012CB114401)、泰山学者"建设工程专项经费"和农业部科研杰出人才和创新团队专项经费共同资助
cDNA Cloning of Coat-Epsilon Gene and Its Tissue Distribution in Fenneropenaeus chinensis
WANG Xiufang1,2, LIU Qinghui2,3, WU Yin1, HUANG Jie2,3
1.Dalian Ocean University, Dalian 116023;2.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266071
Abstract:
Coat-epsilon protein (coat-ε) is a subunit of the coatomer complex that forms COPⅠ. To obtain the full-length sequence of coat-ε of Fenneropenaeus chinensis, we first acquired the sequences of 3' and 5' ends using rapid amplification of cDNA ends (RACE). The results were then spliced by DNAMAN to obtain the full-length 1402 bp sequence. The predicted 5' non-coding region (UTR) had 84 bp and the 3' non-coding region (UTR) had 310 bp. The open reading frame had 1008 bp that was supposed to encode 335 amino acids. The fragment including 230aa to 300aa belonged to the TPR superfamily. Signalp 3.0 server and TMHMM Server Version 2.0 analysis suggested that the amino acid sequence did not contain a signal peptide or a transmembrane structure. PSORTⅡ Prediction showed that coat-epsilon was probably located in mitochondria, cytoplasm, and endoplasmic reticulum. The phylogenetic tree analysis showed that coat-ε of F. chinensis was closely related to that of Arthropoda. We also analyzed the mRNA expression of coat-ε in different tissues with quantitative real-time PCR, and found that it was expressed in all tested tissues including appendage, hepatopancreas, epithelium, heart, stomach, intestine, eyestalk, muscles, gill, lymphoid organ and hemocytes. The expression level was the highest in muscles, followed by the gill and appendage. Our results provided important information for the functional study of coat-ε.
Key words:  Fenneropenaeus chinensis  Coat-ε  Gene clone  Tissue distribution