中国明对虾(Fenneropenaeus chinensis)coat-ε基因全长cDNA克隆及组织分布

cDNA Cloning of Coat-Epsilon Gene and Its Tissue Distribution in Fenneropenaeus chinensis

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,Signal P 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,PSORTⅡPrediction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。

Coat-epsilon protein （coat-ε） is a subunit of the coatomer complex that forms COPⅠ. To obtain the full-length sequence of coat-ε ofFenneropenaeus chinensis, we first acquired the sequences of 3＆#39; and 5＆#39; 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＆#39; non-coding region （UTR） had 84 bp and the 3＆#39; 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 ofF. 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.