| 摘要: |
| 采用RT-PCR和RACE方法克隆了中华绒螯蟹延伸因子EF-2全长cDNA, 序列分析表明EF-2全长2752bp, 编码846个氨基酸。经BLASTX分析表明, EF-2 核苷酸序列与镶边拟蠢蟹EF-2序列的同源性最高, 其相似性为88%; 所编码的氨基酸序列与甲壳动物EF-2的氨基酸序列相似性都在90%以上。聚类分析显示, 中华绒螯蟹EF-2的氨基酸序列与镶边拟蠢蟹EF-2聚为一支, 并与其它甲壳动物聚为一体。荧光定量PCR结果显示, EF-2 在正常成熟中华绒螯蟹肌肉中表达量最高, 精巢、肝胰腺中有少量表达, 心脏、卵巢、胃、肠、鳃中有微量表达。检测了不同发育状态的幼蟹、早熟蟹和正常成熟蟹EF-2在肝胰腺、鳃以及肌肉中的相对表达情况; 同时也检测了在不同pH处理下幼蟹EF-2在肝胰腺和鳃中随时间变化的相对表达情况, 结果显示pH胁迫对幼蟹EF-2表达有一定的诱导效果。 |
| 关键词: 中华绒螯蟹, EF-2, 基因, 克隆, 表达 |
| DOI：10.11693/hyhz201206030030 |
| 分类号: |
| 基金项目:国家高技术研究发展计划(863计划)项目, “主要养殖甲壳类良种培育”, 2012AA100809 号; 上海市教育委员会科研创新项目, 11YZ151 号。 |
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| THE FULL LENGTH cDNA CLONING AND EXPRESSION OF ELONGATION FACTOR 2 FROM THE CHINESE MITTEN CRAB ERIOCHEIR SINENSIS |
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GUO Zi-Hao, YANG Zhi-Gang, LIU Zhi-Wei, JI Lian-Yuan, QUE You-Qing, YANG Xiao-Zhen, CHENG Yong-Xu
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Fisheries and Life Science College of Shanghai Ocean University, Key laboratory of Freshwater Aquatic Genetic Resources Ministry of Agriculture
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| Abstract: |
| In this study, we cloned EF-2 gene from Eriocheir sinensis using reverse transcriptase polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE), the full-length cDNA sequence of EF-2 was 2752bp which coded 846 amino acid residues. Comparation results which was used by BLASTX software showed that the nucleotide homology of EF-2 was 88% similar to Libinia emarginata’s and amino acid homology of EF-2 was higher than 90% similar to others’ crustaceans. The phylogenetic analysis based on amino acid sequence showed that EF-2 had highest similarity with EF-2 of L. emarginata and clustered with other crustaceans. The expression of EF-2 gene in different tissues and stage of normal mature crab were analyzed by real-time fluorescent quantitative PCR. The result showed that EF-2 mRNA was mainly detected in muscle and small amount in testis, hepatopancreas and trace in heart, ovary, stomach, intestine and gill. We examined the expression of the EF-2 in hepatopancreas, gills and muscles in different developmental status of crablet, precocious crab and normal mature crab; and also examined the expression of EF-2 in hepatopancreas and gills in crablet which were exposed to different pH, the results showed that pH stress can induce the expression of EF-2. |
| Key words: Eriocheir sinensis, EF-2, Gene, Cloning, Expression |
