中国明对虾(Fenneropenaeus chinensis)血蓝蛋白基因cDNA的克隆与.pdf

中国明对虾血蓝蛋白基因cDNA的克隆与序列分析1 孙杰1 ，2 ，王宝杰1，2，孙姝娟1，2，李晓华1，姜珊1，2 刘梅1，蒋克勇1，王雷1 (1. 中国科学院海洋研究所 青岛 266071， 2.中国科学院研究生院 北京 100039) 摘要 利用 3’和 5’RACE技术从中国明对虾 Fenneropenaeus chinensis肝胰腺中克隆了一个血 蓝蛋白基因 FCHc, FCHc基因 cDNA全长为 2161bp，其中开放阅读 2034bp，编码 678个氨基酸， 预测分子量为77.59KDa。FCHc序列与凡纳滨对虾Litopenaeus vannamei血蓝蛋白同源性为82%， 与日本囊虾 Marsupenaeus japonicas同源性为 85%。Realtime-PCR实验结果表明，FCHc在肝胰 腺中的相对表达量最高，在心脏和表皮中几乎不表达。该基因在鳗弧菌和对虾白斑综合征病毒 （WSSV）感染后的对虾肝胰腺中的表达量显著增加，并具有不同的时空表达趋势，提示中国 明对虾 FCHc基因在免疫反应中具有重要作用。 关键词：中国明对虾， 血蓝蛋白，基因克隆，序列分析 cDNA cloning and sequence analysis of hemocyanin in Fenneropenaeus chinensis SUN Jie 1, 2 WANG Bao-jie 1 SUN Shu-juan 1, 2 LI Xiao-hua1 JIANG Shan 1, 2 LIU Mei 1 JIANG Ke-yong 1 Wang Lei 1 (1 Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071) (2 Graduate School of Chinese Academy of Sciences, Beijing 100039) ABSTRCT A hemocyanin gene (FCHc) was cloned from hepatopancreas of Chinese shrimp Fenneropenaeus chinensis by Rapid Amplification Complementary DNA Ends (RACE) method. The full length cDNA of FCHc consists of 2,161bp with a 2,034 bp Open Reading Frame (ORF)，which encoded 678 amino acids，and the predicted molecular mass was 77.59kDa. It showed 82% and 85% homology with those of Litopenaeus vannamei and Marsupenaeus japonicas. The real-time PCR results showed that the expression level of FCHc in hepatopancreas was the highest among seven studied tissues．Real-time PCR analysis showed that the expression of FCHc was up-regulated distinctly in the hepatopanereas of shrimp when they were challenged by Vibrio anguillarum and white spot syndrome virus (WSSV). FCHc showed different expression profiles in the hepatopancreas during V. anguillarum or WSSV virus infection．The results implied that FCHc might play an important role in the shrimp immune system． KEY WORDS Fenneropenaeus chinensis Hemocyanin Gene cloning Sequence analysis 分类号：Q178.53 中国明对虾Fenneropenaeus chinensis是我国主要的海水养殖虾类之一。近十年来，由于病毒 病及细菌病的影响，中国明对虾的育苗和养