三疣梭子蟹血细胞全长cDNA文库构建及EST初步分析  

Construction of a full-length cDNA library of Portunus trituberculatus hemocytes and analysis of partial expressed sequence tags

作　　者：申望[1] 叶茂[1] 石戈[1] 王日昕[1] 

机构地区：[1]浙江海洋学院海洋科学学院海洋生物资源及分子工程实验室,舟山316004

出　　处：《海洋渔业》2010年第1期24-29,共6页Marine Fisheries

基　　金：浙江省重大科技专项重大项目(2007C02001);浙江省教育厅项目(20061141)

摘　　要：三疣梭子蟹是我国沿海重要养殖品种之一,近年来养殖病害呈逐年上升趋势,制约了三疣梭子蟹养殖产业的健康可持续发展。为大规模克隆三疣梭子蟹免疫相关基因,研究其免疫基因的功能和免疫机制,奠定三疣梭子蟹病害防治理论基础,采用SMART技术构建了三疣梭子蟹血细胞全长cDNA文库。经测定,文库滴度为1.14×107pfu/mL,文库总容量为5.7×107pfu,重组率达到98%,插入片段平均长度为817.5 bp。文库随机测序获得的70个全长cDNA,从中发现18个已知基因和5个未知基因。已知基因主要为多肽/蛋白质合成相关基因,共9个;免疫相关基因4个,分别是抗菌肽hyastatin、C-reactive prote in-1、profilin和m asquerade-like prote in,且4个免疫相关基因共有48个克隆,占测序总克隆的68.6%,表明血细胞中免疫相关基因表达非常活跃。研究结果证实,构建血细胞全长cDNA文库是大规模克隆三疣梭子蟹免疫相关基因的可靠途径,并为深入研究免疫相关基因的功能和免疫机制奠定基础。Portunus trituberculatus is one of the most important aquaculture crustacean species in China. But the sustainable development of P. trituberculatus aquaculture had been restricted by ascendant aquatic disease. And the immune system of P. trituberculatus has always been an hot issue to researchers. The immunity of P. trituberculatus is related to various immune molecules in plasma. To obtain the full-length cDNA sequences of potential immune-related genes and explore their function, a high-quality cDNA library of P. trituberculatus hemocytes was constructed by SMART technology and evaluated. The library showed a titer of 1.14 ×10^7 pfu/mL of cDNA insets , and contained a total clones of 5.7 ×10^7 pfu. The recombinant rate was about 98% and the average length of the cDNA inserts was 817.5 bp. A number of clones were randomly selected for sequenceing. 70 full-length sequences were processed, assembled and clustered into 23 unigenes, including 6 contigs and 17 singletons. Compared to the NCBI nonredundant （nr） protein databases by performing BLASTX, 18 unigenes were annotated. Half of the annotated genes （9/18） were found to be associated with protein biosynthesis ; four immune-related genes （ hyastatin, C-reactive protein-1, profilin and masquerade-like protein） were obtained, and their clones sumed to 48 （68.6% of the total）. The results indicated that the expression of immune-related genes was very strong in hemocytes, and further confirmed that constructing high-quality cDNA library of the hemocytes was a reliable tool to screen out immune-related functional genes and to probe the functions of immune-related zenes and the immunologic mechanism.

关 键 词：三疣梭子蟹 血细胞 全长CDNA文库 免疫相关基因 表达序列标签 

分 类 号：Q291[生物学—细胞生物学]