【作者】 段亚飞； 刘萍； 李吉涛； 李健； 陈萍；

【Author】 DUAN Ya-fei 12 , LIU Ping 2* , LI Ji-tao 2 , LI Jian 2 , CHEN Ping 2 (1. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, Shandong 266071, PR China 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, PR China)

【机构】 上海海洋大学水产与生命学院； 中国水产科学研究院黄海水产研究所；

【摘要】 采用SMART技术(Switching Mechanism At5′end of RNA Transcript)构建了脊尾白虾(Exopalaemon carincauda)血细胞全长cDNA文库。结果表明:cDNA初级文库滴度为2.8×106pfu/mL,重组率达99%以上,插入片段在400~2000bp之间;扩增文库滴度为4.3×109pfu/mL。随机挑取3411个单克隆进行测序,得到2853条平均长度为436bp的高质量EST序列,序列拼接获得1053条unigenes,包括329条contigs和724条singlets。Blast同源比对发现,其中593条unigenes为与数据库中已知基因同源性较高,22条为未知基因,438条未知序列。通过同源性比较筛选出153条unigenes与免疫相关的功能基因,分为抗菌肽、酚氧化酶激活系统、氧化防御系统、伴侣蛋白、结合蛋白、模式识别受体及其他免疫相关基因等7类。结果表明:构建的脊尾白虾血细胞全长cDNA文库质量较高,可在短期内获得大量脊尾白虾血细胞组织的功能基因表达信息。本研究旨为脊尾白虾功能基因的发掘和分子标记的筛选提供素材。更多还原

【Abstract】 The ridgetail white prawn Exopalaemon carinicauda is one of the most important commercial species in eastern China. However, little information of immune genes in E. carinicauda has been reported. To identify distinctive genes associated with immunity, a cDNA library was constructed from haemocytes of E. carinicauda. The haemocyte cDNA library of Exopalaemon carincauda was constructed by SMART TM ( Switching Mechanism At 5′ end of RNA Transcript) technology. It has been determined that the titer of primary cDNA library was 2.8×10 6 pfu/mL, the recombinant efficiencies was over 99%, the insert size of library was between 400bp and 2000bp; the titer of amplified cDNA library was 4.3 × 10 9 pfu/mL. A total of 3411 clones were sequenced, yielding 2853 ESTs and the average sequence length is 436 bp. The cluster and assembly analysis yielded 1053 unique sequences including 329 contigs and 724 singletons. Blast analysis identified 593 (56.3%) of the unique sequences as orthologs of genes from other organisms (E-value<1e-5). Based on the COG and Gene Ontology (GO), 593 unique sequences were classified. Through comparison with previous studies, 153 genes assembled from 367 ESTs have been identified as possibly involved in defense or immune functions. These genes are categorized into seven categories according to their putative functions in shrimp immune system: antimicrobial peptides, prophenoloxidase activating system, antioxidant defense systems, chaperone proteins, clottable proteins, pattern recognition receptors and other immune-related genes. The quality of haemocyte cDNA library is good enough for getting haemocyte specific expression genes information in haemocyte. The results provided important information of functional genes and layed the groundwork for screening of molecular markers in E. carincauda.更多还原