脊尾白虾血细胞全长cDNA文库的构建及EST序列分析

Construction of a cDNA library from the haemocytes of Exopalaemon carinicauda and analysis of partial expressed sequence tags

采用SMART技术（Switching Mechanism At5’endofRNATranscript）构建了脊尾白虾（Exopalaemon carini-cauda）1~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；扩增文库滴度为4.3×109pfu／mL。随机挑取100个单克隆进行测序，得到95条平均长度为493bp的高质量EST序列，序列拼接获得70条unigenes，包括11条contigs和59条singlets。Blast同源比对发现，其中20条unigenes与数据库中已知基因同源性较高，2条为未知基因，48条未知序列。通过同源性比较筛选出多个与生长、代谢、免疫等相关的功能基因，如肌钙蛋白I、烯醇酶、抗脂多糖因子、溶菌酶等基因。结果证实，所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。本研究旨在为脊尾白虾功能基因的发掘和分子标记的筛选提供素材。

A haemocyte cDNA library of Exopalaemon carinicauda was constructed by SMARTTM technology. We determined that the titer of the primary cDNA library was 2.8 ~ 106 pfu/mL, the recombinant efficiencies were over 99%, the insert size of the library was between 400 bp and 2 000 bp, and the titer of the amplified cDNA li- brary was 4.3 ~ 109 pfu/mL. One hundred single clones were selected randomly to screen for primary detection and a total of 95 high quality ESTs, averaging 493 bp, were obtained. The ESTs were assembled into 70 unigenes, comprising 11 contigs and 59 singlets. When compared with the public database by Blast, 20 unigenes were highly homologous （E-value 〈 le-5） to known genes, two unigenes were unknown genes （hypothetical protein）, and 48 unigenes had no significant homology to any sequence. Several functional genes ofE. carinicauda, including Tro- ponin I, Enolase, Anti-lipopolysaccharide factor and Lysozyme, were identified by homology comparison. The quality of this haemocyte cDNA library is of a high enough standard to obtain haemocyte-specific expression gene information. These results provide important information on functional genes and lay the groundwork for screen- ing of molecular markers in E. carinicauda.