西施舌浮游幼虫cDNA文库构建及EST序列初步分析

Construction of cDNA Library and Preliminary Analysis of EST Sequence in Coelomactra antiquata Planktonic Larva

为了研究西施舌浮游幼虫阶段的基因表达规律,构建了7日龄浮游幼虫的cDNA文库,对文库质量的分析结果表明,cDNA文库的库容量为2.3×10~5,重组率达95%,平均插入片段长度1.0 kb左右.挑取cDNA克隆进行5′端测序,总共进行了304个成功反应,其中294条ESTs长度为100~699 bp,平均长度为492 bp.初步拼接得到158个单基因簇(Unigene),其中包括19个重叠群(Contigs),139个单拷贝ESTs(Singletons).与GenBank非冗余蛋白库进行Blastx比对,发现71.4%ESTs在GenBank中有同源序列(blast score≥50 bits),无明显相似性EST序列占28.6%,提示此文库有许多未知的基因.丰度最高的是组蛋白cDNA(包括组蛋白H1和组蛋白H2A),占总EST序列的39.5%.在116个ESTs中获得组蛋白H1和H2A基因的2个开放阅读框,分别为561和375个碱基,编码186,124个氨基酸残基的肽段.

In order to investigate the patterns of genes expressed in Coelomactra antiquata planktonic larva,we constructed a cDNA library of the seventh instar planktonic larva.Library analysis showed that the original storage capacity of the cDNA library was 2.3×105,the recombination frequency was 95%,with all average fragment size around 1.0 kb.We analyzed the expressed sequence tags(ESTs)obtained from the cDNA library,to examine gene expression profile.A total of 304 successful reactions were carried out in 5'-terminal sequencing of cDNA clones,of which 294 were 100~699 bp in length,with an average length of 492 bp.158 single gene clusters(Unigene)were obtained by preliminary splicing,including 19 Contigs and 139 single copies ESTs(Singletons).Significant similarities(blast score≥50 bits)ESTs to the Genbank data were found for 71.4%,suggesting that the larvae library contains many unknown genes(28.6%).Histone genes were the largest group of ESTs(39.5%)found in our planktonik larva library.Among 116 clones,two ORFs of histone genes were cloned,Histone H1 gene composed of 561 nucleotides with coding 186 amino acids,the sequence of histone H2A gene consists of 375 nucleotides with coding 124 amino acid residues.