厚壳贻贝足腺cDNA文库的构建及部分EST序列分析

为获得厚壳贻贝足腺组织的EST序列标签以及可能的功能基因序列,以pCMVsport6质粒为载体构建了高质量的厚壳贻贝足腺组织cDNA文库,文库滴度为1.31×107pfu/mL,重组率为98%,平均插入片段为1.3kb.通过对文库部分克隆的序列测定,获得了11个新基因和17个功能基因,其中包括部分与厚壳贻贝足丝相关的基因.厚壳贻足腺组织cD-NA文库的成功构建为将来筛选与厚壳贻贝足丝蛋白相关的新基因,系统研究其黏附机制奠定了基础.

The construction of foot gland cDNA library of Mytilus coruscus and the analysis of partial expressed sequence tags

In order to obtain the expressed sequence tags (ESTs) and probe the adhesive related genes in Hard-shell mussel Mytilus coruscus,the cDNA library of foot gland tissues was constructed.The total RNA was extracted by TRIZOL and the mRNA was purified via affinity chromatograph with oligo(dT) cellulose;utilizing gateway technology double-strand cDNA fragments were harvested,concentrated and ligated with pCMV sport6 vector.The titer of amplified library was tested up to 1.31×10~7 pfu/mL and the insert size of randomly picked clones was 1.3 kb.A number of clones were picked up at random,sequenced and analyzed with BLAST on line.The result showed that within the 28 random clones 4 were characterized as rRNA genes,11 were unknown genes which were presumed as potentially noble genes and 13 were functional genes which coded for proteins including precollagen P,precollagen D and precollagen NG etc,some of which were related to the adhesion of Mytilus coruscus.The construction of high-quality cDNA library of Mytilus coruscus foot gland enabled researchers to screen more adhesive related genes and built the foundation for further researches on probing the molecular diversity of adhesive proteins and the mechanism of adhesion.