香鱼肝胰腺cDNA文库的构建和鉴定

Construction and Characterization of a cDNA Library in Hepatopancreas of Ayu Plecoglossus altivelis

以香鱼正常肝胰腺为材料,构建cDNA文库。初始文库库容大于1.3×107cfu/ml,扩增文库的滴度大于1.1×106cfu/ml,平均插入片段约为1.0 kb。从文库中随机挑选297个cDNA克隆测序,得到232个功能已知EST序列,功能包括代谢、蛋白质合成、细胞与机体防御、信号转导以及细胞结构与运动等5个方面。随机测序获得香鱼弹性蛋白酶（Elastase,ELA）编码序列,cDNA序列全长957个核苷酸,3’-末端具有polyA尾,编码一个由268个氨基酸组成28.7 ku大小蛋白。序列分析表明,香鱼ELA的N端15个氨基酸为信号肽,成熟肽具有丝氨酸蛋白酶家族的典型结构特征。香鱼ELA与斜带石斑鱼、大西洋鳕和金头鲷的ELA氨基酸序列同源性最高,达76%~77%。系统进化树分析表明,香鱼ELA与其他鱼类ELA紧密成簇。RT-PCR检测显示,香鱼ELA基因mRNA在香鱼肝胰腺、脾、肠组织中均大量表达。

The cDNA library was constructed in hepatopancreas of healthy ayu,Plecoglossus altivelis.The primary cDNA library storage capacity was found greater than 1.3×107 cfu/ml,the titer of amplified library greater than 1.1×106 cfu/ml,and the average size of inserted fragments 1.0 kb.A random set of 297 clones was sequenced and 232 ESTs with known functions obtained were found to be involved in the biological processes including metabolism,protein synthesis,defence of cells and organisms,signal transport,structure and movement of cell and so on.For 297 sequenced clones,the cDNA sequence of ayu elastase（ELA） gene was obtained,and consisted of 957 bp nucleotides.The open reading frame encoded a 268-amino acids-long,and 28.7 ku protein and had a polyA tail in the 3′-end.The sequence analysis showed that the ayu ELA had a 15-amino acids-long signal peptide,the mature peptide being of the typical features of serine protease family.The comparative analysis of ELA amino acid sequences and the phylogenetic analysis showed that the ELAs were relatively conserved,similar to those of E.coioides,G.morhua and S.aurata,up to 76%～77%.Phylogenetic tree analysis indicated that ayu ELA closely clustered with other fish ELA.The ayu ELA gene was expressed in the hepatopancreas,spleen and intestine by RT-PCR.