斜带石斑鱼胰蛋白酶原和淀粉酶全长cDNA的克隆与序列分析

Molecular cloning and sequence analysis of pancreatic trypsinogen and amylase from orange-spotted grouper (Epinephelus coioides)

采用RT-PCR及RACE法从斜带石斑鱼Epinephelus coioides肝胰脏克隆得到胰蛋白酶原(trypsinogen,TRY)与淀粉酶(amylase,AMY)基因cDNA全序列。斜带石斑鱼肝胰脏TRY基因cDNA全长911bp,其中5'非翻译区(5'-UTR)为55bp,3'-UTR为127bp,开放阅读框(ORF)为729bp,编码242个氨基酸,包含所有丝氨酸蛋白酶中共有的高度保守的催化活性中心。序列一致性分析发现,斜带石斑鱼与牙鲆Paralichthys olivaceus、金头鲷Sparus aurata、鳎Solea senegalensis、石鲽Pleuronectes bicoloratus的TRY序列相似性高达86.8%-89.7%,与人Homo sapiens、小鼠Mus musculus、斑马鱼Danio rerio的TRY相似性较低为59.9%-64.5%。斜带石斑鱼AMY基因cDNA全长1657bp,其中5'-UTR为41bp,3'-UTR为77bp,ORF为1539bp,编码512个氨基酸,包含与哺乳动物α-AMY二级结构相似的8个α螺旋和8个β折叠。序列一致性分析发现,斜带石斑鱼与澳洲肺鱼Neoceratodus forsteri、美洲拟鲽Limanda americanus、大西洋鲑Salmo salar、斑马鱼AMY基因序列相似性高达82.4%-91.8%,与人、小鼠、鸡G.Gallus的AMY基因相似性较低为70.1%-72.3%。斜带石斑鱼TRY和AMY基因cDNA全序列的成功克隆为进一步研究其表达调控机理及研发有效提高其表达水平的饲料添加剂奠定基础。

Two full-length cDNA sequence encoding pancreatic trypsinogen（TRY） and amylase（AMY） genes were isolated from orange-spotted grouper（Epinephelus coioides） by RT-PCR and RACE methods.The sequences were deposited in the GenBank database under accession number EU715402 and EU715401.The cDNA of TRY was 911bp,encoding 242 amino acids.The deduced amino acid sequence of trypsinogen has a catalytic triad,and sequence analysis reveals that the identities of trypsinogen amino acid are 59.9%-89.7% between fish and mammals.The obtained AMY was 1657bp in length,encoding 512 amino acids.The deduced amino acid of amylase contains eight α-helical and eight β-sheet structures,and the identities of amylase amino acid among fish,mammals and bird are 70.1%-91.8%,which suggests that amylase is relatively more conserved.This research will help to further study the regulation of the two genes and to develop dietary additives to stimulate their expression levels.