鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法，克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明，鳜鱼Try基因cDNA全长为896 bp，其中开放阅读框 (open reading frame,ORF)为744 bp，编码247个氨基酸. 序列同源性分析发现，鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp，其中ORF为1 539 bp，编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明，鳜鱼Try基因由4个内含子和5个外显子组成，全长1 362 bp；鳜鱼Amy基因由8个内含子和9个外显子组成，全长4 267 bp；鳜鱼Pep基因由8个内含子和9个外显子组成，全长 4 032 bp，与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列，并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究，为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.

Molecular Cloning and Sequences Analysis of Trypsin, Amylase and Pepsinogen Genes from Chinese Perch (Siniperca chuatsi )

Two full-length cDNA sequences encoding trypsin (Try) and amylase (Amy) genes were isolated from Chinese perch (Siniperca chuatsi) by RT-PCR and RACEs methods. The Try cDNA was 896 bp, and contained a 744 bp open reading frame (ORF) encoding 247 amino acids. The deduced Try amino acid sequence shared some homology with those of zebrafish (Danio rerio) (81.4%), African clawed frog(Xenopus laevis) (75.3% ), mouse (74.5%) and human (71.4%). The AmycDNA was 1 647 bp, contained a 1 539 bp ORF encoding 512 amino acids. The deduced Amy amino acid sequence shared some homology with those of zebrafish (79.7%), African clawed frog (75.4%), mouse (71.9%) and human (70.9%). Try, Amy and Pepgenomic sequences obtained by PCR from Chinese perch genome, displayed the similar genomic structure as other vertebrates (Try: 4 introns and 5 exons；Amy: 8 introns and 9 exons； Pep: 8 introns and 9 exons) spanning 1.4 kb, 4.3 kb and 3.3 kb, respectively. Through genome walker method, the 5′-flanking regions of Try, Amy and Pepand 3′-flanking regions of Pepwere obtained, which spanned 1 189 bp, 413 bp, 527 bp and 704 bp, respectively. Several potential regulatory elements were identified in the promoter regions. These results will provide an important basis for studying the function of digestion enzyme genes in fish.