鳜鱼(Siniperca chuatsi)幽门垂中两种胰蛋白酶的cDNA克隆及其生物信息学分析

CLONING AND BIOINFORMATIC ANALYSIS OF TWO TRYPSIN GENES FROM MANDARIN FISH SINIPERCA CHUATSI

采用RT-PCR、3′-RACE及5′-RACE技术从鳜鱼幽门垂组织中克隆了两种胰蛋白酶(分别命名为TRS-A与TRS-B)cDNA全长序列。将测序结果递交GenBank数据库,分别获得登录号为ACD70339和ACD70340。采用生物信息学的方法和工具预测了鳜鱼胰蛋白酶的理化参数及其高级结构,并构建了系统进化树。结果表明:(1)TRS-A和TRS-B的cDNA开放阅读框编码的蛋白序列中均含有一段长度为15个氨基酸残基的信号肽、5个氨基酸残基组成的激活肽、222个氨基酸残基组成的成熟蛋白区域;(2)TRS-A成熟蛋白的相对分子量为24.1kDa、理论等电点为5.69,TRS-B的相对分子量为24.2kDa、理论等电点为5.75;(3)建立了TRS-A和TRS-B的三维结构模型,预测了其活性位点及12个半胱氨酸残基形成二硫键的位置。

The cDNAs of two isoforms of trypsins, designated as TRS-A and TRS-B, were isolated from the pyloric caeca of mandarin fish Siniperca chuatsi through RT-PCR, 3＇-RACE and 5＇-RACE. Sequence analysis indicated that both trypsin cDNAs had an open reading frame of 729bp encoding 242 amino acid residues. The two sequences had been deposited into GenBank under accession number of ACD70339 and ACD70340. Alignment of two amino acid sequences for fish trypsinogens and construction of the corresponding phylogenetic trees were performed based on amino acid sequences of the prepro-forms （pretrpsinogens）. The results suggested that TRS-A was more closely related to trypsin IA from Atlantic salmon （Salmo salar） while TRS-B was more closely related to trypsins from vertebrates lives on land. Some physical and chemical parameters and three-dimensional structures of the two trypsins were predicted by bioinformatics. The results indicated that： （1） both the amino acid sequences of the prepro-forms of TRS-A and TRS-B were composed of a signal peptide （15 residues）, a propeptide （5 residues） and a trpsin moiety （222 residues）; （2） TRS-A had a molecular mass of 24.1kDa and its theoretical pI was 5.96. TRS-B had a molecular mass of 24.2kDa and its theoretical pI was 5.75; （3） the three-dimensional structures of TRS-A and TRS-B were constructed by homology modeling to predict their active sites and disulfide bonds formed by twelve cysteine residues. This study provides an insight into the relationship between structure and function of fish trypsins and established a foundation for further research in fish trypsins using site-directed mutagenesis.