猪Cathepsin B基因cDNA分子克隆、序列分析及遗传多态性分析

Molecular Cloning,Characterization and Polymorphism of the Porcine (Sus scrofa) Cathepsin B Gene

采用RT-PCR结合克隆测序的方法,从猪肌肉组织总RNA中克隆出了猪组织蛋白酶B(Cathepsin B,CTSB)基因的cDNA序列,并推导出其编码的氨基酸序列。猪CTSB基因开放阅读框(ORF)全长1008 bp,编码335个氨基酸。同源性分析结果表明,猪与人、鼠、牛的CTSB基因cDNA编码区(CDS)同源性分别为85%、81%、90%,推测的氨基酸序列同源性分别为81%、79%、91%。利用同源性结合序列特征预测表明该蛋白具有信号肽和前肽序列。蛋白质结构同源建模分析表明,该蛋白具有木瓜蛋白酶家族的典型空间结构,包括1个底物结合凹槽和3个相互靠近的活性位点。另外采用PCR-SSCP方法,在147头个体中分析了CTSB基因第6内含子内的SS-CP位点多态性,检测到3个等位基因6种基因型。FF基因型个体的各项嫩度指标最高,其最大剪切力与硬度值分别为6.56 kg和23.55 kg.s,极显著地高于EE和EF基因型个体(P<0.01),平均剪切力为4.85 kg,显著地高于EF基因型个体(P<0.05)。

The porcine cathepsin B （CTSB） nucleotide and amino acid sequence were obtained by RT-PCR and sequencing. The open reading frames （ORFs） were 1 008 bp long （encoding for 335 amino acids）. The nucleotide sequence identities of porcine cathepsin B ORFs were 85%, 81% and 90% comparing to that of the human, rat and bovine, respectively. The deduced amino acid sequence identities of the porcine cathepsin B protein were 81 %, 79%, and, 91% comparing to that of the rat, human and bovine, respectively. The structure of cathepsin B protein was predicted by homologous modelling which was similar to other papains. The substrate-binding cleft and the catalytic triad were found in the structure model. The polymorphisms of porcine cathepsin B gene was detected in 147 pigs by PCR-SSCP. There were six genotypes be detected on the SSCP locus. The genotype FF on CTSB gene SSCP locus had the highest tenderness traits, it had significantly higher shear force （6.56 kg） and toughness （23.55 kg · s） than genotype EE and EF （P〈0. 01）, and it had significantly higher mean shear force （4. 85 kg） than genotype EF （P〈 0.05）.