金鱼胰蛋白酶原基因的克隆及镉和过氧化氢暴露对其基因表达的影响（英文）

MOLECULAR CLONING AND CHARACTERIZATION OF TRYPSINOGEN GENE IN GOLDFISH(CARASSIUS AURATUS) AND ITS EXPRESSION PROFILES IN RESPONSE TO CADMIUM AND OXIDATIVE STRESS

为深入研究胰蛋白酶在鱼类中的蛋白结构和生理功能,利用RT-PCR和RACE方法,从金鱼肝胰脏中成功克隆获得了一种全长864 bp的胰蛋白酶原cD NA序列(gfT ryp)。gfT rypc DNA包含21 bp的5′-非翻译区、114 bp的3′-非翻译区和729 bp的开放读码框,编码242个氨基酸组成的胰蛋白酶原(gf Tryp)。gf Tryp含有15个氨基酸的信号肽和5个氨基酸(LDDDK)的激活肽。氨基酸序列分析表明,gf Tryp具备胰蛋白酶原的保守结构特征,如含有催化三联体氨基酸(His-57、Asp-102和Ser-195),12个半胱氨酸,位于底物结合口袋底部Asp-189和口袋开口处的Gly-216、Gly-226等,提示其可能具有保守的蛋白消化功能。RT-PCR结果显示,gf Tryp mRNA在所检测的各个组织中均有表达,其中在肝胰脏、肠和脂肪中表达量为最高。进一步研究发现,相较于摄食前,肝胰脏gf Tryp mRNA在金鱼摄食后显著升高。在0.5和5μg/m L镉暴露处理后,肝胰脏gf Tryp mRNA显著升高(与未处理组相比,分别约为3.2和4.7倍)。随着镉浓度增加到10μg/mL后,gfT ryp mR NA表达量下降。经100μmol/L过氧化氢处理3h、6h、12h和24h后,金鱼肝胰脏gf Tryp mRNA的表达水平均显著下降,在6h达到最大效应(约为对照组的0.21倍)。研究结果证实了重金属镉和过氧化氢处理能调控胰蛋白酶原基因表达,为进一步探讨鱼类消化生理提供了新的视角。

Trypsin is a serine protease that plays a major role in protein digestion.It is synthesized and secreted by pancreas as a prepro enzyme,trypsinogen.In the present study,a full-length c DNA of goldfish trypsinogen（gf Tryp）was successfully cloned from the hepatopancreas by rapid amplification of c DNA ends technique.The obtained gf Tryp c DNA was 864 bp long with a 21 bp 5′-untranslated region（UTR）,a 114 bp3′-UTR containing the consensus polyadenylation signal AATAAA,and a 729 bp open reading frame encoding a protein of 242 amino acid residues.Sequence alignment showed that the gf Tryp possessed all the characteristic features of trypsin family,suggesting its conserved function in protein digestion.Its m RNA expression was observed in all tissues examined,and the relatively higher levels were detected in hepatopancreas,intestine and fat.Hepatopancreatic gf Tryp m RNA level decreased significantly after fasting for one week.Further periprandial expression analysis showed that gf Tryp m RNA expression level in hepatopancreas dramatically up-regulated after meal.Hepatopancreatic gf Tryp m RNA expression level increased significantly with 0.5 and 5μg/m L cadmium（3.2 and 4.7 fold,respectively）,and decreased with cadmium concentration up to 10μg/m L.Strikingly,H_2O_2 exposure significantly decreased gf Tryp m RNA expression in hepatopancreas at 3h,6h,12h and 24h.Our study provides the evidence that nutritional status,cadmium and oxidative stress can regulate the trypsinogen expression at the transcript level,and sheds new light on the physiological expression of trypsinogen in fish.