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陈文波, 闫芳芳, 秦少宗, 娄本杰. 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[J]. 水生生物学报, 2018, 42(1): 68-76. DOI: 10.7541/2018.009
引用本文: 陈文波, 闫芳芳, 秦少宗, 娄本杰. 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[J]. 水生生物学报, 2018, 42(1): 68-76. DOI: 10.7541/2018.009
Wen-Bo CHEN, Fang-Fang YAN, Shao-Zong QIN, Ben-Jie LOU. MOLECULAR CLONING AND CHARACTERIZATION OF TRYPSINOGEN GENE IN GOLDFISH (CARASSIUS AURATUS) AND ITS EXPRESSION PROFILES IN RESPONSE TO CADMIUM AND OXIDATIVE STRESS[J]. ACTA HYDROBIOLOGICA SINICA, 2018, 42(1): 68-76. DOI: 10.7541/2018.009
Citation: Wen-Bo CHEN, Fang-Fang YAN, Shao-Zong QIN, Ben-Jie LOU. MOLECULAR CLONING AND CHARACTERIZATION OF TRYPSINOGEN GENE IN GOLDFISH (CARASSIUS AURATUS) AND ITS EXPRESSION PROFILES IN RESPONSE TO CADMIUM AND OXIDATIVE STRESS[J]. ACTA HYDROBIOLOGICA SINICA, 2018, 42(1): 68-76. DOI: 10.7541/2018.009

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

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

  • 摘要: 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cDNA序列(gfTryp)。gfTrypc 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(gfTryp)。gfTryp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, gfTryp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, gfTryp 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gfTryp mRNA在金鱼摄食后显著升高。在0.5和5 μg/mL镉暴露处理后, 肝胰脏gfTryp 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10 μg/mL后, gfTryp mRNA表达量下降。经100 μmol/L过氧化氢处理3h、6h、12h和24h后, 金鱼肝胰脏gfTryp 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。

     

    Abstract: 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 cDNA of goldfish trypsinogen (gfTryp) was successfully cloned from the hepatopancreas by rapid amplification of cDNA ends technique. The obtained gfTryp cDNA was 864 bp long with a 21 bp 5′-untranslated region (UTR), a 114 bp 3′-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 gfTryp possessed all the characteristic features of trypsin family, suggesting its conserved function in protein digestion. Its mRNA expression was observed in all tissues examined, and the relatively higher levels were detected in hepatopancreas, intestine and fat. Hepatopancreatic gfTryp mRNA level decreased significantly after fasting for one week. Further periprandial expression analysis showed that gfTryp mRNA expression level in hepatopancreas dramatically up-regulated after meal. Hepatopancreatic gfTryp mRNA expression level increased significantly with 0.5 and 5 μg/mL cadmium (3.2 and 4.7 fold, respectively), and decreased with cadmium concentration up to 10 μg/mL. Strikingly, H2O2 exposure significantly decreased gfTryp mRNA 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.

     

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