草鱼Aminopeptidase N基因的克隆与表达研究
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摘要
蛋白质是水产动物营养的重要组成部分,而氨肽酶N在蛋白质的消化和代谢平衡方面起着重要作用。本实验采用RACE技术克隆草鱼氨肽酶NcDNA全序列,对其结构与功能进行分析。并利用实时荧光定量PCR方法对氨肽酶N基因在草鱼发育过程中表达量的变化、在不同组织的表达差异及肠道氨肽酶N昼夜表达变化进行了研究分析。此外,研究了不同蛋白水平和蛋白源对APN基因在前肠的表达影响的特征。本研究将为氨肽酶N基因功能与结构的关系和揭示水产动物对蛋白质消化代谢的分子机理研究奠定基础。本实验的主要研究内容及结果如下：
1、对水产动物营养代谢相关基因进行分子克隆,是开展水产动物分子营养学研究的前提。本实验采用RT-PCR和RACE等技术克隆草鱼氨肽酶NcDNA全序列,并对其进行了生物信息学分析。结果发现,APN cDNA全长为3258bp,包含2679bp的开放阅读框,编码892个氨基酸；草鱼与斑马鱼基因同源性和编码氨基酸同源性分别为81.5%和75.4%,与其他动物同源性分别为58.8%-61.2%和54.3%-60.2%。系统进化分析表明,草鱼APN基因与斑马鱼(Danio rerio)的亲缘关系最近。应用生物信息学分析,发现APN基因编码蛋白的分子量为100.61ku,等电点为5.18；该蛋白无信号肽,而具有与哺乳动物十分相似的1个螺旋跨膜结构,但跨膜区氨基酸同源性较低。
2、为了进一步深入了解氨肽酶N与草鱼蛋白质消化代谢生理各方面存在着怎样的相互关系。本实验采用Real time PCR技术在基因表达水平上对氨肽酶N基因在草鱼发育过程中表达量的变化、在不同组织的表达差异及肠道氨肽酶N昼夜表达变化进行了研究分析。基因的发育表达,结果显示：草鱼胚胎发育时期,APN mRNA表达量相对较低,在原肠期最高；胚后发育时期,从出膜期到出膜第7d,总体上呈现递增的趋势；而从出膜第14d到35d基因表达变化有波动,总体上低于第7d的表达丰度；组织差异表达,结果显示APN基因在草鱼前肠、中肠和后肠均有较高的表达量,以前肠组织表达量最高；昼夜节律研究发现,肠道APN基因06：00-18：00的表达量较18：00-06：00高。
3、为了研究不同蛋白水平和蛋白种类饲料对草鱼肠道APN基因表达的影响的特征。本研究利用草鱼生长实验,设计2种蛋白源和5种蛋白水平饲料饲养草鱼鱼种,实时定量PCR检测草鱼前肠APN mRNA相对表达量。不同蛋白源对草鱼前肠APN mRNA表达影响结果表明,豆粕组除第7d草鱼前肠APN mRNA相对表达丰度与鱼粉组无显著差异外,其他时间都有显著差异(p<0.05),且都比鱼粉组要低。不同蛋白水平对草鱼肠道组织APN mRNA表达影响实验结果显示总体上,不同时间段不同蛋白水平从22.3%CP到27.3%CP前肠APN mRNA相对表达丰度有升高趋势,而从27.3%CP到42.1%CP相对表达丰度呈现一种降低趋势。因此认为,草鱼氨肽酶N表达量与草鱼生长发育密切相关,具有组织差异性,且呈现昼夜节律。草鱼肠道氨肽酶N基因表达对饲料蛋白营养存在一定的适应反应。
Protein is an important part of aquatic animal nutrition,and aminopeptidase N plays an important role in the digestion of protein and the metabolic balance.In this study, aminopeptidase N cDNA's full-length sequences has been cloned,and its structure and functions has been analyzed.The changes of APN gene expression in the grass carp development process and the APN gene expression differences of the different tissues, and the changes of APN gene expression of the circadian in the intestinal have been studied by a real-time PCR technology.In this study,also,the different protein level and protein source influencing on the APN gene expression in the foregut has been studied.Therefore, this study could lay the foundation for the reserch of the relationship between gene function and structure of aminopeptidase N and revealing the molecular mechanism of protein digestion and metabolism in aquatic animals.The major results are as follows:
1.The means of RT-PCR、RACE techniques were used to clone the APN full-length cDNA gene sequence. The bioinformatic analysis of APN gene was carried out.The results showed that:the full length of cDNA sequence of APN had3258nucleotides, including27nucleotides at5'UTR and552nucleotides at3'UTR; its open reading frame had2679nucleotides encoding a892-amino-acid peptide;the deduced amino acid sequences of APN gene from C. idellus displayed the highest similarity with Danio rerio (75.4%), but varied to other animals from61.2%to58.8%;the encoded protein molecular weight was predicted at100.61ku with p1at5.18; phylogenetic analysis showed that the sequence of APN gene was clustered with D. rerio as its closest neighbor, which shared a sequence similarity of81.5%, and had lower similarity with other animals from60.2%to54.3%;the APN protein had no signal peptide and one helix trans-membrane region, but its amino acid sequence of the region demonstrated a low homology relationship to other vertebrates.
2.The mRNA abundance of APN gene was assayed using real-time PCR.The results showed that:during embryonic development, the APN mRNA expression is relatively low,while the APN gene was expressed at highest level in the gastrula stage;during post-embryonic,the mRNA abundance of APN gene, in general, showed a consistent increasing trend from hatching stage to7d post-hatch; and.the APN gene expression changes with some range of fluctuation during14d post-hatch to35d post-hatch,but lower than he7d post-hatch, in general; the APN gene was differentially expressed at different tissues with a gradient from higher to lower among the tissues of fore-intestine, hind-intestine, liver, mid-intestine, kidney, muscle, spleen and heart, respectivelty.The effects of the circadian rhythms on APN expression of C. Idellus showed that there was a time-dependent pattern at higher rhythm during06:00-18:00and lower rhythm during18:00-06:00.
3.The effects of the feeds containing different protein sources on the APN gene expression in the foregut showed that there were no significant differences on the APN mRNA expression in the foregut between the FM group and SM Group at7d; there were significant differences between the FM group and SM Group from14to56d(P<0.05);and the APN mRNA expression of the SM Group was lower than that of the FM group. The effects of the feeds containing different protein levels on the APN gene expression in the foregut showed that, in general, there was a consistent increasing trend from the22.3%CP to27.3%CP group; and there was a consistent downward trend from the27.3%CP to42.1%CP group at different time periods. The results indicated that the APN mRNA expression of Ctenopharyngodon idella correlates tightly to the developmental stage, and showed tissue variability and circadian rhythms.The APN mRNA expression of Ctenopharyngodon idella had adaptive reaction to protein nutrition.
1、对水产动物营养代谢相关基因进行分子克隆,是开展水产动物分子营养学研究的前提。本实验采用RT-PCR和RACE等技术克隆草鱼氨肽酶NcDNA全序列,并对其进行了生物信息学分析。结果发现,APN cDNA全长为3258bp,包含2679bp的开放阅读框,编码892个氨基酸；草鱼与斑马鱼基因同源性和编码氨基酸同源性分别为81.5%和75.4%,与其他动物同源性分别为58.8%-61.2%和54.3%-60.2%。系统进化分析表明,草鱼APN基因与斑马鱼(Danio rerio)的亲缘关系最近。应用生物信息学分析,发现APN基因编码蛋白的分子量为100.61ku,等电点为5.18；该蛋白无信号肽,而具有与哺乳动物十分相似的1个螺旋跨膜结构,但跨膜区氨基酸同源性较低。
2、为了进一步深入了解氨肽酶N与草鱼蛋白质消化代谢生理各方面存在着怎样的相互关系。本实验采用Real time PCR技术在基因表达水平上对氨肽酶N基因在草鱼发育过程中表达量的变化、在不同组织的表达差异及肠道氨肽酶N昼夜表达变化进行了研究分析。基因的发育表达,结果显示：草鱼胚胎发育时期,APN mRNA表达量相对较低,在原肠期最高；胚后发育时期,从出膜期到出膜第7d,总体上呈现递增的趋势；而从出膜第14d到35d基因表达变化有波动,总体上低于第7d的表达丰度；组织差异表达,结果显示APN基因在草鱼前肠、中肠和后肠均有较高的表达量,以前肠组织表达量最高；昼夜节律研究发现,肠道APN基因06：00-18：00的表达量较18：00-06：00高。
3、为了研究不同蛋白水平和蛋白种类饲料对草鱼肠道APN基因表达的影响的特征。本研究利用草鱼生长实验,设计2种蛋白源和5种蛋白水平饲料饲养草鱼鱼种,实时定量PCR检测草鱼前肠APN mRNA相对表达量。不同蛋白源对草鱼前肠APN mRNA表达影响结果表明,豆粕组除第7d草鱼前肠APN mRNA相对表达丰度与鱼粉组无显著差异外,其他时间都有显著差异(p<0.05),且都比鱼粉组要低。不同蛋白水平对草鱼肠道组织APN mRNA表达影响实验结果显示总体上,不同时间段不同蛋白水平从22.3%CP到27.3%CP前肠APN mRNA相对表达丰度有升高趋势,而从27.3%CP到42.1%CP相对表达丰度呈现一种降低趋势。因此认为,草鱼氨肽酶N表达量与草鱼生长发育密切相关,具有组织差异性,且呈现昼夜节律。草鱼肠道氨肽酶N基因表达对饲料蛋白营养存在一定的适应反应。
Protein is an important part of aquatic animal nutrition,and aminopeptidase N plays an important role in the digestion of protein and the metabolic balance.In this study, aminopeptidase N cDNA's full-length sequences has been cloned,and its structure and functions has been analyzed.The changes of APN gene expression in the grass carp development process and the APN gene expression differences of the different tissues, and the changes of APN gene expression of the circadian in the intestinal have been studied by a real-time PCR technology.In this study,also,the different protein level and protein source influencing on the APN gene expression in the foregut has been studied.Therefore, this study could lay the foundation for the reserch of the relationship between gene function and structure of aminopeptidase N and revealing the molecular mechanism of protein digestion and metabolism in aquatic animals.The major results are as follows:
1.The means of RT-PCR、RACE techniques were used to clone the APN full-length cDNA gene sequence. The bioinformatic analysis of APN gene was carried out.The results showed that:the full length of cDNA sequence of APN had3258nucleotides, including27nucleotides at5'UTR and552nucleotides at3'UTR; its open reading frame had2679nucleotides encoding a892-amino-acid peptide;the deduced amino acid sequences of APN gene from C. idellus displayed the highest similarity with Danio rerio (75.4%), but varied to other animals from61.2%to58.8%;the encoded protein molecular weight was predicted at100.61ku with p1at5.18; phylogenetic analysis showed that the sequence of APN gene was clustered with D. rerio as its closest neighbor, which shared a sequence similarity of81.5%, and had lower similarity with other animals from60.2%to54.3%;the APN protein had no signal peptide and one helix trans-membrane region, but its amino acid sequence of the region demonstrated a low homology relationship to other vertebrates.
2.The mRNA abundance of APN gene was assayed using real-time PCR.The results showed that:during embryonic development, the APN mRNA expression is relatively low,while the APN gene was expressed at highest level in the gastrula stage;during post-embryonic,the mRNA abundance of APN gene, in general, showed a consistent increasing trend from hatching stage to7d post-hatch; and.the APN gene expression changes with some range of fluctuation during14d post-hatch to35d post-hatch,but lower than he7d post-hatch, in general; the APN gene was differentially expressed at different tissues with a gradient from higher to lower among the tissues of fore-intestine, hind-intestine, liver, mid-intestine, kidney, muscle, spleen and heart, respectivelty.The effects of the circadian rhythms on APN expression of C. Idellus showed that there was a time-dependent pattern at higher rhythm during06:00-18:00and lower rhythm during18:00-06:00.
3.The effects of the feeds containing different protein sources on the APN gene expression in the foregut showed that there were no significant differences on the APN mRNA expression in the foregut between the FM group and SM Group at7d; there were significant differences between the FM group and SM Group from14to56d(P<0.05);and the APN mRNA expression of the SM Group was lower than that of the FM group. The effects of the feeds containing different protein levels on the APN gene expression in the foregut showed that, in general, there was a consistent increasing trend from the22.3%CP to27.3%CP group; and there was a consistent downward trend from the27.3%CP to42.1%CP group at different time periods. The results indicated that the APN mRNA expression of Ctenopharyngodon idella correlates tightly to the developmental stage, and showed tissue variability and circadian rhythms.The APN mRNA expression of Ctenopharyngodon idella had adaptive reaction to protein nutrition.
引文
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