达氏鳇谷氨酸脱氢酶基因的克隆及双低菜粕添加对其表达的影响
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摘要
谷氨酸脱氢酶(glutamate dehydrogenase,GDH)可通过逆催化谷氨酸脱氨参与氨基酸代谢,也可生产合成氨,参与氮代谢。实验克隆得到达氏鳇(Huso dauricus)谷氨酸脱氢酶基因(gdh)的开放阅读框序列。该序列全长为1 635 bp,编码544个氨基酸。氨基酸序列多重比对结果显示达氏鳇GDH与其它脊椎动物的序列一致性较高。进一步的系统进化树分析发现,达氏鳇GDH与两栖类、爬行类及哺乳类聚为一支。组织表达分析结果表明,达氏鳇gdh的mRNA在各组织中都有分布,并且在肠中转录水平最高。随后,采用荧光定量PCR分析了饲料中添加双低菜粕对达氏鳇蛋白质代谢相关基因(gdh、氨肽酶N apN、小肽转运载体pepT1)及应激反应相关的热休克蛋白(hsp90)基因表达的影响。在肝脏中,不同饲料投喂组gdh和hsp90的转录水平没有显著差异。在肠中,菜粕组gdh及apN基因的转录水平显著高于基础饲料组;菜粕组pepT1基因的转录水平也高于基础饲料组,但是没有显著性差异。以上结果表明,植物蛋白原料菜粕添加可引起达氏鳇肠道中蛋白质代谢水平上升,而不引起鱼体的应激反应。
Glutamate dehydrogenase(GDH) plays vital roles in amino acid metabolism by reversely catalyzing glutamate deamination, or participates in the nitrogen metabolism cycle by producing ammonia. The open reading frame cDNA sequences of gdh was cloned in Kaluga Huso dauricus, which was 1,635 bp encoding 544 amino acids. Multiple amino acid sequence alignment showed that GDH of Kaluga shared high sequence similarities with its counterpart of other vertebrates. Phylogenetic tree analysis indicated that GDH of Kaluga clustered with amphibians, reptiles and mammals. Besides, tissue expression detection presented extensive distribution of gdh in all tissues analyzed, with the highest transcription in intestine. In addition, the transcription changes of three digestion-related genes(gdh, aminopeptidase N apN and peptide transporter pepT1) and the stress-related gene hsp90 caused by the addition of double-low rapeseed meal were analyzed by relative real-time PCR. In the liver, the transcription levels of both gdh and hsp90 were not changed in these two groups. However, in the intestine, the transcription levels of both gdh and apN were significantly higher in the rapeseed meal group than that in the basal control meal group. The transcription level of pepT1 in the rapeseed meal group was also higher than that in the basal control meal group, but with no statistical significance. The above result indicates that the plant-sourced rapeseed meal addition promotes the protein metabolism level with no stress response in the Kaluga.
Glutamate dehydrogenase(GDH) plays vital roles in amino acid metabolism by reversely catalyzing glutamate deamination, or participates in the nitrogen metabolism cycle by producing ammonia. The open reading frame cDNA sequences of gdh was cloned in Kaluga Huso dauricus, which was 1,635 bp encoding 544 amino acids. Multiple amino acid sequence alignment showed that GDH of Kaluga shared high sequence similarities with its counterpart of other vertebrates. Phylogenetic tree analysis indicated that GDH of Kaluga clustered with amphibians, reptiles and mammals. Besides, tissue expression detection presented extensive distribution of gdh in all tissues analyzed, with the highest transcription in intestine. In addition, the transcription changes of three digestion-related genes(gdh, aminopeptidase N apN and peptide transporter pepT1) and the stress-related gene hsp90 caused by the addition of double-low rapeseed meal were analyzed by relative real-time PCR. In the liver, the transcription levels of both gdh and hsp90 were not changed in these two groups. However, in the intestine, the transcription levels of both gdh and apN were significantly higher in the rapeseed meal group than that in the basal control meal group. The transcription level of pepT1 in the rapeseed meal group was also higher than that in the basal control meal group, but with no statistical significance. The above result indicates that the plant-sourced rapeseed meal addition promotes the protein metabolism level with no stress response in the Kaluga.
引文
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[14]周发林,陈劲松,黄建华,等.斑节对虾谷氨酸脱氢酶基因克隆及氨氮胁迫对其时空表达的影响[J].中国水产科学,2016,23(6):1236-1246.
[15]刘臻.不同倍性鲫鲤蛋白质代谢相关基因的克隆与表达研究[D].长沙:湖南师范大学,2012.
[16]钟文英,普雄明.热休克蛋白的分子生物学研究进展[J].医学综述,2005,11(2):148-150.
[17] Sherriff R M,Broom M F,Chadwick V S.Isolation and purification of N-formylmethionine aminopeptidase from rat intestine [J].BBA-Protein Struct M,1992,1119:275-280.
[18] Tang J Z,Qu F F,Tang X B,et al.Molecular characterization and dietary regulation of aminopeptidase N(APN) in the grass carp(Ctenopharyngodo nidella) [J].Gene,2016,582:77-84.
[19] Brandsch M,Knütter I,Leibach FH.The intestinal H+/peptide symporter PEPT1:structure affinity relationships [J].Euro J Pharm Sci,2004,21:53-60.
[2] Higgs D A,McBride J R,Markert J R,et al.Evaluation of tower and candle rapeseed(canola) meal and bronowski rapeseed protein concentrate as protein supplements in practical dry diets for juvenile chinook salmon(Oncorhynchus tshawytscha) [J].Aquaculture,1982,29(2):1-31.
[3] Che J,Su B,Tang B,et al.Apparent digestibility coefficients of animal and plant feed ingredients for juvenile Pseudobagrus ussuriensis [J].Aquacul Nutr,2016,23:1128-1135.
[4]李会涛,麦康森,艾庆辉,等.大黄鱼对几种饲料蛋白原料消化率的研究[J].饲料工业,2007,31(3):370-375.
[5] Safari O,Naserizadeh M,Mohammadi A.Digestibility of selected feedstuffs in sub-adult Caspian great sturgeon,Huso huso using settlement faecal collection and stripping methods [J].Aquacul Nutr,2016,22:293-303.
[6] Anderson J S,Jackson A J,MattyA J,et al.Effect of dietary carbohydrate and fiber on the tilapia Orechromis niloticus (Linn.) [J].Aquaculture,1984,37:303-314.
[7]谢小军,邓利,张波.饥饿对鱼类生理生态学影响的研究进展[J].水生生物学报,1998,22(2):181-183.
[8]王镜岩,朱圣庚,徐长法.生物化学[M].北京:高等教育出版社,2002.
[9]韩骥,王云山,李文龙.黑龙江达氏鳇苗种培育技术[J].河北渔业,2018,(1):39-40.
[10]吴金平,禇志鹏,陈细华,等.达氏鳇幼鱼对六种蛋白质原料中营养物质的表观消化率[J].动物营养学报,2018,30(11):4482-4489.
[11] Peng G G,Zhao W,Shi Z G,et al.Cloning HSP70 and HSP90 genes of kaluga(Huso dauricus) and the effects of temperature and salinity stress on their gene expression [J].Cell Stress Chaperone,2016,21:349-359.
[12] Li E,Arena L,Chen L,et al.Characterization and tissue-specific expression of the two glutamate dehydrogenase cDNAs in Pacific white shrimp,Litopenaeus vannamei [J].J Crust Biol,2009,29(3):379-386.
[13]李少飞.中国对虾氨氮代谢酶基因的cDNA克隆及其在氨氮解毒代谢过程中的作用[D].大连:大连海洋大学,2014.
[14]周发林,陈劲松,黄建华,等.斑节对虾谷氨酸脱氢酶基因克隆及氨氮胁迫对其时空表达的影响[J].中国水产科学,2016,23(6):1236-1246.
[15]刘臻.不同倍性鲫鲤蛋白质代谢相关基因的克隆与表达研究[D].长沙:湖南师范大学,2012.
[16]钟文英,普雄明.热休克蛋白的分子生物学研究进展[J].医学综述,2005,11(2):148-150.
[17] Sherriff R M,Broom M F,Chadwick V S.Isolation and purification of N-formylmethionine aminopeptidase from rat intestine [J].BBA-Protein Struct M,1992,1119:275-280.
[18] Tang J Z,Qu F F,Tang X B,et al.Molecular characterization and dietary regulation of aminopeptidase N(APN) in the grass carp(Ctenopharyngodo nidella) [J].Gene,2016,582:77-84.
[19] Brandsch M,Knütter I,Leibach FH.The intestinal H+/peptide symporter PEPT1:structure affinity relationships [J].Euro J Pharm Sci,2004,21:53-60.