鲫鱼(Carassius auratus)半胱亚磺酸脱酸酶CSAD基因克隆及其表达研究
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摘要
本研究以鲫鱼(Carassius auratus)为研究对象,采用巢式PCR和RACE技术克隆鲫鱼半胱亚磺酸脱酸酶CSAD基因c DNA序列,并且利用实时荧光定量PCR检测了CSAD m RNA在鲫鱼不同组织和昼夜节律中的相对表达水平,同时还研究了牛磺酸对鲫鱼肠道CSAD m RNA表达的影响。结果表明:(1)鲫鱼CSAD基因c DNA序列包含186bp的5?UTR序列,675bp的3?UTR序列,1503bp开放阅读框,编码500个氨基酸。同源性分析表明,鲫鱼和鲤鱼的同源性为97.2%。系统发育分析表明,鲫鱼与鲤鱼之间的亲缘关系最接近,置信度为100。经预测,其编码的蛋白质的分子量和等电点分别为56.82k Da和5.77;(2)Ca CSAD m RNA在肌肉、心脏、肠道及肝脏中的表达水平较高,在脑和鳃组织中的相对表达量较低;(3)鲫鱼肠道CSAD m RNA的相对表达量在6:00am点时最高,9:00pm点时相对表达量最低;(4)鲫鱼CSAD的相对表达丰度随着牛磺酸添加量的增加而逐渐下降。本研究结果不仅有助于理解鲫鱼CSAD基因的分子特征,同时将为进一步研究鱼类CSAD营养调控功能提供理论依据。
The CSAD(cysteine sulfinic acid decarboxylase) c DNA sequence in Carassius auratus was cloned using nested PCR(polymerase chain reaction) and RACE(rapid amplification of c DNA ends) techniques, and the relative expression levels of CSAD m RNA in different tissues and circadian rhythms fish and in the intestine of taurine-feed C. auratus were analyzed. The results show the following findings.(1) The cloned c DNA sequence of CSAD was 2364 bp in length, including 186 bp 5′UTR sequence, 675 bp 3′UTR sequence and 1503 bp open reading frame encoding 500 amino acid polypeptide. The homology analysis showed that CSAD gene from C. auratus had 97.2% uniformity with C. carpio. Phylogenetic analysis indicated that the C. auratus CSAD was most closely related to CSAD of C. carpio in confidence level of 100. The encoded protein molecular weight was predicted for 56.82 k Da with a p I of 5.77.(2) The highest expression of the CSAD m RNA was observed in the muscle, heart, intestine, and liver, and the lowest in the brain and gill.(3) The highest expression of intestine CSAD m RNA was found at 6:00 am, and the lowest at 9:00 pm.(4) The relative abundance of the CSAD m RNA decreased gradually with the increase of taurine. Therefore, this work could deepen understanding on the molecular characteristics of CSAD in C. auratus, and provide a theoretical basis for future study on the nutritional regulation of fish CSAD genes.
The CSAD(cysteine sulfinic acid decarboxylase) c DNA sequence in Carassius auratus was cloned using nested PCR(polymerase chain reaction) and RACE(rapid amplification of c DNA ends) techniques, and the relative expression levels of CSAD m RNA in different tissues and circadian rhythms fish and in the intestine of taurine-feed C. auratus were analyzed. The results show the following findings.(1) The cloned c DNA sequence of CSAD was 2364 bp in length, including 186 bp 5′UTR sequence, 675 bp 3′UTR sequence and 1503 bp open reading frame encoding 500 amino acid polypeptide. The homology analysis showed that CSAD gene from C. auratus had 97.2% uniformity with C. carpio. Phylogenetic analysis indicated that the C. auratus CSAD was most closely related to CSAD of C. carpio in confidence level of 100. The encoded protein molecular weight was predicted for 56.82 k Da with a p I of 5.77.(2) The highest expression of the CSAD m RNA was observed in the muscle, heart, intestine, and liver, and the lowest in the brain and gill.(3) The highest expression of intestine CSAD m RNA was found at 6:00 am, and the lowest at 9:00 pm.(4) The relative abundance of the CSAD m RNA decreased gradually with the increase of taurine. Therefore, this work could deepen understanding on the molecular characteristics of CSAD in C. auratus, and provide a theoretical basis for future study on the nutritional regulation of fish CSAD genes.
引文
伍琴,唐建洲,刘臻等,2015.牛磺酸对鲫鱼(Carassius auratus)生长、肠道细胞增殖及蛋白消化吸收相关基因表达的影响.海洋与湖沼,46(6):1516-1523
齐国山,2012.饲料中牛磺酸、蛋氨酸、胱氨酸、丝氨酸和半胱胺对大菱鲆生长性能及牛磺酸合成代谢的影响.青岛:中国海洋大学博士学位论文,43-53
徐亚沙,李书俊,吴芹等,2015.小鼠肝脏胆酸合成及转运基因表达的昼夜节律.遵义医学院学报,38(5):474-478
Dominy J,Eller S,Dawson Jr R,2004.Building biosynthetic schools:reviewing compartmentation of CNS taurine synthesis.Neurochemical Research,29(1):97-103
Goto T,Endou Y,Kitaoka Y et al,2012.Distribution of cysteinesulfinate decarboxylase and cysteamine dioxygenase activities in internal organs of common carp Cyprinus carpio.Aquaculture Science,56(4):607-608
Haga Y,Kondo H,Kumagai A et al,2015.Isolation,molecular characterization of cysteine sulfinic acid decarboxylase(CSD)of red sea bream Pagrus major and yellowtail Seriola quinqueradiata and expression analysis of CSD from several marine fish species.Aquaculture,449:8-17
Honjoh K,Matsuura K,Machida T et al,2010.Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine:co-expression of c DNA clones,from Cyprinus carpio,for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae.Amino Acids,38(4):1173-1183
Hope D B,1955.Pyridoxal phosphate as the coenzyme of the mammalian decarboxylase for L-cysteine sulphinic and L-cysteic acids.Biochemical Journal,59(3):497-500
Ide T,Kushiro M,Takahashi Y et al,2002.m RNA expression of enzymes involved in taurine biosynthesis in rat adipose tissues.Metabolism Clinical and Experimental,51(9):1191-1197
Kim H W,Yoon S H,Park T et al,2006.gene expressions of taurine transporter and taurine biosynthetic enzyme during mouse and chicken embryonic development.Advances in Experimental Medicine and Biology,583:69-77
Li J H,Ling Y Q,Fan J J et al,2006.Expression of cysteine sulfinate decarboxylase(CSD)in male reproductive organs of mice.Histochemistry and Cell Biology,125(6):607-613
Lin C T,Songg X,Wu J Y,1985.Ultrastructural demonstration of L-glutamate decarboxylase and cysteinesulfinic acid decarboxylase in rat retina by immunocytochemistry.Brain Research,331(1):71-80
Matsunari H,Hamada K,Mushiake K et al,2006.Effects of taurine levels in broodstock diet on reproductive performance of yellowtail Seriola quinqueradiata.Fisheries Science,72(5):955-960
Park S Y,Schuller-Levisg,Park E,2015.A novel cysteine sulfinic acid decarboxylase knock-out mouse:immune function.In:Marcinkiewicz J,Schaffer S eds.Taurine 9.Advances in Experimental Medicine and Biology.Cham:Springer International Publishing,803:89-98
Strausberg R L,Feingold E A,Grouse L H et al,2002.generation and initial analysis of more than 15,000 full-length human and mouse c DNA sequences.Proceedings of the National Academy of Sciences of the United States of America,99(26):16899-16903
Takagi S,Murata H,Goto T et al,2006.Hemolytic suppression roles of taurine in yellowtail Seriola quinqueradiata fed non-fishmeal diet based on soybean protein.Fisheries Science,72(3):546-555
Winge I,Teigen K,Fossbakk A et al,2015.Mammalian CSADandg ADL1 have distinct biochemical properties and patterns of brain expression.Neurochemistry International,90:173-184
Xie Zg,Wang F R,Liu H Y et al,2014.Effect of dietary taurine levels ongrowth performance and taurine content of Nibea albiflora larvae.Aquaculture International,22(6):1851-1862
Yamamoto T,Unuma T,Akiyama T,2000.The influence of dietary protein sources on tissue free amino acid levels of fingerling rainbow trout.Fisheries Science,66(2):310-320
Yang J C,Wug F,Feng Y et al,2010.CSD m RNA expression in rat testis and the effect of taurine on testosterone secretion.Amino Acids,39(1):155-160
齐国山,2012.饲料中牛磺酸、蛋氨酸、胱氨酸、丝氨酸和半胱胺对大菱鲆生长性能及牛磺酸合成代谢的影响.青岛:中国海洋大学博士学位论文,43-53
徐亚沙,李书俊,吴芹等,2015.小鼠肝脏胆酸合成及转运基因表达的昼夜节律.遵义医学院学报,38(5):474-478
Dominy J,Eller S,Dawson Jr R,2004.Building biosynthetic schools:reviewing compartmentation of CNS taurine synthesis.Neurochemical Research,29(1):97-103
Goto T,Endou Y,Kitaoka Y et al,2012.Distribution of cysteinesulfinate decarboxylase and cysteamine dioxygenase activities in internal organs of common carp Cyprinus carpio.Aquaculture Science,56(4):607-608
Haga Y,Kondo H,Kumagai A et al,2015.Isolation,molecular characterization of cysteine sulfinic acid decarboxylase(CSD)of red sea bream Pagrus major and yellowtail Seriola quinqueradiata and expression analysis of CSD from several marine fish species.Aquaculture,449:8-17
Honjoh K,Matsuura K,Machida T et al,2010.Enhancement of menadione stress tolerance in yeast by accumulation of hypotaurine and taurine:co-expression of c DNA clones,from Cyprinus carpio,for cysteine dioxygenase and cysteine sulfinate decarboxylase in Saccharomyces cerevisiae.Amino Acids,38(4):1173-1183
Hope D B,1955.Pyridoxal phosphate as the coenzyme of the mammalian decarboxylase for L-cysteine sulphinic and L-cysteic acids.Biochemical Journal,59(3):497-500
Ide T,Kushiro M,Takahashi Y et al,2002.m RNA expression of enzymes involved in taurine biosynthesis in rat adipose tissues.Metabolism Clinical and Experimental,51(9):1191-1197
Kim H W,Yoon S H,Park T et al,2006.gene expressions of taurine transporter and taurine biosynthetic enzyme during mouse and chicken embryonic development.Advances in Experimental Medicine and Biology,583:69-77
Li J H,Ling Y Q,Fan J J et al,2006.Expression of cysteine sulfinate decarboxylase(CSD)in male reproductive organs of mice.Histochemistry and Cell Biology,125(6):607-613
Lin C T,Songg X,Wu J Y,1985.Ultrastructural demonstration of L-glutamate decarboxylase and cysteinesulfinic acid decarboxylase in rat retina by immunocytochemistry.Brain Research,331(1):71-80
Matsunari H,Hamada K,Mushiake K et al,2006.Effects of taurine levels in broodstock diet on reproductive performance of yellowtail Seriola quinqueradiata.Fisheries Science,72(5):955-960
Park S Y,Schuller-Levisg,Park E,2015.A novel cysteine sulfinic acid decarboxylase knock-out mouse:immune function.In:Marcinkiewicz J,Schaffer S eds.Taurine 9.Advances in Experimental Medicine and Biology.Cham:Springer International Publishing,803:89-98
Strausberg R L,Feingold E A,Grouse L H et al,2002.generation and initial analysis of more than 15,000 full-length human and mouse c DNA sequences.Proceedings of the National Academy of Sciences of the United States of America,99(26):16899-16903
Takagi S,Murata H,Goto T et al,2006.Hemolytic suppression roles of taurine in yellowtail Seriola quinqueradiata fed non-fishmeal diet based on soybean protein.Fisheries Science,72(3):546-555
Winge I,Teigen K,Fossbakk A et al,2015.Mammalian CSADandg ADL1 have distinct biochemical properties and patterns of brain expression.Neurochemistry International,90:173-184
Xie Zg,Wang F R,Liu H Y et al,2014.Effect of dietary taurine levels ongrowth performance and taurine content of Nibea albiflora larvae.Aquaculture International,22(6):1851-1862
Yamamoto T,Unuma T,Akiyama T,2000.The influence of dietary protein sources on tissue free amino acid levels of fingerling rainbow trout.Fisheries Science,66(2):310-320
Yang J C,Wug F,Feng Y et al,2010.CSD m RNA expression in rat testis and the effect of taurine on testosterone secretion.Amino Acids,39(1):155-160