卵形鲳鲹AQP1a分子特征及其对急性盐度胁迫的表达响应
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摘要
水通道蛋白(aquaporin,AQPs)是由内在膜蛋白组成的超家族,介导水分子的跨膜运输,对渗透压调节具有重要作用。为研究AQP1a在急性盐度胁迫下对卵形鲳鲹(Trachinotus ovatus)的渗透压调节作用,该研究获得AQP1a基因。基因全长1 078 bp,开放阅读框786 bp,编码261个氨基酸,具有MIP家族特有序列(HINPAVTLG)和2个天冬酰胺-脯氨酸-丙氨酸蛋白基序。q RT-PCR结果显示,AQP1a在11个被测组织中均有表达,在性腺中的表达量最高,其次是鳃和肠,在肌肉中表达量最低。在急性盐度胁迫下,当转入淡水时,AQP1a在鳃的表达量在第4小时显著上升,而在肾和肠中没有显著变化;当转入盐度10和20海水时,鳃中AQP1a的表达量在第2和第4小时显著升高,然后下降趋于稳定;转入盐度10海水时,肠中AQP1a的表达量均上升,肾中AQP1a的表达量呈先上升后下降趋势;转入盐度20海水时,肠中AQP1a的表达量仅在第4、第8和第48小时显著上升,肾中AQP1a的表达量在第12小时达到最大;转入盐度40海水中,鳃中AQP1a的表达量明显下降,相反,在肾和肠中AQP1a的表达量均明显上调。这些结果反映了AQP1a在不同组织中的功能特异性,证实了AQP1a在卵形鲳鲹盐度适应中起重要作用。
Aquaporin, a superfamily of internal membrane proteins that mediate transmembrane transport of water molecules, plays an important role in osmotic adjustment. AQP1 a gene was obtained in order to study the role of AQP1 a in osmoregulation of Trachinotus ovatus under acute salinity stress. The sequence of AQP1 a gene was 1 078 bp with an open reading frame of 786 bp encoding 261 amino acids. The structural analysis shows that it has the structural characteristics of MIP family-specific sequence(HINPAVTLG) and two asparagine-proline-alanine(NPA) motifs. The q RT-PCR results show that AQP1 a was distributed in the 11 tested tissues, highest in gonads and then in gill, intestine and liver, lowest in muscle. Under acute salinity stress, after being transfered to fresh water, the expression of AQP1 a in gill increased at 4 th hour, while there was no significant change in the expression in kidney and intestine. After being transfered to 10‰ and 20‰ salinity seawater, the expression of AQP1 a in gill increased significantly at 2 nd and 4 th hour, and then decreased gradually. When being transferred to 10‰ salinity seawater, the expression of AQP1 a in intestine increased. In kidney, the expression of AQP1 a first increased and then decreased. When being transferred to 20‰ salinity seawater, the expression of AQP1 a in intestine only increased significantly at 4 th, 8 th and 48 th hour. In kidney, the expression of AQP1 a reached the maximum at 12 th hour. In 40‰ salinity seawater, the expression of AQP1 a in gill decreased significantly. On the contrary, the expression of AQP1 a in kidney and intestine were significantly up-regulated. The results reveal that the specificity of AQP1 a functions in different tissues and plays an important role in T.ovatus salinity adaptation.
Aquaporin, a superfamily of internal membrane proteins that mediate transmembrane transport of water molecules, plays an important role in osmotic adjustment. AQP1 a gene was obtained in order to study the role of AQP1 a in osmoregulation of Trachinotus ovatus under acute salinity stress. The sequence of AQP1 a gene was 1 078 bp with an open reading frame of 786 bp encoding 261 amino acids. The structural analysis shows that it has the structural characteristics of MIP family-specific sequence(HINPAVTLG) and two asparagine-proline-alanine(NPA) motifs. The q RT-PCR results show that AQP1 a was distributed in the 11 tested tissues, highest in gonads and then in gill, intestine and liver, lowest in muscle. Under acute salinity stress, after being transfered to fresh water, the expression of AQP1 a in gill increased at 4 th hour, while there was no significant change in the expression in kidney and intestine. After being transfered to 10‰ and 20‰ salinity seawater, the expression of AQP1 a in gill increased significantly at 2 nd and 4 th hour, and then decreased gradually. When being transferred to 10‰ salinity seawater, the expression of AQP1 a in intestine increased. In kidney, the expression of AQP1 a first increased and then decreased. When being transferred to 20‰ salinity seawater, the expression of AQP1 a in intestine only increased significantly at 4 th, 8 th and 48 th hour. In kidney, the expression of AQP1 a reached the maximum at 12 th hour. In 40‰ salinity seawater, the expression of AQP1 a in gill decreased significantly. On the contrary, the expression of AQP1 a in kidney and intestine were significantly up-regulated. The results reveal that the specificity of AQP1 a functions in different tissues and plays an important role in T.ovatus salinity adaptation.
引文
[1]AN K W,KIM N N,CHOI C Y.Cloning and expression of aquaporin 1 and arginine vasotocin receptor m RNA from the black porgy,Acanthopagrus schlegeli:effect of freshwater acclimation[J].Fish Physiol Biochem,2008,34(2):185-194.
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[11]MARTINEZ A S,CUTLER C P,WILSON G D,et al.Regulation of expression of two aquaporin homologs in the intestine of the European eel:effects of seawater acclimation and cortisol treatment[J].Am J Physiol Regul Integr Comp Physiol,2005,288(6):R1733-R1743.
[12]JEONG S Y,KIM J H,LEE W O,et al.Salinity changes in the anadromous river pufferfish,Takifugu obscurus,mediate gene regulation[J].Fish Physiol Biochem,2014,40(1):205-219.
[13]KIM Y K,LEE S Y,KIM B S,et al.Isolation and m RNA expression analysis of aquaporin isoforms in marine medaka Oryzias dancena,a euryhaline teleost[J].Comp Biochem Physiol A,2014,171(5):1-8.
[14]IP Y K,SOH M M,CHEN X L,et al.Molecular characterization of branchial aquaporin 1aa and effects of seawater acclimation,emersion or ammonia exposure on its m RNA expression in the gills,gut,kidney and skin of the freshwater climbing perch,Anabas testudineus[J].PLo S One,2013,8(4):e61163.
[15]GIFFARD-MENA I,BOULO V,AUJOULAT F,et al.Aquaporin molecular characterization in the sea-bass(Dicentrarchus labrax):the effect of salinity on AQP1 and AQP3 expression[J].Comp Biochem Physiol A,2007,148(2):430-444.
[16]于文博,朱克诚,郭华阳,等.卵形鲳鲹MHCⅡβ基因的克隆与表达分析[J].南方水产科学,2017,13(4):69-79.
[17]GEYER R R,MUSA-AZIZ R,QIN X,et al.Relative CO(2)/NH(3)selectivity's of mammalian aquaporins 0-9[J].Am JPhysiol Cell Physiol,2013,304(10):C985-C994.
[18]FINN R N,CHAUVIGNéF,HLIDBERG J B,et al.The lineagespecific evolution of aquaporin gene clusters facilitated tetrapod terrestrial adaptation[J].PLo S One,2014,9(11):e113686.
[19]KONG Y,MA J.Dynamic mechanisms of the membrane water channel aquaporin-1(AQP1)[J].Proc Natl Acad Sci USA,2001,98(25):14345-14349.
[20]FUJIYOSHI Y,MITSUOKA K,de GROOT B L,et al.Structure and function of water channels[J].Curr Opin Struct Biol,2002,12(4):509-515.
[21]De GROOT B L,GRUBMüLLER H.Water permeation across biological membranes:mechanism and dynamics of aquaporin-1and Glp F[J].Science,2001,294(5550):2353-2357.
[22]BEITZ E,WU B,HOLM L M,et al.Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea,glycerol,ammonia,and protons[J].Proc Natl Acad Sci USA,2006,103(2):269-274.
[23]姜勇,麻彤辉.NPA motif在水通道蛋白AQP1表达和转水功能中的重要性[J].科学通报,2007,52(4):426-431.
[24]WALZ T,HIRAI T,MURATA K,et al.The three-dimensional structure of aquaporin-1[J].Nature,1997,387(6633):624-627.
[25]丁炜东.黄鳝性别分化相关候选基因的筛选及相关功能研究[D].南京:南京农业大学,2016:76-83
[26]姚小皓,李学军.水孔蛋白1的结构与功能[J].生理科学进展,2000,31(4):345-348.
[27]BOJ M,CHAUVIGN?F,ZAPATER C,et al.Gonadotropin-activated androgen-dependent and independent pathways regulate aquaporin expression during teleost(Sparus aurata)spermatogenesis[J].PLo S One,2015,10(11):e0142512.
[28]AOKI M,KANEKO T,KATOH F,et al.Intestinal water absorption through aquaporin 1 expressed in the apical membrane of mucosal epithelial cells in seawater-adapted Japanese eel[J].J Exp Biol,2003,206(19):3495-3505.
[29]丁炜东,曹丽萍,曹哲明,等.黄鳝AQP1 c DNA的克隆与表达分析[J].华北农学报,2012,27(S1):6-11.
[30]SANG Y L,NAM Y K,YI K K.Characterization and expression profiles of aquaporins(AQPs)1a and 3a in mud loach Misgurnus mizolepis after experimental challenges[J].Fish Aquat Sci,2017,20(1):23.
[31]王美垚,杨健,徐跑,等.刀鲚水通道蛋白1的分子克隆及高盐作用下的表达分析[J].中国水产科学,2017,24(3):449-458.
[2]CHNG Y R,ONG J L,CHING B,et al.Molecular characterization of aquaporin 1 and aquaporin 3 from the gills of the African lungfish,Protopterus annectens,and changes in their branchial m RNA expression levels and protein abundance during three phases of aestivation[J].Front Physiol,2016,7:532.
[3]ABASCAL F,IRISARRI I,ZARDOYA R.Diversity and evolution of membrane intrinsic proteins[J].Biochim Biophys Acta,2014,1840(5):1468-1481.
[4]FINN R N,CERDàJ.Evolution and functional diversity of aquaporins[J].Biol Bull,2015,229(1):6-23.
[5]AGRE P,SASAKI S,CHRISPEELS M J.Aquaporins:a family of water channel proteins[J].Am J Physiol,1993,265(3 Pt 2):F461.
[6]TINGAUD-SEQUEIRA A,CHAUVIGNéF,FABRA M,et al.Structural and functional divergence of two fish aquaporin-1 water channels following teleost-specific gene duplication[J].BMCEvol Biol,2008,8(1):259.
[7]CHEN L M,ZHAO J,MUSA-AZIZ R,et al.Cloning and characterization of a zebrafish homologue of human AQP1:a bifunctional water and gas channel[J].Am J Physiol Regul Integr Comp Physiol,2010,299(5):R1163-R1174.
[8]DEANE E E,LUK J C Y,WOO N Y S.Aquaporin 1a expression in gill,intestine,and kidney of the euryhaline silver sea bream[J].Front Physiol,2011,2:39.
[9]GUO H,WEI M,LIU Y,et al.Molecular cloning and expression analysis of the aqp1aa gene in half-smooth tongue sole(Cynoglossus semilaevis)[J].PLo S One,2017,12(4):e175033.
[10]TIPSMARK C K,S?RENSEN K J,MADSEN S S.Aquaporin expression dynamics in osmoregulatory tissues of Atlantic salmon during smoltification and seawater acclimation[J].J Exp Biol,2010,213(3):368-379.
[11]MARTINEZ A S,CUTLER C P,WILSON G D,et al.Regulation of expression of two aquaporin homologs in the intestine of the European eel:effects of seawater acclimation and cortisol treatment[J].Am J Physiol Regul Integr Comp Physiol,2005,288(6):R1733-R1743.
[12]JEONG S Y,KIM J H,LEE W O,et al.Salinity changes in the anadromous river pufferfish,Takifugu obscurus,mediate gene regulation[J].Fish Physiol Biochem,2014,40(1):205-219.
[13]KIM Y K,LEE S Y,KIM B S,et al.Isolation and m RNA expression analysis of aquaporin isoforms in marine medaka Oryzias dancena,a euryhaline teleost[J].Comp Biochem Physiol A,2014,171(5):1-8.
[14]IP Y K,SOH M M,CHEN X L,et al.Molecular characterization of branchial aquaporin 1aa and effects of seawater acclimation,emersion or ammonia exposure on its m RNA expression in the gills,gut,kidney and skin of the freshwater climbing perch,Anabas testudineus[J].PLo S One,2013,8(4):e61163.
[15]GIFFARD-MENA I,BOULO V,AUJOULAT F,et al.Aquaporin molecular characterization in the sea-bass(Dicentrarchus labrax):the effect of salinity on AQP1 and AQP3 expression[J].Comp Biochem Physiol A,2007,148(2):430-444.
[16]于文博,朱克诚,郭华阳,等.卵形鲳鲹MHCⅡβ基因的克隆与表达分析[J].南方水产科学,2017,13(4):69-79.
[17]GEYER R R,MUSA-AZIZ R,QIN X,et al.Relative CO(2)/NH(3)selectivity's of mammalian aquaporins 0-9[J].Am JPhysiol Cell Physiol,2013,304(10):C985-C994.
[18]FINN R N,CHAUVIGNéF,HLIDBERG J B,et al.The lineagespecific evolution of aquaporin gene clusters facilitated tetrapod terrestrial adaptation[J].PLo S One,2014,9(11):e113686.
[19]KONG Y,MA J.Dynamic mechanisms of the membrane water channel aquaporin-1(AQP1)[J].Proc Natl Acad Sci USA,2001,98(25):14345-14349.
[20]FUJIYOSHI Y,MITSUOKA K,de GROOT B L,et al.Structure and function of water channels[J].Curr Opin Struct Biol,2002,12(4):509-515.
[21]De GROOT B L,GRUBMüLLER H.Water permeation across biological membranes:mechanism and dynamics of aquaporin-1and Glp F[J].Science,2001,294(5550):2353-2357.
[22]BEITZ E,WU B,HOLM L M,et al.Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea,glycerol,ammonia,and protons[J].Proc Natl Acad Sci USA,2006,103(2):269-274.
[23]姜勇,麻彤辉.NPA motif在水通道蛋白AQP1表达和转水功能中的重要性[J].科学通报,2007,52(4):426-431.
[24]WALZ T,HIRAI T,MURATA K,et al.The three-dimensional structure of aquaporin-1[J].Nature,1997,387(6633):624-627.
[25]丁炜东.黄鳝性别分化相关候选基因的筛选及相关功能研究[D].南京:南京农业大学,2016:76-83
[26]姚小皓,李学军.水孔蛋白1的结构与功能[J].生理科学进展,2000,31(4):345-348.
[27]BOJ M,CHAUVIGN?F,ZAPATER C,et al.Gonadotropin-activated androgen-dependent and independent pathways regulate aquaporin expression during teleost(Sparus aurata)spermatogenesis[J].PLo S One,2015,10(11):e0142512.
[28]AOKI M,KANEKO T,KATOH F,et al.Intestinal water absorption through aquaporin 1 expressed in the apical membrane of mucosal epithelial cells in seawater-adapted Japanese eel[J].J Exp Biol,2003,206(19):3495-3505.
[29]丁炜东,曹丽萍,曹哲明,等.黄鳝AQP1 c DNA的克隆与表达分析[J].华北农学报,2012,27(S1):6-11.
[30]SANG Y L,NAM Y K,YI K K.Characterization and expression profiles of aquaporins(AQPs)1a and 3a in mud loach Misgurnus mizolepis after experimental challenges[J].Fish Aquat Sci,2017,20(1):23.
[31]王美垚,杨健,徐跑,等.刀鲚水通道蛋白1的分子克隆及高盐作用下的表达分析[J].中国水产科学,2017,24(3):449-458.