大黄鱼Lc-UBE2D4基因的克隆及其表达分析
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摘要
泛素缀合酶E2是蛋白质泛素化系统中的关键酶,对降解的靶蛋白的特异性选择起决定性作用,参与了细胞内80%以上的蛋白降解,对于细胞周期调控、细胞凋亡、基因转录及表达等生理活动具有重要的调控作用。本研究从已构建的大黄鱼(Larimichthys crocea)性腺线性化c DNA文库中筛选出泛素缀合酶(Ubiquitin-conjugating enzymes E2 D4,Lc-UBE2D4)同源基因片段,利用SMART-RACE方法克隆出其全长c DNA序列,并利用荧光定量PCR技术检测其在大黄鱼不同组织和性腺不同发育阶段的差异表达情况。结果显示,Lc-UBE2D4基因全长798 bp,其中ORF为441 bp,可编码147个氨基酸,含有一段典型的泛素缀合酶UBC结构域及其半胱氨酸激活位点。定量PCR结果分析发现,Lc-UBE2D4在脾脏和性腺中表达量较高,在脾脏和精巢的表达水平极显著高于其他各组织(P<0.01),同时在卵巢中的表达亦显著高于除脾脏和精巢外的其他各组织(P<0.05);通过对Lc-UBE2D4基因在性腺不同发育阶段表达模式的研究发现,该基因在精巢3个时期中的表达水平显著高于各发育阶段的卵巢(P<0.05),推测Lc-UBE2D4基因在大黄鱼性腺发育过程中起着重要的调节作用,脾脏中的高表达也暗示其可能参与免疫机能。
Protein ubiquitination is a fundamental post-translational modification event that serves as a signaling function in diversebiological processes,including cell-cycle progression,cell apoptosis,gene transcriptional regulation and expression,etc. In these processesof ubiquitin-proteasome pathway,ubiquitin-conjugating enzyme E2 is of pivotal importance that mediates more than 80 percent of proteindegradation of eukaryotic cells,and it plays a key role on the specific selection of degraded of target protein. In this study,a homologous genefragment of ubiquitin-conjugating enzymes E2D4(Lc-UBE2D4)was screened from previous constructed normalized gonad cDNA library oflarge yellow croaker Larimichthys crocea was obtained,and its full length cDNA was cloned by using SMART-RACE technology,and thenthe differential expression profiles in different tissues and different gonadal development stages were analyzed using quantitative real time PCR(q RT-PCR). The full length cDNA of Lc-UBE2 D4 gene was of 798 bp,with 441 bp of ORF encoding a protein of 147 amino acids,and itcontained the highly conserved ubiquitin-conjugating enzymes UBC domain and an active site cysteine. The q RT-PCR results revealed that thehighest expression level of Lc-UBE2 D4 presented in spleen and testis which were extremely higher than those in other tissues(P < 0.01),andits expression level in ovary was significantly higher than other organs(excluding spleen and testis)(P < 0.05). Moreover,its expressionlevels in three stages of testis were higher than in the same growth stages of ovary(P < 0.05). These results suggested that Lc-UBE2 D4 playedessential roles in the gonad development,especially in testis development. Meanwhile,Lc-UBE2 D4 may be involved in immune function dueto highest expression in spleen.
Protein ubiquitination is a fundamental post-translational modification event that serves as a signaling function in diversebiological processes,including cell-cycle progression,cell apoptosis,gene transcriptional regulation and expression,etc. In these processesof ubiquitin-proteasome pathway,ubiquitin-conjugating enzyme E2 is of pivotal importance that mediates more than 80 percent of proteindegradation of eukaryotic cells,and it plays a key role on the specific selection of degraded of target protein. In this study,a homologous genefragment of ubiquitin-conjugating enzymes E2D4(Lc-UBE2D4)was screened from previous constructed normalized gonad cDNA library oflarge yellow croaker Larimichthys crocea was obtained,and its full length cDNA was cloned by using SMART-RACE technology,and thenthe differential expression profiles in different tissues and different gonadal development stages were analyzed using quantitative real time PCR(q RT-PCR). The full length cDNA of Lc-UBE2 D4 gene was of 798 bp,with 441 bp of ORF encoding a protein of 147 amino acids,and itcontained the highly conserved ubiquitin-conjugating enzymes UBC domain and an active site cysteine. The q RT-PCR results revealed that thehighest expression level of Lc-UBE2 D4 presented in spleen and testis which were extremely higher than those in other tissues(P < 0.01),andits expression level in ovary was significantly higher than other organs(excluding spleen and testis)(P < 0.05). Moreover,its expressionlevels in three stages of testis were higher than in the same growth stages of ovary(P < 0.05). These results suggested that Lc-UBE2 D4 playedessential roles in the gonad development,especially in testis development. Meanwhile,Lc-UBE2 D4 may be involved in immune function dueto highest expression in spleen.
引文
[1]韩坤煌,张子平,王艺磊,等.Cyclin-CDK-CKI及UPP参与生殖调控及在甲壳动物性腺发育中的研究进展[J].生物技术通报,2010(7):48-54.
[2]Varshavsky A.The Ubiquitin System[J].Trends Biochem Sci,1997,22(10):383-387.
[3]Baarends WM,Roest HP,Grootegoed JA.The ubiquitin system ingametogenesis[J].Mol Cell Endocrinol,1999,151(1-2):5-16.
[4]国果,吴沁怡,吴建伟,等.家蝇泛素结合酶基因的生物信息学分析[J].生物技术通报,2014(2):107-111.
[5]Schwob E,B?hm T,Mendenhall MD,et al.The B-type cyclinkinase inhibitor p40SIC1 controls the G1 to S transition in S.cerevisiae[J].Cell,1994,79(2):233-244.
[6]Yew PR,Kirschner MW.Proteolysis and DNA replication:theCDC34 requirement in the Xenopus egg cell cycle[J].Science,1997,277(5332):1672-1676.
[7]Reymond F,Wirbelauer C,Krek W.Association of humanubiquitin-conjugating enzyme CDC34 with the mitotic spindle inanaphase[J].J Cell Sci,2000,113(Pt 10):1687-1694.
[8]Pati D,Meistrich ML,Plon SE.Human Cdc34 and Rad6B ubiquitinconjugating enzymes target repressors of cyclic AMP-inducedtranscription for proteolysis[J].Mol Cell Biol,1999,19(7):5001-5013.
[9]Von Hofsten J,Olsson PE.Zebrafish sex determination anddifferentiation:involvement of FTZ-F1 genes[J].Reprod BiolEndocrinol,2005,3(1):63.
[10]刘家富.大黄鱼养殖与生物学[M].厦门:厦门大学出版社,2013.
[11]农业部渔业渔政管理局.2016年中国渔业统计年鉴[M].北京:中国农业出版社,2016.
[12]Manku G,Wing SS,Culty M.Expression of the ubiquitinproteasome system in neonatal rat gonocytes and spermatogonia:role in gonocyte differentiation[J].Biol Reprod,2001,87(2):44.
[13]Shen B,Zhang Z,Wang Y,et al.Differential expression ofubiquitin-conjugating enzyme E2r in the developing ovary andtestis of penaeid shrimp Marsupenaeus japonicus[J].Mol BiolRep,2009,36(5):1149-1157.
[14]Koenig PA,Nicholls PK,Schmidt FI,et al.The E2 ubiquitinconjugating enzyme UBE2J1 is required for spermiogenesis inmice[J].J Biol Chem,2014,289(50):34490-34502.
[15]钱照君,祝天谅,姜虎成,等.克氏原螯虾泛素结合酶E2r基因的克隆及表达分析[J].上海海洋大学学报,2016,5:641-651.
[16]唐蕾,傅明骏,刘文生,等.斑节对虾UBE2H基因克隆及其表达[J].南方农业学报,2016,47(11):1958-1965.
[17]向云龙,张亮,陈卓.过表达泛素偶联酶(UBE2C)对小鼠胚胎发育的影晌[J].黑龙江动物繁殖,2014,22(1):3-7.
[18]胡月阳.UBE2C基因在斑马鱼胚胎发育中的时空表达规律[D].石家庄:河北医科大学,2015.
[19]Zhou P,Zhang ZP,Wang YL,et al.EST analysis and identificationof gonad-related genes from the normalized c DNA library of largeyellow croaker,Larimichthys crocea[J].Comp Biochem PhysiolPart D Genomics Proteomics,2010,5(2):89-97.
[20]陈芸,周鹏,张子平,等.大黄鱼性腺线性化c DNA文库的构建及生殖免疫相关基因的筛选[J].生物技术通报,2011(7):180-185.
[21]周鹏,张子平,王艺磊,等.大黄鱼ubc9基因的克隆和组织表达[J].生物技术通报,2009(8):76-82.
[22]Jiang Y,Han K,Chen S,et al.Characterization and expressionanalysis of Lc-Sox4 in large yellow croaker Larimichthys crocea[J].Gene,2015,574(2):287-301.
[23]Zhang Z,Wu RS,Mok HO,et al.Isolation,characterization andexpression analysis of a hypoxia-responsive glucose transportergene from the grass carp,Ctenopharyngodon idellus[J].Eur JBiochem,2003,270(14):3010-3017.
[24]周鹏.若干大黄鱼性腺发育相关基因的克隆与表达[D].厦门:集美大学,2009.
[25]姚杨,苏杰,魏迪,等.泛素结合酶基因生物信息学与功能分析[J].医学研究生学报,2014,11:1156-1159.
[26]Hochstrasser M.Ubiquitin-Dependent Protein Degradation[J].Annu Rev Genet,1996,30:405-439.
[27]van Wijk SJ,Timmers HT.The family of ubiquitin-conjugatingenzymes(E2s):deciding between life and death ofproteins[J].FASEB J,2010,24(4):981-993.
[28]Criqui MC,de Almeida Engler J,Camasses A,et al.Molecularcharacterization of plant ubiquitin-conjugating enzymes belongingto the Ubc P4/E2-C/UBCx/Ubc H10 gene family[J].PlantPhysiol,2002,130:1230-1240.
[29]Michelle C,Vourc’h P,Mignon L,et al.What was the set ofubiquitin and ubiquitin-like conjugating enzymes in the eukaryotecommon ancestor?[J].J Mol Evol,2009,68(6):616-628.
[30]Jentsch S.The ubiquitin-conjugation system[J].Annu RevGenet,1992,26(1):179-207.
[31]唐海蓉,陈仕均,王选年.鱼类免疫组织的研究进展[J].现代畜牧兽医,2006,9:52-54.
[32]张晓研.鲤鱼Akirin基因的分子克隆及免疫功能研究[D].济南:山东师范大学,2010.
[33]Zheng W,Liu G,Ao J,et al.Expression analysis of immunerelevant genes in the spleen of large yellow croaker(Pseudosciaena crocea)stimulated with poly I:C[J].Fish Shellfish Immunol,2006,21(4):414-430.
[34]张宇,韩芳,刘岚萍,等.大黄鱼Rab11基因的克隆与表达分析[J].集美大学学报:自然科学版,2016,21(3):167-174.
[35]郑维兵,慕鹏飞,丁连帅,等.大黄鱼(Larimichthys crocea)IL-10基因的克隆与表达分析[J].海洋学报,2017,39(4):50-60.
[36]王少鑫,巩伟丽,陈媛,等.UBE2W抑制TNFα诱导的NF-κB转录活性[J].科学技术与工程,2012,12(35):9457-9460.
[37]Broomfield S,Hryciw T,Xiao W.DNA postreplication repair andmutagenesis in Saccharomyces cerevisiae[J].Mutat Res,2001,486(3):167-184.
[38]陈学昭,张雷,于珊珊,等.松江鲈(Trachidermus fasciatus)泛素结合酶E2-D2基因的分子克隆及组织表达分析[J].海洋学报,2015,37(10):133-140.
[39]王红梅,祝诚.泛素—蛋白水解酶复合体通路在生殖系统中的研究进展[J].生物化学与生物物理进展,2002,29(1):31-34.
[40]Cenci G,Rawson RB,Belloni G,et al.Ubc D1,a Drosophilaubiquitin-conjugating enzyme required for proper telomerebehavior[J].Genes Dev,1997,11(7):863-875.
[41]张强,牟丽莎,桂耀庭,等.UBE2B基因与男性不育的研究进展[J].中华男科学杂志,2014,20(4):367-371.
[42]Roest HP,van Klaveren J,de Wit J,et al.Inactivation of the HR6Bubiquitin-conjugating DNA repair enzyme in mice causes malesterility associated with chromatin modification[J].Cell,1996,86(5):799-810.
[43]Baarends WM,Wassenaar E,Hoogerbrugge JW,et al.Lossof HR6B ubiquitin-conjugating activity results in damagedsynaptonemal complex structureand increased crossing-overfrequency during the male meiotic prophase[J].Mol Cell Biol,2003,23(4):1151-1162.
[44]Escalier D,Bai XY,Silvius D,et al.Spermatid nuclear and spermperiaxonemal anomalies in the mouse Ube2b null mutant[J].Mol Reprod Dev,2003,65(3):298-308.
[45]Yatsenko AN,Georgiadis AP,Murthy LJ,et al.UBE2B m RNAalterations are associated with severe oligozoospermia in infertilemen[J].Mol Hum Reprod,2013,19(6):388-394.
[46]蔡明夷,周鹏,韩坤煌,等.大黄鱼cyclin B1和cdc2 c DNA序列特征及组织表达分析[J].厦门大学学报:自然科学版,2014,53(1):132-141.
[47]罗辉玉,张东玲,叶坤,等.大黄鱼Lc DCAF17基因的克隆与表达分析[J].集美大学学报:自然科学版,2017,22(1):1-9.
[48]林丹军,张健,骆嘉,等.人工养殖的大黄鱼性腺发育及性周期研究[J].福建师范大学学报:自然科学版,1992,8(3):81-87.
[2]Varshavsky A.The Ubiquitin System[J].Trends Biochem Sci,1997,22(10):383-387.
[3]Baarends WM,Roest HP,Grootegoed JA.The ubiquitin system ingametogenesis[J].Mol Cell Endocrinol,1999,151(1-2):5-16.
[4]国果,吴沁怡,吴建伟,等.家蝇泛素结合酶基因的生物信息学分析[J].生物技术通报,2014(2):107-111.
[5]Schwob E,B?hm T,Mendenhall MD,et al.The B-type cyclinkinase inhibitor p40SIC1 controls the G1 to S transition in S.cerevisiae[J].Cell,1994,79(2):233-244.
[6]Yew PR,Kirschner MW.Proteolysis and DNA replication:theCDC34 requirement in the Xenopus egg cell cycle[J].Science,1997,277(5332):1672-1676.
[7]Reymond F,Wirbelauer C,Krek W.Association of humanubiquitin-conjugating enzyme CDC34 with the mitotic spindle inanaphase[J].J Cell Sci,2000,113(Pt 10):1687-1694.
[8]Pati D,Meistrich ML,Plon SE.Human Cdc34 and Rad6B ubiquitinconjugating enzymes target repressors of cyclic AMP-inducedtranscription for proteolysis[J].Mol Cell Biol,1999,19(7):5001-5013.
[9]Von Hofsten J,Olsson PE.Zebrafish sex determination anddifferentiation:involvement of FTZ-F1 genes[J].Reprod BiolEndocrinol,2005,3(1):63.
[10]刘家富.大黄鱼养殖与生物学[M].厦门:厦门大学出版社,2013.
[11]农业部渔业渔政管理局.2016年中国渔业统计年鉴[M].北京:中国农业出版社,2016.
[12]Manku G,Wing SS,Culty M.Expression of the ubiquitinproteasome system in neonatal rat gonocytes and spermatogonia:role in gonocyte differentiation[J].Biol Reprod,2001,87(2):44.
[13]Shen B,Zhang Z,Wang Y,et al.Differential expression ofubiquitin-conjugating enzyme E2r in the developing ovary andtestis of penaeid shrimp Marsupenaeus japonicus[J].Mol BiolRep,2009,36(5):1149-1157.
[14]Koenig PA,Nicholls PK,Schmidt FI,et al.The E2 ubiquitinconjugating enzyme UBE2J1 is required for spermiogenesis inmice[J].J Biol Chem,2014,289(50):34490-34502.
[15]钱照君,祝天谅,姜虎成,等.克氏原螯虾泛素结合酶E2r基因的克隆及表达分析[J].上海海洋大学学报,2016,5:641-651.
[16]唐蕾,傅明骏,刘文生,等.斑节对虾UBE2H基因克隆及其表达[J].南方农业学报,2016,47(11):1958-1965.
[17]向云龙,张亮,陈卓.过表达泛素偶联酶(UBE2C)对小鼠胚胎发育的影晌[J].黑龙江动物繁殖,2014,22(1):3-7.
[18]胡月阳.UBE2C基因在斑马鱼胚胎发育中的时空表达规律[D].石家庄:河北医科大学,2015.
[19]Zhou P,Zhang ZP,Wang YL,et al.EST analysis and identificationof gonad-related genes from the normalized c DNA library of largeyellow croaker,Larimichthys crocea[J].Comp Biochem PhysiolPart D Genomics Proteomics,2010,5(2):89-97.
[20]陈芸,周鹏,张子平,等.大黄鱼性腺线性化c DNA文库的构建及生殖免疫相关基因的筛选[J].生物技术通报,2011(7):180-185.
[21]周鹏,张子平,王艺磊,等.大黄鱼ubc9基因的克隆和组织表达[J].生物技术通报,2009(8):76-82.
[22]Jiang Y,Han K,Chen S,et al.Characterization and expressionanalysis of Lc-Sox4 in large yellow croaker Larimichthys crocea[J].Gene,2015,574(2):287-301.
[23]Zhang Z,Wu RS,Mok HO,et al.Isolation,characterization andexpression analysis of a hypoxia-responsive glucose transportergene from the grass carp,Ctenopharyngodon idellus[J].Eur JBiochem,2003,270(14):3010-3017.
[24]周鹏.若干大黄鱼性腺发育相关基因的克隆与表达[D].厦门:集美大学,2009.
[25]姚杨,苏杰,魏迪,等.泛素结合酶基因生物信息学与功能分析[J].医学研究生学报,2014,11:1156-1159.
[26]Hochstrasser M.Ubiquitin-Dependent Protein Degradation[J].Annu Rev Genet,1996,30:405-439.
[27]van Wijk SJ,Timmers HT.The family of ubiquitin-conjugatingenzymes(E2s):deciding between life and death ofproteins[J].FASEB J,2010,24(4):981-993.
[28]Criqui MC,de Almeida Engler J,Camasses A,et al.Molecularcharacterization of plant ubiquitin-conjugating enzymes belongingto the Ubc P4/E2-C/UBCx/Ubc H10 gene family[J].PlantPhysiol,2002,130:1230-1240.
[29]Michelle C,Vourc’h P,Mignon L,et al.What was the set ofubiquitin and ubiquitin-like conjugating enzymes in the eukaryotecommon ancestor?[J].J Mol Evol,2009,68(6):616-628.
[30]Jentsch S.The ubiquitin-conjugation system[J].Annu RevGenet,1992,26(1):179-207.
[31]唐海蓉,陈仕均,王选年.鱼类免疫组织的研究进展[J].现代畜牧兽医,2006,9:52-54.
[32]张晓研.鲤鱼Akirin基因的分子克隆及免疫功能研究[D].济南:山东师范大学,2010.
[33]Zheng W,Liu G,Ao J,et al.Expression analysis of immunerelevant genes in the spleen of large yellow croaker(Pseudosciaena crocea)stimulated with poly I:C[J].Fish Shellfish Immunol,2006,21(4):414-430.
[34]张宇,韩芳,刘岚萍,等.大黄鱼Rab11基因的克隆与表达分析[J].集美大学学报:自然科学版,2016,21(3):167-174.
[35]郑维兵,慕鹏飞,丁连帅,等.大黄鱼(Larimichthys crocea)IL-10基因的克隆与表达分析[J].海洋学报,2017,39(4):50-60.
[36]王少鑫,巩伟丽,陈媛,等.UBE2W抑制TNFα诱导的NF-κB转录活性[J].科学技术与工程,2012,12(35):9457-9460.
[37]Broomfield S,Hryciw T,Xiao W.DNA postreplication repair andmutagenesis in Saccharomyces cerevisiae[J].Mutat Res,2001,486(3):167-184.
[38]陈学昭,张雷,于珊珊,等.松江鲈(Trachidermus fasciatus)泛素结合酶E2-D2基因的分子克隆及组织表达分析[J].海洋学报,2015,37(10):133-140.
[39]王红梅,祝诚.泛素—蛋白水解酶复合体通路在生殖系统中的研究进展[J].生物化学与生物物理进展,2002,29(1):31-34.
[40]Cenci G,Rawson RB,Belloni G,et al.Ubc D1,a Drosophilaubiquitin-conjugating enzyme required for proper telomerebehavior[J].Genes Dev,1997,11(7):863-875.
[41]张强,牟丽莎,桂耀庭,等.UBE2B基因与男性不育的研究进展[J].中华男科学杂志,2014,20(4):367-371.
[42]Roest HP,van Klaveren J,de Wit J,et al.Inactivation of the HR6Bubiquitin-conjugating DNA repair enzyme in mice causes malesterility associated with chromatin modification[J].Cell,1996,86(5):799-810.
[43]Baarends WM,Wassenaar E,Hoogerbrugge JW,et al.Lossof HR6B ubiquitin-conjugating activity results in damagedsynaptonemal complex structureand increased crossing-overfrequency during the male meiotic prophase[J].Mol Cell Biol,2003,23(4):1151-1162.
[44]Escalier D,Bai XY,Silvius D,et al.Spermatid nuclear and spermperiaxonemal anomalies in the mouse Ube2b null mutant[J].Mol Reprod Dev,2003,65(3):298-308.
[45]Yatsenko AN,Georgiadis AP,Murthy LJ,et al.UBE2B m RNAalterations are associated with severe oligozoospermia in infertilemen[J].Mol Hum Reprod,2013,19(6):388-394.
[46]蔡明夷,周鹏,韩坤煌,等.大黄鱼cyclin B1和cdc2 c DNA序列特征及组织表达分析[J].厦门大学学报:自然科学版,2014,53(1):132-141.
[47]罗辉玉,张东玲,叶坤,等.大黄鱼Lc DCAF17基因的克隆与表达分析[J].集美大学学报:自然科学版,2017,22(1):1-9.
[48]林丹军,张健,骆嘉,等.人工养殖的大黄鱼性腺发育及性周期研究[J].福建师范大学学报:自然科学版,1992,8(3):81-87.