白三叶TrMYB1R1全长克隆及转录表达分析
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摘要
R1-MYB是MYB转录因子家族中的一亚类,可能参与植物非生物胁迫应答。通过RT-PCR和RACE技术,在白三叶(Trifolium repens)中成功克隆出一个MYB转录因子TrMYB1R1,基因全长1 403 bp,编码297个氨基酸残基。通过生物信息学分析,发现TrMYB1R1蛋白含有一个MYB结构域,属于MYB-related蛋白,保守域形成3个α-螺旋;亚细胞定位预测该蛋白可能位于细胞核中。同源进化分析结果显示TrMYB1R1蛋白和豆科植物MYB蛋白亲缘较近,蛋白进化符合植物进化分类。非生物胁迫和外源激素处理结果表明,该基因在白三叶根和叶中均受到诱导调节。在聚乙二醇(PEG)水分胁迫、镉胁迫、4℃冷处理及35℃高温处理下,白三叶TrMYB1R1表达受到抑制,且该基因对镉、冷及热处理响应强烈,对PEG处理响应较弱;而在NaCl胁迫处理下该基因受到诱导表达;同时,脱落酸(ABA)处理前期抑制TrMYB1R1表达,但细胞分裂素(CTK)显著诱导该基因表达。这些结果表明,该基因可能参与多种非生物胁迫应答并可能受激素调控,但对TrMYB1R1在白三叶中的功能及调控作用仍需进一步研究。
R1-MYB,a subtribe of the MYB family,may be involved in responses to abiotic stress in plants.In this study,a MYB transcription factor ‘TrMYB1R1' was cloned from white clover(Trifolium repens) through RT-PCR and RACE.The complete sequence of TrMYB1R1 is 1 403 bp encoding 297 amino acids.Based on the analysis of information biology,TrMYB1R1 contains a MYB structural domain and its conservative domain could form 3 alpha-helixes,indicating that TrMYB1R1 is a MYB-related protein.TrMYB1R1 was located in the nucleus through prediction of the subcellular localization.The result of homologous evolution analysis showed that TrMYB1R1 has closer genetic relationships with MYB of Leguminosae.The TrMYB1R1 could be induced in leaves and roots of white clover under abiotic stress and phytohormone treatments.In detail,the expression of TrMYB1R1 was inhibited under water stress(PEG),cadmium,cold(4 °C),and heat(35 °C) stress.More importantly,cadmium and cold had stronger effects on the expression of TrMYB1R1 than PEG treatment.Differently,salt(NaCl) stress could significantly induce the increase in the expression of TrMYB1R1.For the phytohormone treatments,abscisic acid(ABA) inhibited the expression of TrMYB1R1,whereas cytomin(CTK) induced its expression.All of these results indicated that TrMYB1R1 could be involved in responses to abiotic stress and phytohormones,but the function of TrMYB1R1 still requires further investigation in white clover.
R1-MYB,a subtribe of the MYB family,may be involved in responses to abiotic stress in plants.In this study,a MYB transcription factor ‘TrMYB1R1' was cloned from white clover(Trifolium repens) through RT-PCR and RACE.The complete sequence of TrMYB1R1 is 1 403 bp encoding 297 amino acids.Based on the analysis of information biology,TrMYB1R1 contains a MYB structural domain and its conservative domain could form 3 alpha-helixes,indicating that TrMYB1R1 is a MYB-related protein.TrMYB1R1 was located in the nucleus through prediction of the subcellular localization.The result of homologous evolution analysis showed that TrMYB1R1 has closer genetic relationships with MYB of Leguminosae.The TrMYB1R1 could be induced in leaves and roots of white clover under abiotic stress and phytohormone treatments.In detail,the expression of TrMYB1R1 was inhibited under water stress(PEG),cadmium,cold(4 °C),and heat(35 °C) stress.More importantly,cadmium and cold had stronger effects on the expression of TrMYB1R1 than PEG treatment.Differently,salt(NaCl) stress could significantly induce the increase in the expression of TrMYB1R1.For the phytohormone treatments,abscisic acid(ABA) inhibited the expression of TrMYB1R1,whereas cytomin(CTK) induced its expression.All of these results indicated that TrMYB1R1 could be involved in responses to abiotic stress and phytohormones,but the function of TrMYB1R1 still requires further investigation in white clover.
引文
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[14]MATYS V,KELMARGOULIS O V,FRICKE E,LIEBICH I,LAND S,BARRE-DIRRIE A,REUTER I,CHEKMENEV D,KRULL M,HORNISCHER K,VOSS N,STEGMAIER P,LEWICKI-POTAPOV B,SAXEL H,KEL A E,WINGENDER E.TRANSFAC and its module TRANSCompel:Transcriptional gene regulation in eukaryotes.Nucleic Acids Research,2006,34:108-110.
[15]LIANG Q,WANG P.Summarization on the progress in protein phosphorylation.Science&Technology Review,2012,30(31):75-81.
[16]DHAUBHADEL S,LI X.A new client for 14-3-3 proteins:GmMYB176,an R1 MYB transcription factor.Plant Signaling&Behavior,2010,5(7):921-923.
[17]JUN J H,LIU C G,XIAO X R,DIXON R A.The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula.Plant Cell,2015,27(10):2860-2879.
[18]高友菲.不同大豆品种GmFT2a基因的多态性分析及其转录因子的筛选.北京:中国农业科学院硕士学位论文,2014.GAO Y F.Polymorphism analysis of GmFT2a in different soybean varieties and screening of its transcription factor.Master Thesis.Beijing:Chinese Academy of Agricultural Sciences,2014.
[19]RAMALINGAM A,KUDAPA H,PAZHAMALA L T,GARG V,VARSHNEY R K.Gene expression and yeast two-hybrid studies of 1R-MYB transcription factor mediating drought stress response in chickpea(Cicer arietinum L.).Frontiers in Plant Science,2015,6:1117.
[20]XIONG H,LI J J,LIU P L,DUAN J Z,ZHAO Y,GUO X,LI Y,ZHANG H L,ALI J,LI Z C.Overexpression of OsMYB48-1,a novel MYB-related transcription factor,enhances drought and salinity tolerance in rice.PLoS One,2014,9(3):e92913.
[21]WEI M L,PEI L L,LIU J M,MIN D H.Isolation and molecular characteristics analysis of soybean transcription factor gene GmMYB174.Journal of Plant Genetic Resources,2015,16(1):94-99.
[22]URAO T,YAMAGUCHI-SHINOZAKI K,URAO S,SHINOZAKI K.An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.Plant Cell,1993,5:1529-1539.
[23]彭志红.拟南芥芸苔素调控花青素的积累及分子机理.长沙:湖南农业大学博士学位论文,2013.PENG Z H.Anthocyanin accumulation and molecular mechanism for brassinolide-induced in Arabidopsis.PhD Thesis.Changsha:Hunan Agricultural University,2013.
[2]GUO J.Advances in impacts of climate change on agricultural production in China.Journal of Applied Meteorological Science,2015,26(1):1-11.
[3]李文誉,李德明.盐碱及重金属对植物生长发育的影响.北方园艺,2010,8:221-224.LI W Y,LI D M.Effects of salinization and heavy metals stress on plant growth and development.Northen Horticulture,2010,8:221-224.
[4]吴永洁,王元素,李莉.国内白三叶研究进展.种子,2015,34(7):46-51.WU Y J,WANG Y S,LI L.Research advancement on Trifolium repens in China.Seed,2015,34(7):46-51.
[5]HIRAYAMA T,SHINOZAKI K.Research on plant abiotic stress responses in the post-genome era:Past,present and future.Plant Journal for Cell&Molecular Biology,2010,61(6):1041-1052.
[6]GOLLDACK D,LüKING I,YANG O.Plant tolerance to drought and salinity:Stress regulating transcription factors and their functional significance in the cellular transcriptional network.Plant Cell Reports,2011,30(8):1383-1391.
[7]HUSSAIN S S,KAYANI M A,AMJAD M.Transcription factors as tools to engineer enhanced drought stress tolerance in plants.Biotechnology Progress,2011,27(2):297-306.
[8]左然,徐美玲,柴国华,周功克.植物MYB转录因子功能及调控机制研究进展.生命科学,2012,24(10):1133-1140.ZUO R,XU M L,CHAI G H,ZHOU G K.Function and regulation mechanism of plant MYB transcription factors.Chinese Bulletin of Life Sciecnes,2012,24(10):1133-1140.
[9]DUBOS C,STRACKE R,GROTEWOLD E,WEISSHAAR B,MARTIN C,LEPINIEC L.MYB transcription factors in Arabidopsis.Trends in Plant Science,2010,15(10):573-581.
[10]冯盼盼,陈鹏,洪文杰,赵小英,刘选明.拟南芥MYB转录因子家族研究进展.生命科学研究,2016,20(6):555-560.FENG P P,CHEN P,HONG W J,ZHAO X Y,LIU X M.Research progress of MYB transcription factor family in Arabidopsis thaliana.Life Science Research,2016,20(6):555-560.
[11]PAZ-ARES J,GHOSAL D,WIENAND U,PETERSON P A,SAEDLER H.The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.The EMBO Journal,1987,6(12):3553-3558.
[12]KRANZ H D,DENEKAMP M,GRECO R,JIN H,LEYVA A,MEISSNER R C,PETRONI K,URZAINQUI A,BEVAN M,MARTIN C,SMEEKENS S,TONELLI C,PAZ-ARES J,WEISSHAAR B.Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana.Plant Journal for Cell&Molecular Biology,1998,16(2):263-276.
[13]FELLER A,MACHEMER K,BRAUN E L,GROTEWOLD E.Evolutionary and comparative analysis of MYB and bHLH plant transcription factors.Plant Journal,2011,66(1):94-116.
[14]MATYS V,KELMARGOULIS O V,FRICKE E,LIEBICH I,LAND S,BARRE-DIRRIE A,REUTER I,CHEKMENEV D,KRULL M,HORNISCHER K,VOSS N,STEGMAIER P,LEWICKI-POTAPOV B,SAXEL H,KEL A E,WINGENDER E.TRANSFAC and its module TRANSCompel:Transcriptional gene regulation in eukaryotes.Nucleic Acids Research,2006,34:108-110.
[15]LIANG Q,WANG P.Summarization on the progress in protein phosphorylation.Science&Technology Review,2012,30(31):75-81.
[16]DHAUBHADEL S,LI X.A new client for 14-3-3 proteins:GmMYB176,an R1 MYB transcription factor.Plant Signaling&Behavior,2010,5(7):921-923.
[17]JUN J H,LIU C G,XIAO X R,DIXON R A.The transcriptional repressor MYB2 regulates both spatial and temporal patterns of proanthocyandin and anthocyanin pigmentation in Medicago truncatula.Plant Cell,2015,27(10):2860-2879.
[18]高友菲.不同大豆品种GmFT2a基因的多态性分析及其转录因子的筛选.北京:中国农业科学院硕士学位论文,2014.GAO Y F.Polymorphism analysis of GmFT2a in different soybean varieties and screening of its transcription factor.Master Thesis.Beijing:Chinese Academy of Agricultural Sciences,2014.
[19]RAMALINGAM A,KUDAPA H,PAZHAMALA L T,GARG V,VARSHNEY R K.Gene expression and yeast two-hybrid studies of 1R-MYB transcription factor mediating drought stress response in chickpea(Cicer arietinum L.).Frontiers in Plant Science,2015,6:1117.
[20]XIONG H,LI J J,LIU P L,DUAN J Z,ZHAO Y,GUO X,LI Y,ZHANG H L,ALI J,LI Z C.Overexpression of OsMYB48-1,a novel MYB-related transcription factor,enhances drought and salinity tolerance in rice.PLoS One,2014,9(3):e92913.
[21]WEI M L,PEI L L,LIU J M,MIN D H.Isolation and molecular characteristics analysis of soybean transcription factor gene GmMYB174.Journal of Plant Genetic Resources,2015,16(1):94-99.
[22]URAO T,YAMAGUCHI-SHINOZAKI K,URAO S,SHINOZAKI K.An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence.Plant Cell,1993,5:1529-1539.
[23]彭志红.拟南芥芸苔素调控花青素的积累及分子机理.长沙:湖南农业大学博士学位论文,2013.PENG Z H.Anthocyanin accumulation and molecular mechanism for brassinolide-induced in Arabidopsis.PhD Thesis.Changsha:Hunan Agricultural University,2013.
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