蒺藜苜蓿MtGT1基因克隆、表达及亚细胞定位分析
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摘要
目前,尚未见关于模式植物蒺藜苜蓿Trihelix转录因子家族成员的功能性研究,本研究在蒺藜苜蓿中发现一个Trihelix转录因子家族的成员MtGT1基因。为了初步鉴定该基因的功能,本研究以蒺藜苜蓿为试验材料,根据MtGT1基因序列设计引物,通过基因克隆、生物信息学分析、荧光定量等手段,从蒺藜苜蓿中分离MtGT1基因。生物信息学技术分析结果显示,该基因属于Trihelix家族的GT1亚家族,与三叶草的同源性较高,可能为脂溶性疏水蛋白,不含有跨膜结构和信号肽;二级结构显示其由α螺旋、延伸链和无规则卷曲3种结构组成;三级结构发现其部分残基与拟南芥GT1的DNA结合结构域相似度极高。荧光定量表达发现,该基因在根中表达量最高,且响应IAA和6-BA两种植物激素及干旱、低温等非生物胁迫;亚细胞定位发现该蛋白是核定位蛋白。该研究可为深入挖掘MtGT1基因的功能提供理论依据。
At pres ent, no functional studies have been performed on the members of the Trihelix family of transcription factors in the model plant. In this study, a member of the Trihelix family of transcription factors, the MtGT1 gene, was discovered. In order to identify the function of the gene, this study took wolfberry as the experimental material. According to the design in MtGT1 gene sequence, we isolated MtGT1 gene from loquat by gene cloning, bioinformatics analysis, and fluorescence quantification. The results of bioinformatics analysis showed that the gene belongs to the GT1 subfamily of Trihelix family and has high homology with clover. It might be a liposoluble hydrophobin, did not contain transmembrane structure and signal peptide. And its secondary structure showed that it might be composed of α. helix, extended strand, and random coil three structures; The tertiary structure found that some of its residues were highly similar to the DNA-binding domain of Arabidopsis GT1. Fluorescent quantitative expression revealed that the gene expressed the highest amount in roots, and responded to two plant hormones IAA and 6-BA and abiotic stresses such as drought and hypothermia. Subcellular localization revealed that the gene might be a nuclear localization protein. This study might provide a theoretical basis for the further exploration of the function of the MtGT1 gene.
At pres ent, no functional studies have been performed on the members of the Trihelix family of transcription factors in the model plant. In this study, a member of the Trihelix family of transcription factors, the MtGT1 gene, was discovered. In order to identify the function of the gene, this study took wolfberry as the experimental material. According to the design in MtGT1 gene sequence, we isolated MtGT1 gene from loquat by gene cloning, bioinformatics analysis, and fluorescence quantification. The results of bioinformatics analysis showed that the gene belongs to the GT1 subfamily of Trihelix family and has high homology with clover. It might be a liposoluble hydrophobin, did not contain transmembrane structure and signal peptide. And its secondary structure showed that it might be composed of α. helix, extended strand, and random coil three structures; The tertiary structure found that some of its residues were highly similar to the DNA-binding domain of Arabidopsis GT1. Fluorescent quantitative expression revealed that the gene expressed the highest amount in roots, and responded to two plant hormones IAA and 6-BA and abiotic stresses such as drought and hypothermia. Subcellular localization revealed that the gene might be a nuclear localization protein. This study might provide a theoretical basis for the further exploration of the function of the MtGT1 gene.
引文
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Bonnin I.,Prosperi J.M.,and Olivieri I.,1997,Comparison of quantitative genetic parameters between two natural populations of a selfing plant species,Medicago truncatula Gaertn,Theor.Appl.Genet.,94(5):641-651
Chao Y.H.,Li S.S.,Teng K.,Xu L.X.,Xiao G.Z.,Guo T.,and Han L.B.,2016,Cloning of MsNAP gene from Medicago sativa L.and transformation to tobacco(Nicotiana tabacum),Zhongguo Caodi Xuebao(Chinese Journal of Grassland),38(2):13-19(晁跃辉,李珊珊,滕珂,许立新,肖国增,郭涛,韩烈保,2016,紫花苜蓿MsNAP基因克隆及烟草转化,中国草地学报,38(2):13-19)
Endre G.,Kereszt A.,Kevei Z.,Mihacea S.,KalóP.,and Kiss G.B.,2002,A receptor kinase gene regulating symbiotic nodule de-velopment,Nature,417(6892):962-966
Ji J.H.,Zhou Y.J.,Wu H.H.,and Yang L.M.,2015,Genomewide analysis and functional prediction of the Trihelix transcription factor family in rice,Yichuan(Hereditas),37(12):1228-1241(纪剑辉,周颖君,吴贺贺,杨立明,2015,水稻Trihelix转录因子家族全基因组分析及功能预测,遗传,37(12):1228-1241)
Kaplan-Levy R.N.,Brewer P.B.,Quon T.,and Smyth D.R.,2012,T he tri helix family of transcription factors-light,stress and devel-opment,Trends Plant Sci.,17(3):163-171
Li H.,Huang W.,Liu Z.W.,Wu Z.J.,and Zhuang J.,2017,Trihelix family transcription factors in tea plant(Camellia sinensis):iden-tification,classification,and expression profiles response to abiotic stress,Acta Physiol.Plant,39(10):217
Li Y.,Liu X.D.,Dong Y.M.,Xie Z.M.,and Chen S.Y.,2015a,Cloning and functional analysis of the cotton Trihelix transcription factor GhGT29,Yichuan(Hereditas),37(12):1218-1227(李月,刘晓东,董永梅,谢宗铭,陈受宜,2015a,棉花Trihelix转录因子GhGT29基因的克隆及功能分析,遗传,37(12):1218-1227)
Li Y.,Liu X.D.,Xie Z.M.,Dong Y.M.,Wu D.M.,and Chen S.Y.,2015b,Cloning and function analysis of cotton transcription factor GhGT-2,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(24):4872-4884(李月,刘晓东,谢宗铭,董永梅,武冬梅,陈受宜,2015b,棉花GhGT-2转录因子的克隆及其功能分析,中国农业科学,48(24):4872-4884)
Li Y.,Sun J.,Chen S.Y.,and Xie Z.M.,2013,Cloning and transcription function analysis of cotton transcription factor GhGT30 gene,Zuowu Xuebao(Acta Agronomica Sinica),39(5):806-815(李月,孙杰,陈受宜,谢宗铭,2013,棉花转录因子GhGT30基因的克隆及转录功能分析,作物学报,39(5):806-815)
Li Y.K.,Wang X.M.,Wang Z.,Li J.,Vladimir C.,Nicolay D.,Sun G.Z.,and Gao H.W.,2012,Cloning and analysis of the dehydrin(DHN)gene from Galega orientalis,Caoye Xuebao(Acta Prataculturae Sinica),21(1):176-183(李玉坤,王学敏,王赞,李俊,Vladimir C.,Nicolay D.,孙桂枝,高洪文,2012,东方山羊豆脱水蛋白基因的克隆及初步分析,草业学报,21(1):176-183)
Liang S.M.,Luo D.L.,Cheng Y.J.,Yu L.A.,Yang Y.J.,Chen J.Y.,Lu W.J.,and Kuang J.F.,2017,Identification and characterization of a trihelix transcription factor MaGTL1a from banana,Guoshu Xuebao(Journal of Fruit Science),34(8):935-945(梁淑敏,罗冬兰,程玉瑾,俞乐安,杨亚洁,陈建业,陆旺金,邝健飞,2017,香蕉trihelix转录因子MaGTL1a的分离及特性,果树学报,34(8):935-945)
Liu J.L.,Yun T.Y.,Cao J.M.,and Ren X.,2008,Effect of acid treatment on seed germination of Medicago truncatula Gaert,Zhongzi(Seed),27(9):87-88(刘建利,云天运,曹君迈,任贤,2008,酸处理对蒺藜状苜蓿种子萌发的影响,种子,27(9):87-88)
Mao L.J.,and Lin W.F.,2008,The application of auxin in agriculture,Hebei Nongye Kexue(Journal of Hebei Agricultural Sciences),12(2):80-81(毛丽君,林位夫,2008,植物生长素在农业中的应用,河北农业科学,12(2):80-81)
Nagano Y.,Inaba T.,Furuhashi H.,and Sasaki Y.,2001,Trihelix DNA-binding protein with specificities for two distinctcis-elements both important for light down-regulated and dark-inducible gene expression in higher plants,J.Biol.Chem.,276(25):22238-22243
Song A.P.,Wu D.,Fan Q.Q.,Tian C.,Chen S.M.,Guan Z.Y.,Xin J.J.,Zhao K.K.,and Chen F.D.,2016,Transcriptomewide iden-tification and expression profiling analysis of chrysanthemum trihelix transcription factors,Int.J.Mol.Sci.,17(2):198
Stacey G.,Libault M.,Brechenmacher L.,Wan J.,and May G.D.,2006,Genetics and functional genomics of legume nodulation,Curr.Opin.Plant Biol.,9(2):110-121
Tan P.H.,Li J.,Chen X.Y.,Zhang L.,Teng K.,and Yin S.X.,2017,Cloning,subcellular localization and expression analysis of PpGA2ox gene in kentucky bluegrass,Zhongguo Caodi Xuebao(Chinese Journal of Grassland),39(3):8-14(檀鹏辉,李俊,陈小云,张兰,滕珂,尹淑霞,2017,草地早熟禾PpGA2ox基因的克隆,亚细胞定位及表达分析,中国草地学报,39(3):8-14)
Tu D.P.,Wei Z.W.,Wu Z.N.,Lei Y.F.,Zhang D.,and Qiu W.W.,2011,Distribution characteristics and marker exploitation of EST-SSRs in Medicago truncatula,Caoye Kexue(Pratacultural Science),28(5):746-752(屠德鹏,魏臻武,武自念,雷艳芳,张栋,邱伟伟,2011,蒺藜苜蓿EST-SSRs分布特征及标记的开发,草业科学,28(5):746-752)
Wei Z.W.,and Gai J.Y.,2008,Model legume:Medicago truncatula,Caoye Xuebao(Acta Prataculturae Sinica),17(1):114-120(魏臻武,盖钧镒,2008,豆科模式植物-蒺藜苜蓿,草业学报,17(1):114-120)
Xie B.X.,2014,Bioinformatic analysis of SPO11 gene in Arabidopsis,Thesis for M.S.,Zhengzhou University,Supervisors:Cao G.Q.,and Wei F.,pp.1-12(谢冰心,2014,拟南芥SPO11基因的生物信息学分析,硕士学位论文,郑州大学,导师:曹刚强,位芳,pp.1-12)
Yu C.Y.,Cai X.F.,Ye Z.B.,and Li H.X.,2015,Genome-wide identification and expression profiling analysis of trihelix gene family in tomato,Biochem.Biophys.Res.Commun.,468(4):653-659
Zhou H.,Qian J.,Li R.F.,Chen D.D.,Fang R.J.,and Zhao W.G.,2017,Research of the Arabidopsis trihelix transcription factor gene family,Jiangsu Keji Daxue Xuebao(Journal of Jiangsu University of Science and Technology),31(2):231-236(周宏,钱娇,李荣芳,陈丹丹,方荣俊,赵卫国,2017,拟南芥Trihelix转录因子基因家族研究,江苏科技大学学报,31(2):231-236)
Bonnin I.,Prosperi J.M.,and Olivieri I.,1997,Comparison of quantitative genetic parameters between two natural populations of a selfing plant species,Medicago truncatula Gaertn,Theor.Appl.Genet.,94(5):641-651
Chao Y.H.,Li S.S.,Teng K.,Xu L.X.,Xiao G.Z.,Guo T.,and Han L.B.,2016,Cloning of MsNAP gene from Medicago sativa L.and transformation to tobacco(Nicotiana tabacum),Zhongguo Caodi Xuebao(Chinese Journal of Grassland),38(2):13-19(晁跃辉,李珊珊,滕珂,许立新,肖国增,郭涛,韩烈保,2016,紫花苜蓿MsNAP基因克隆及烟草转化,中国草地学报,38(2):13-19)
Endre G.,Kereszt A.,Kevei Z.,Mihacea S.,KalóP.,and Kiss G.B.,2002,A receptor kinase gene regulating symbiotic nodule de-velopment,Nature,417(6892):962-966
Ji J.H.,Zhou Y.J.,Wu H.H.,and Yang L.M.,2015,Genomewide analysis and functional prediction of the Trihelix transcription factor family in rice,Yichuan(Hereditas),37(12):1228-1241(纪剑辉,周颖君,吴贺贺,杨立明,2015,水稻Trihelix转录因子家族全基因组分析及功能预测,遗传,37(12):1228-1241)
Kaplan-Levy R.N.,Brewer P.B.,Quon T.,and Smyth D.R.,2012,T he tri helix family of transcription factors-light,stress and devel-opment,Trends Plant Sci.,17(3):163-171
Li H.,Huang W.,Liu Z.W.,Wu Z.J.,and Zhuang J.,2017,Trihelix family transcription factors in tea plant(Camellia sinensis):iden-tification,classification,and expression profiles response to abiotic stress,Acta Physiol.Plant,39(10):217
Li Y.,Liu X.D.,Dong Y.M.,Xie Z.M.,and Chen S.Y.,2015a,Cloning and functional analysis of the cotton Trihelix transcription factor GhGT29,Yichuan(Hereditas),37(12):1218-1227(李月,刘晓东,董永梅,谢宗铭,陈受宜,2015a,棉花Trihelix转录因子GhGT29基因的克隆及功能分析,遗传,37(12):1218-1227)
Li Y.,Liu X.D.,Xie Z.M.,Dong Y.M.,Wu D.M.,and Chen S.Y.,2015b,Cloning and function analysis of cotton transcription factor GhGT-2,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(24):4872-4884(李月,刘晓东,谢宗铭,董永梅,武冬梅,陈受宜,2015b,棉花GhGT-2转录因子的克隆及其功能分析,中国农业科学,48(24):4872-4884)
Li Y.,Sun J.,Chen S.Y.,and Xie Z.M.,2013,Cloning and transcription function analysis of cotton transcription factor GhGT30 gene,Zuowu Xuebao(Acta Agronomica Sinica),39(5):806-815(李月,孙杰,陈受宜,谢宗铭,2013,棉花转录因子GhGT30基因的克隆及转录功能分析,作物学报,39(5):806-815)
Li Y.K.,Wang X.M.,Wang Z.,Li J.,Vladimir C.,Nicolay D.,Sun G.Z.,and Gao H.W.,2012,Cloning and analysis of the dehydrin(DHN)gene from Galega orientalis,Caoye Xuebao(Acta Prataculturae Sinica),21(1):176-183(李玉坤,王学敏,王赞,李俊,Vladimir C.,Nicolay D.,孙桂枝,高洪文,2012,东方山羊豆脱水蛋白基因的克隆及初步分析,草业学报,21(1):176-183)
Liang S.M.,Luo D.L.,Cheng Y.J.,Yu L.A.,Yang Y.J.,Chen J.Y.,Lu W.J.,and Kuang J.F.,2017,Identification and characterization of a trihelix transcription factor MaGTL1a from banana,Guoshu Xuebao(Journal of Fruit Science),34(8):935-945(梁淑敏,罗冬兰,程玉瑾,俞乐安,杨亚洁,陈建业,陆旺金,邝健飞,2017,香蕉trihelix转录因子MaGTL1a的分离及特性,果树学报,34(8):935-945)
Liu J.L.,Yun T.Y.,Cao J.M.,and Ren X.,2008,Effect of acid treatment on seed germination of Medicago truncatula Gaert,Zhongzi(Seed),27(9):87-88(刘建利,云天运,曹君迈,任贤,2008,酸处理对蒺藜状苜蓿种子萌发的影响,种子,27(9):87-88)
Mao L.J.,and Lin W.F.,2008,The application of auxin in agriculture,Hebei Nongye Kexue(Journal of Hebei Agricultural Sciences),12(2):80-81(毛丽君,林位夫,2008,植物生长素在农业中的应用,河北农业科学,12(2):80-81)
Nagano Y.,Inaba T.,Furuhashi H.,and Sasaki Y.,2001,Trihelix DNA-binding protein with specificities for two distinctcis-elements both important for light down-regulated and dark-inducible gene expression in higher plants,J.Biol.Chem.,276(25):22238-22243
Song A.P.,Wu D.,Fan Q.Q.,Tian C.,Chen S.M.,Guan Z.Y.,Xin J.J.,Zhao K.K.,and Chen F.D.,2016,Transcriptomewide iden-tification and expression profiling analysis of chrysanthemum trihelix transcription factors,Int.J.Mol.Sci.,17(2):198
Stacey G.,Libault M.,Brechenmacher L.,Wan J.,and May G.D.,2006,Genetics and functional genomics of legume nodulation,Curr.Opin.Plant Biol.,9(2):110-121
Tan P.H.,Li J.,Chen X.Y.,Zhang L.,Teng K.,and Yin S.X.,2017,Cloning,subcellular localization and expression analysis of PpGA2ox gene in kentucky bluegrass,Zhongguo Caodi Xuebao(Chinese Journal of Grassland),39(3):8-14(檀鹏辉,李俊,陈小云,张兰,滕珂,尹淑霞,2017,草地早熟禾PpGA2ox基因的克隆,亚细胞定位及表达分析,中国草地学报,39(3):8-14)
Tu D.P.,Wei Z.W.,Wu Z.N.,Lei Y.F.,Zhang D.,and Qiu W.W.,2011,Distribution characteristics and marker exploitation of EST-SSRs in Medicago truncatula,Caoye Kexue(Pratacultural Science),28(5):746-752(屠德鹏,魏臻武,武自念,雷艳芳,张栋,邱伟伟,2011,蒺藜苜蓿EST-SSRs分布特征及标记的开发,草业科学,28(5):746-752)
Wei Z.W.,and Gai J.Y.,2008,Model legume:Medicago truncatula,Caoye Xuebao(Acta Prataculturae Sinica),17(1):114-120(魏臻武,盖钧镒,2008,豆科模式植物-蒺藜苜蓿,草业学报,17(1):114-120)
Xie B.X.,2014,Bioinformatic analysis of SPO11 gene in Arabidopsis,Thesis for M.S.,Zhengzhou University,Supervisors:Cao G.Q.,and Wei F.,pp.1-12(谢冰心,2014,拟南芥SPO11基因的生物信息学分析,硕士学位论文,郑州大学,导师:曹刚强,位芳,pp.1-12)
Yu C.Y.,Cai X.F.,Ye Z.B.,and Li H.X.,2015,Genome-wide identification and expression profiling analysis of trihelix gene family in tomato,Biochem.Biophys.Res.Commun.,468(4):653-659
Zhou H.,Qian J.,Li R.F.,Chen D.D.,Fang R.J.,and Zhao W.G.,2017,Research of the Arabidopsis trihelix transcription factor gene family,Jiangsu Keji Daxue Xuebao(Journal of Jiangsu University of Science and Technology),31(2):231-236(周宏,钱娇,李荣芳,陈丹丹,方荣俊,赵卫国,2017,拟南芥Trihelix转录因子基因家族研究,江苏科技大学学报,31(2):231-236)