大豆GmMYB46基因的克隆、定位及表达分析
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摘要
[目的]本文旨在研究MYB类转录因子基因GmMYB46的结构特征和定位,并阐述其对不同胁迫的响应,为明确其在逆境胁迫下的作用奠定基础。[方法]从大豆品种‘晋豆21’中克隆出GmMYB46的CDS序列,采用生物信息学方法对该基因及其编码蛋白进行分析,并利用PlantCARE软件分析GmMYB46基因启动子元件。通过洋葱表皮细胞瞬时表达系统对GmMYB46蛋白质全长及不同结构域M1(aa1～123)和M2(aa124～334)进行亚细胞定位分析。采用实时荧光定量PCR检测GmMYB46在不同逆境处理下的表达情况。[结果]GmMYB46 CDS序列全长为1 005 bp,编码334个氨基酸,蛋白质相对分子质量为82.53×10~3,氨基酸序列中含有2个高度保守的SANT结构域。进化树分析表明该基因编码的蛋白与野生大豆Gs MYB46亲缘关系最近。GmMYB46包含MBS、ABRE、GARE、TCA、LTR等逆境胁迫应答元件。亚细胞定位结果显示,p BIN-GmMYB46-GFP及p BIN-GmMYB46M1-GFP融合蛋白在细胞核中表达,p BIN-GmMYB46M2-GFP绿色荧光遍布整个细胞。实时荧光定量PCR分析表明,在干旱、盐(200 mmol·L~(-1)NaCl)、低温(4℃)、ABA(200μmol·L~(-1))、SA(500μmol·L~(-1))、GA(100μmol·L~(-1))处理下均能诱导GmMYB46基因在大豆根、茎、叶中的上调表达。[结论]GmMYB46基因的保守结构域对亚细胞定位起决定性作用,该基因可能参与大豆对非生物胁迫的响应。
[Objectives]In this study,the structural features and localization of the MYB transcription factor gene GmMYB46 were analyzed. Expression patterns of GmMYB46 under different stresses were tested,which can be applied to the further study of stress effect of soybean. [Methods]The CDS sequence of GmMYB46 was cloned from the soybean variety‘Jindou 21',and the gene and its encoding protein were analyzed by bioinformatics methods. The GmMYB46 gene promoter motif element was analyzed by using the PlantCARE software. The subcellular localizations of the full-length and different domains of M1( aa1-123) and M2( aa124-334) of GmMYB46 protein were analyzed by the onion epidermal cell transient expression system. The expression of GmMYB46 under different stress treatments were studied by RT-qPCR. [Results]Full-length CDS sequence of GmMYB46 was 1 005 bp,encoding 334 amino acids,and the relative molecular mass of MYB46 protein was 82. 53 × 10~3. Domain analysis showed that the amino acid sequence contained two highly conserved SANT domains. Phylogenetic tree analysis showed that GmMYB46 was highly homologous to Gs MYB46 from Glycine soja. The promoter element analysis showed that GmMYB46 contained stress responsive elements,such as MBS,ABRE,GARE,TCA and LTR. The subcellular localization results showed that the fusion protein of p BIN-GmMYB46-GFP and p BIN-GmMYB46 M1-GFP were located in the nucleus,but the fusion protein of p BIN-GmMYB46 M2-GFP was expressed in the whole cell. Quantitative PCR( q PCR) analysis showed that the transcription level of GmMYB46 in soybean root,stem and leaves was upregulated under the drought,salt( 200 mmol·L~(-1) NaCl),cold( 4 ℃),ABA( 200 μmol·L~(-1)),SA( 500 μmol·L~(-1)) and GA( 100μmol·L~(-1)) treatments. [Conclusions]The conserved domain of GmMYB46 played a crucial role in subcellular localization and the gene might be involved in stress response.
[Objectives]In this study,the structural features and localization of the MYB transcription factor gene GmMYB46 were analyzed. Expression patterns of GmMYB46 under different stresses were tested,which can be applied to the further study of stress effect of soybean. [Methods]The CDS sequence of GmMYB46 was cloned from the soybean variety‘Jindou 21',and the gene and its encoding protein were analyzed by bioinformatics methods. The GmMYB46 gene promoter motif element was analyzed by using the PlantCARE software. The subcellular localizations of the full-length and different domains of M1( aa1-123) and M2( aa124-334) of GmMYB46 protein were analyzed by the onion epidermal cell transient expression system. The expression of GmMYB46 under different stress treatments were studied by RT-qPCR. [Results]Full-length CDS sequence of GmMYB46 was 1 005 bp,encoding 334 amino acids,and the relative molecular mass of MYB46 protein was 82. 53 × 10~3. Domain analysis showed that the amino acid sequence contained two highly conserved SANT domains. Phylogenetic tree analysis showed that GmMYB46 was highly homologous to Gs MYB46 from Glycine soja. The promoter element analysis showed that GmMYB46 contained stress responsive elements,such as MBS,ABRE,GARE,TCA and LTR. The subcellular localization results showed that the fusion protein of p BIN-GmMYB46-GFP and p BIN-GmMYB46 M1-GFP were located in the nucleus,but the fusion protein of p BIN-GmMYB46 M2-GFP was expressed in the whole cell. Quantitative PCR( q PCR) analysis showed that the transcription level of GmMYB46 in soybean root,stem and leaves was upregulated under the drought,salt( 200 mmol·L~(-1) NaCl),cold( 4 ℃),ABA( 200 μmol·L~(-1)),SA( 500 μmol·L~(-1)) and GA( 100μmol·L~(-1)) treatments. [Conclusions]The conserved domain of GmMYB46 played a crucial role in subcellular localization and the gene might be involved in stress response.
引文
[1] Baldoni E,Genga A,Cominelli E,et al. Plant MYB transcription factors:their role in drought response mechanisms[J]. Intl J Mol Sci,2015,16(7):15811-15851.
[2] Paz-Ares J,Ghosal D,Wienand U,et al. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators[J]. EMBO J,1987,6(12):3553-3558.
[3] Dubos C,Stracke R,Grotewold E,et al. MYB transcription factors in Arabidopsis[J]. Trends Plant Sci,2010,15(10):573-581.
[4] Katiyar A,Smita S,Lenka S K,et al. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis[J]. BMC Genomics,2012,13:544.
[5] Liu J,Osbourn A,Ma P. MYB transcription factors as regulators of phenylpropaniod metabolism in plants[J]. Mol Plant,2015,8:689-708.
[6] Wang H Y,Wang H L,Shao H B,et al. Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology[J]. Front Plant Sci,2016,7:67.
[7]许玲,卫培培,张大勇,等.大豆转录因子基因GmMYB111的克隆及功能分析[J].中国农业科学,2015,48(15):3079-3089.Xu L,Wei P P,Zhang D Y,et al. Expression and function analysis of the transcription factor GmMYB111 in soybean[J]. Scientia Agricultura Sinica,2015,48(15):3079-3089(in Chinese with English abstract).
[8] Wang N,Zhang W X,Qin M Y,et al. Drought tolerance conferred in soybean(Glycine max L.)by GmMYB84,a novel R2R3-MYB transcription factor[J]. Plant Cell Physiol,2017,58(10):1764-1776.
[9] Guo H Y,Wang Y C,Wang L Q,et al. Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla[J]. Plant Biotechnology Journal,2017,15:107-121.
[10]王春霞,黄晨辉,吴光耀,等.转基因烟草分析COP1亚细胞定位及各结构域的功能[J].科学通报,2002,47(7):501-507.Wang C X,Huang C H,Wu G Y,et al. Transgenic tobacco analysis of subcellular localization of COP1 and function of each domain[J]. Chinese Science Bulletin,2002,47(7):501-507(in Chinese with English abstract).
[11]潘婷,邱全胜. N-端结构域决定拟南芥KEA1和KEA2的亚细胞定位及定位与功能的关系[J].中国科技论文,2017,12(18):2124-2129.Pan T,Qiu Q S. N-terminal domain of Arabidopsis KEA1 and KEA2 determines their subcellular localization and relation between localization and function[J]. China Science Paper,2017,12(18):2124-2129(in Chinese with English abstract).
[12] Huang C J,Hu G J,Li F F,et al. NbPHAN,a MYB transcriptional factor,regulates leaf development and affects drought tolerance in Nicotiana benthamiana[J]. Physiologia Plantarum,2013,149:297-309.
[13] Kim W C,Ko J H,Kim J Y,et al. MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis[J].Plant J,2013,73(1):26-36.
[14] Zhong R Q,Ye Z H. MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes[J]. Plant Cell Physiol,2012,53(2):368-380.
[15]朱秋萍,郭春苗,王娟,等.扁桃MYB46转录因子基因的克隆及其表达模式分析[J].植物生理学报,2018,54(4):669-676.Zhu Q P,Guo C M,Wang J,et al. Cloning and expression patterns analysis of MYB46 transcription factor gene in almond[J]. Plant Physiology Journal,2018,54(4):669-676(in Chinese with English abstract).
[16]杨文杰.大豆MYB转录因子基因的克隆及其表达研究[D].雅安:四川农业大学,2007.Yang W J. Cloning and expression of soybean MYB transcription factor gene[D]. Ya’an:Sichuan Agricultural University,2007(in Chinese with English abstract).
[17]杜海,杨文杰,刘蕾,等.大豆MYB转录因子基因GmMYBJ6和GmMYBJ7的克隆及表达分析[J].作物学报,2008,34(7):1179-1187.Du H,Yang W J,Liu L,et al. Cloning and functional identification of the two MYB transcription factors Gm MYBJ6 and Gm MYBJ7 in soybean[J]. Acta Agronomica Sinica,2008,34(7):1179-1187(in Chinese with English abstract).
[18] Zhao M Z,Wang T L,Wu P,et al. Isolation and characterization of GmMYBJ3,an R2R3-MYB transcription factor that affects isoflavonoids biosynthesis in soybean[J]. PLoS One,2017,12(6):e0179990.
[19] Feng K,Xu Z S,Que F,et al. An R2R3-MYB transcription factor,Oj MYB1,functions in anthocyanin biosynthesis in Oenanthe javanica[J].Planta,2018,247(2):301-315.
[20]曹蕾.西瓜ClMYB46基因的耐低温功能鉴定及调控作用分析[D].武汉:华中农业大学,2017.Cao L. Functional identification and regulation analysis of low temperature responsive CLMYB46 in watermelon[D]. Wuhan:Huazhong Agricultural University,2017(in Chinese with English abstract).
[21] Wei Q H,Luo Q C,Wang R B,et al. A wheat R2R3-type MYB transcription factor Ta ODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants[J]. Front Plant Sci,2017,8:1374.
[22] Prabu G,Ppasad D T. Functional characterization of sugarcane MYB transcription factor gene promoter(PScMYBASl)in response to abiotic stresses and hormones[J]. Plant Cell Reports,2012,31(4):661-669.
[23] Chen W Q,Provart N J,Glazebrook J,et al. Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses[J]. Plant Cell,2002,14(3):559-574.
[24] Rubio V,Linhares F,Solano R,et al. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae[J]. Genes&Development,2001,15(16):2122-2133.
[25] Tang Z,Sadka A,Morishige D T,et al. Homeodomain leucine zipper proteins bind to the phosphate response domain of the soybean Vsp B tripartite promoter[J]. Plant Physiology,2001,125(2):797-809.
[26] Wu P,Ma L,Hou X,et al. Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves[J]. Plant Physiology,2003,132(3):1260-1271.
[27] Qin Y X,Wang M C,Tian Y C,et al. Over-expression of Ta MYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis[J]. Mol Biol Rep,2012,39(6):7183-7192.
[28] Liang Y K,Dubos C,Dodd I C,et al. At MYB61,an R2R3-MYB transcription factor controlling stomatal aperture in Arabidopsis thaliana[J].Curr Biol,2008,15(13):1201-1206.
[29] Jung C,Seo J S,Han S W,et al. Overexpression of At MYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis[J].Plant Physiol,2008,146(2):623-635.
[30] Su L T,Li J W,Liu D Q,et al. A novel MYB transcription factor,Gm MYBJ1,from soybean confers drought and cold tolerance in Arabidopsis thaliana[J]. Gene,2014,538(1):46-55.
[31] Vannini C,Locatelli F,Bracale M,et al. Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants[J]. Plant J,2004,37(1):115-127.
[32] Ma Q,Dai X,Xu Y,et al. Enhanced tolerance to chilling stress in Os MYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes[J]. Plant Physiol,2009,150(1):244-256.
[33] Al-Attala M N,Wang X,Abou-Attia M A,et al. A novel TaMYB4 transcription factor involved in the defence response against Puccinia striiformis f. sp. tritici and abiotic stresses[J]. Plant Mol Biol,2014,84(4/5):589.
[34]冯莹莹,董先娟,刘晓,等.白木香转录因子As MYB1和As MYB2克隆及表达分析[J].中国中药杂志,2017,42(17):3305-3311.Feng Y Y,Dong X J,Liu X,et al. Cloning and expression analysis of transcription factor As MYB1 and As MYB2 from Aquilaria sinensis[J]. China Journal of Chinese Materia Medica,2017,42(17):3305-3311(in Chinese with English abstract).
[2] Paz-Ares J,Ghosal D,Wienand U,et al. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators[J]. EMBO J,1987,6(12):3553-3558.
[3] Dubos C,Stracke R,Grotewold E,et al. MYB transcription factors in Arabidopsis[J]. Trends Plant Sci,2010,15(10):573-581.
[4] Katiyar A,Smita S,Lenka S K,et al. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis[J]. BMC Genomics,2012,13:544.
[5] Liu J,Osbourn A,Ma P. MYB transcription factors as regulators of phenylpropaniod metabolism in plants[J]. Mol Plant,2015,8:689-708.
[6] Wang H Y,Wang H L,Shao H B,et al. Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology[J]. Front Plant Sci,2016,7:67.
[7]许玲,卫培培,张大勇,等.大豆转录因子基因GmMYB111的克隆及功能分析[J].中国农业科学,2015,48(15):3079-3089.Xu L,Wei P P,Zhang D Y,et al. Expression and function analysis of the transcription factor GmMYB111 in soybean[J]. Scientia Agricultura Sinica,2015,48(15):3079-3089(in Chinese with English abstract).
[8] Wang N,Zhang W X,Qin M Y,et al. Drought tolerance conferred in soybean(Glycine max L.)by GmMYB84,a novel R2R3-MYB transcription factor[J]. Plant Cell Physiol,2017,58(10):1764-1776.
[9] Guo H Y,Wang Y C,Wang L Q,et al. Expression of the MYB transcription factor gene BplMYB46 affects abiotic stress tolerance and secondary cell wall deposition in Betula platyphylla[J]. Plant Biotechnology Journal,2017,15:107-121.
[10]王春霞,黄晨辉,吴光耀,等.转基因烟草分析COP1亚细胞定位及各结构域的功能[J].科学通报,2002,47(7):501-507.Wang C X,Huang C H,Wu G Y,et al. Transgenic tobacco analysis of subcellular localization of COP1 and function of each domain[J]. Chinese Science Bulletin,2002,47(7):501-507(in Chinese with English abstract).
[11]潘婷,邱全胜. N-端结构域决定拟南芥KEA1和KEA2的亚细胞定位及定位与功能的关系[J].中国科技论文,2017,12(18):2124-2129.Pan T,Qiu Q S. N-terminal domain of Arabidopsis KEA1 and KEA2 determines their subcellular localization and relation between localization and function[J]. China Science Paper,2017,12(18):2124-2129(in Chinese with English abstract).
[12] Huang C J,Hu G J,Li F F,et al. NbPHAN,a MYB transcriptional factor,regulates leaf development and affects drought tolerance in Nicotiana benthamiana[J]. Physiologia Plantarum,2013,149:297-309.
[13] Kim W C,Ko J H,Kim J Y,et al. MYB46 directly regulates the gene expression of secondary wall-associated cellulose synthases in Arabidopsis[J].Plant J,2013,73(1):26-36.
[14] Zhong R Q,Ye Z H. MYB46 and MYB83 bind to the SMRE sites and directly activate a suite of transcription factors and secondary wall biosynthetic genes[J]. Plant Cell Physiol,2012,53(2):368-380.
[15]朱秋萍,郭春苗,王娟,等.扁桃MYB46转录因子基因的克隆及其表达模式分析[J].植物生理学报,2018,54(4):669-676.Zhu Q P,Guo C M,Wang J,et al. Cloning and expression patterns analysis of MYB46 transcription factor gene in almond[J]. Plant Physiology Journal,2018,54(4):669-676(in Chinese with English abstract).
[16]杨文杰.大豆MYB转录因子基因的克隆及其表达研究[D].雅安:四川农业大学,2007.Yang W J. Cloning and expression of soybean MYB transcription factor gene[D]. Ya’an:Sichuan Agricultural University,2007(in Chinese with English abstract).
[17]杜海,杨文杰,刘蕾,等.大豆MYB转录因子基因GmMYBJ6和GmMYBJ7的克隆及表达分析[J].作物学报,2008,34(7):1179-1187.Du H,Yang W J,Liu L,et al. Cloning and functional identification of the two MYB transcription factors Gm MYBJ6 and Gm MYBJ7 in soybean[J]. Acta Agronomica Sinica,2008,34(7):1179-1187(in Chinese with English abstract).
[18] Zhao M Z,Wang T L,Wu P,et al. Isolation and characterization of GmMYBJ3,an R2R3-MYB transcription factor that affects isoflavonoids biosynthesis in soybean[J]. PLoS One,2017,12(6):e0179990.
[19] Feng K,Xu Z S,Que F,et al. An R2R3-MYB transcription factor,Oj MYB1,functions in anthocyanin biosynthesis in Oenanthe javanica[J].Planta,2018,247(2):301-315.
[20]曹蕾.西瓜ClMYB46基因的耐低温功能鉴定及调控作用分析[D].武汉:华中农业大学,2017.Cao L. Functional identification and regulation analysis of low temperature responsive CLMYB46 in watermelon[D]. Wuhan:Huazhong Agricultural University,2017(in Chinese with English abstract).
[21] Wei Q H,Luo Q C,Wang R B,et al. A wheat R2R3-type MYB transcription factor Ta ODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants[J]. Front Plant Sci,2017,8:1374.
[22] Prabu G,Ppasad D T. Functional characterization of sugarcane MYB transcription factor gene promoter(PScMYBASl)in response to abiotic stresses and hormones[J]. Plant Cell Reports,2012,31(4):661-669.
[23] Chen W Q,Provart N J,Glazebrook J,et al. Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses[J]. Plant Cell,2002,14(3):559-574.
[24] Rubio V,Linhares F,Solano R,et al. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae[J]. Genes&Development,2001,15(16):2122-2133.
[25] Tang Z,Sadka A,Morishige D T,et al. Homeodomain leucine zipper proteins bind to the phosphate response domain of the soybean Vsp B tripartite promoter[J]. Plant Physiology,2001,125(2):797-809.
[26] Wu P,Ma L,Hou X,et al. Phosphate starvation triggers distinct alterations of genome expression in Arabidopsis roots and leaves[J]. Plant Physiology,2003,132(3):1260-1271.
[27] Qin Y X,Wang M C,Tian Y C,et al. Over-expression of Ta MYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis[J]. Mol Biol Rep,2012,39(6):7183-7192.
[28] Liang Y K,Dubos C,Dodd I C,et al. At MYB61,an R2R3-MYB transcription factor controlling stomatal aperture in Arabidopsis thaliana[J].Curr Biol,2008,15(13):1201-1206.
[29] Jung C,Seo J S,Han S W,et al. Overexpression of At MYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis[J].Plant Physiol,2008,146(2):623-635.
[30] Su L T,Li J W,Liu D Q,et al. A novel MYB transcription factor,Gm MYBJ1,from soybean confers drought and cold tolerance in Arabidopsis thaliana[J]. Gene,2014,538(1):46-55.
[31] Vannini C,Locatelli F,Bracale M,et al. Overexpression of the rice Osmyb4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants[J]. Plant J,2004,37(1):115-127.
[32] Ma Q,Dai X,Xu Y,et al. Enhanced tolerance to chilling stress in Os MYB3R-2 transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes[J]. Plant Physiol,2009,150(1):244-256.
[33] Al-Attala M N,Wang X,Abou-Attia M A,et al. A novel TaMYB4 transcription factor involved in the defence response against Puccinia striiformis f. sp. tritici and abiotic stresses[J]. Plant Mol Biol,2014,84(4/5):589.
[34]冯莹莹,董先娟,刘晓,等.白木香转录因子As MYB1和As MYB2克隆及表达分析[J].中国中药杂志,2017,42(17):3305-3311.Feng Y Y,Dong X J,Liu X,et al. Cloning and expression analysis of transcription factor As MYB1 and As MYB2 from Aquilaria sinensis[J]. China Journal of Chinese Materia Medica,2017,42(17):3305-3311(in Chinese with English abstract).