铁皮石斛GRAS转录因子家族基因SCL3克隆及表达分析
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摘要
GRAS家族是一类植物特有的转录调控因子,参与调控植物生长发育及抵御逆境胁迫,并且在赤霉素(gibberellins, GAs)信号转导过程中具有重要作用。本研究采用RT-PCR结合RACE方法从铁皮石斛(Dendrobium officinale Kimura et Migo)原球茎中克隆到了一个转录因子GRAS家族基因SCL3 (scarecrow-like 3)并对其进行了表达分析。该基因cDNA序列全长2 278 bp,命名为DoSCL3 (GenBank注册号:MG252261),其编码425个氨基酸,分子量为47.88 kD,等电点(PI)为6.21。DoSCL3基因编码的蛋白不含跨膜域和信号肽,具有GRAS转录因子家族特有的保守结构域(22～422);多序列比对表明,DoSCL3与小兰屿蝴蝶兰的SCL3蛋白高度同源(相似性达到83%),同时进化树分析结果显示其与小兰屿蝴蝶兰亲缘关系非常相近。qRT-PCR实验结果显示,DoSCL3基因在铁皮石斛种子共生萌发中随着萌发进程的变化(1～3级,萌发至原球茎阶段)其表达量逐渐升高,并且在种子萌发至2级时,共生萌发组表达量显著高于无菌萌发组(为无菌组2.2倍),表明DoSCL3在兰科药用植物铁皮石斛种子共生萌发中菌根共生关系的建立过程起到重要的调控作用。
The GRAS family is a type of plant-specific transcriptional factors that is involved in the regulation of plant growth and development and defense against environmental stress, as well as plays an important role in the signal transduction of gibberellins(GAs) in plant and mycorrhizal signaling. In this study, a transcription factor GRAS family of gene SCL3(scarecrow-like 3) was cloned from the protocorm of Dendrobium officinale(Kimura et Migo)by RT-PCR and RACE method, and its expression was analyzed. The full-length cDNA of the gene was 2 278 bp,named DoSCL3(GenBank accession number: MG252261). It encoded 425 amino acids with a molecular weight of47.88 kD and an isoelectric point(PI) of 6.21. The DoSCL3 contained a conserved domain(22～422) specific to the GRAS family of transcription factors protein but without transmembrane domain and signal peptide; multiple sequence alignments showed that DoSCL3 shared the highest homology with the SCL3 protein of Phalaenopsis equestris(83%). The results of qRT-PCR showed that the expression level of DoSCL3 gene increased gradually in symbiotic germination with the change of germination process(grade 1-3, germination to protocorm stage). At the stage 2 of seed germination, the expression level in symbiotic germination group was significantly higher than that in aseptic germination group(2.2 times as much as that in aseptic germination group). It would be suggested that DoSCL3 might play an important role in the establishment of mycorrhizal symbiotic relationship in the symbiotic germination of D. officinale seeds.
The GRAS family is a type of plant-specific transcriptional factors that is involved in the regulation of plant growth and development and defense against environmental stress, as well as plays an important role in the signal transduction of gibberellins(GAs) in plant and mycorrhizal signaling. In this study, a transcription factor GRAS family of gene SCL3(scarecrow-like 3) was cloned from the protocorm of Dendrobium officinale(Kimura et Migo)by RT-PCR and RACE method, and its expression was analyzed. The full-length cDNA of the gene was 2 278 bp,named DoSCL3(GenBank accession number: MG252261). It encoded 425 amino acids with a molecular weight of47.88 kD and an isoelectric point(PI) of 6.21. The DoSCL3 contained a conserved domain(22～422) specific to the GRAS family of transcription factors protein but without transmembrane domain and signal peptide; multiple sequence alignments showed that DoSCL3 shared the highest homology with the SCL3 protein of Phalaenopsis equestris(83%). The results of qRT-PCR showed that the expression level of DoSCL3 gene increased gradually in symbiotic germination with the change of germination process(grade 1-3, germination to protocorm stage). At the stage 2 of seed germination, the expression level in symbiotic germination group was significantly higher than that in aseptic germination group(2.2 times as much as that in aseptic germination group). It would be suggested that DoSCL3 might play an important role in the establishment of mycorrhizal symbiotic relationship in the symbiotic germination of D. officinale seeds.
引文
Bolle C.,Koncz C.,and Chua N.H.,2000,PAT1,a new member of the GRAS family,is involved in phytochrome a signa transduction,Genes Dev.,14(10):1269-1278
Brenner S.E.,Chothia C.,and Hubbard T.J.P.,1998,Assessing sequence comparison methods with reliable structurally identified distant evolutionary relationships,Proc.Natl.Acad Sci.USA,95(11):6073-7078
Chen X.M.,Li Y.Y.,and Guo S.X.,2013,Research progress on seed germination of Dendrobium species,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),48(19):1629-1633(陈晓梅,李媛媛,郭顺星,2013,石斛属植物种子萌发的研究进展,中国药学杂志,48(19):1629-1633)
Cenci A.,and Rouard M.,2017,Evolutionary analyses of gras transcription factors in angiosperms,Frontiers in Plant Science,8:273
Di Laurenzio L.,Wysocka-Diller J.,Malamy J.E.,Pysh L.,Helariutta Y.,Freshour G.,Hahn M.G.,Feldmann K.A.,and Benfey P.N.,1996,The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radia organization of the Arabidopsis root,Cell,86(3):423-433
Dong L.L.,Zhao H.S.,Li L.C.,Sun H.Y.,Wang L.L.,and Gao ZM.,2016,Expression characteristics and promoter activity analysis of PeSCL3 gene from Phyllostachys edulis,Reda Yaredai Zhiwu Xuebao(Journal of Tropical and Subtropica Botany),24(3):252-258(董丽莉,赵韩生,李利超,孙化雨王丽丽,高志民,2016,毛竹PeSCL3基因表达特征及其启动子活性研究,热带亚热带植物学报,24(3):252-258)
Gobbato E.,Marsh J.F.,VerniéT.,Wang E.,Maillet F.,Kim J.,Miller J.B.,Sun J.,Bano A.S.,Ratet P.,MysoreK.S.,Deénarie J.,Schultze M.,and Oldroyd G.E.D.,2012,A GRAS-type transcription factor with a specific function in mycorrhizal signaling,Current Biology,22(23):2236-2241
Hirano Y.,Nakagawa M.,Suyama T.,Murase K.,Shirakawa M.,Takayama S.,Sun T.P.,and Hakoshima T.,2017,Structure of the shr-scr heterodimer bound to the bird/idd transcriptional factor jkd,Nat.Plants,3:17010
Hirsch S.,Kim J.,Munoz A.,Heckmann A.B.,Downie J.A.,and Oldroyd G.E.D.,2009,GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula,Plant Cell,21:545-557
KalóP.,Gleason C.,Edwards A.,Marsh J.,Mitra R.M.,Hirsch S.,Jakab J.,Sims S.,Long S.R.,Rogers J.,Kiss G.B.,Downie J.A.,and Oldroyd G.E.D.,2005,Nodulation signaling in legumes requires NSP2,a member of the GR-AS family of transcriptional regulators,Science,308(5729):1786-1789
Kozak M.,1987,An analysis of 5'-noncoding sequences from699 vertebrate messenger RNAs,Nucleic Acids Res.,15(20):8125
Liu S.S.,Chen J.,and Guo S.X.,2015,Review on germination of Orchid seeds,Zhongzi(Seed),34(6):43-50(刘思思,陈娟,郭顺星,2015,兰科植物种子萌发的研究进展,种子,34(6):43-50)
Liu S.S.,Zhang G.,Chen X.M.,Li S.C.,Chen J.,and Guo S.X.,2016,Cloning and expression analysis of gibberellins 3-oxidase gene in Dendrobium officinale,Zhongcaoyao(Chinese Traditional and Herbal Drug),47(6):990-996(刘思思,张岗,陈晓梅,李淑超,陈娟,郭顺星,2016,铁皮石斛赤霉素3-氧化酶基因的克隆及表达分析,中草药,47(6):990-996)
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative pcr and the2-ΔΔctmethod,Methods,25(4):402-408
Llave C.,Xie Z.,Kasschau K.D.,and Carrington J.C.,2002,Cleavage of scarecrow-like m RNA targets directed by a class of Arabidopsis mi RNA,Science,297(5589):2053-2056
Ma H.S.,Liang D.,Shuai P.,Xia X.L.,and Yin W.L.,2010,The salt-and drought-inducible poplar gras protein scl7 confers salt and drought tolerance in Arabidopsis thaliana,J.Exp.Bot.,61(14):4011-4019
Niu Y.L.,Jiang X.M.,and Xu X.Y.,2016,Cloning and VIGSvector construction of GRAS transcription factors family gene SIGLD1 from tomato,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(8):2155-2160(牛义岭,姜秀明,许向阳,2016,番茄GRAS转录因子家族基因SIGLD1的克隆及VIGS载体构建,基因组学与应用生物学,35(8):2155-2160)
Peng J.,Carol P.,Richards D.E.,King K.E.,Cowling R.J.,Murphy G.P.,and Harberd N.P.,1997,The Arabidopsis GAIgene defines a signaling pathway that negatively regulates gibberellin responses,Genes Dev.,11(23):3194-3205
Pimprikar P.,Carbonnel S.,Paries M.,Katzer K.,Klingl V.,Bohmer M.J.,Karl L.,Floss D.S.,Harrison M.J.,Parniske M.,and Gutjahr C.,2016,A ccamk-cyclops-della complex activates transcription of ram1 to regulate arbuscule branching,Current Biology,26(8):987-998
Pysh L.D.,Wysocka-Diller J.W.,Camilleri C.,Bouchez D.,and Benfey P.N.,1999,The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes,The Plant Journal,18(1):111-119
Silverstone A.L.,Ciampaglio C.N.,and Sun T.,1998,The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway,Plant Cell,10(2):155-169
Smit P.,Raedts J.,Portyanko V.,DebelléF.,Gough C.,Bisseling T.,and Geurts R.,2005,NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription,Science,308(5729):1789-1791
Sun X.,Xie Z.,Zhang C.,Mu Q.,Wu W.,Wang B.,and Fang J.,2016,A characterization of grapevine of GRAS domain transcription factor gene family,Functional and Integrative Genomics,16(4):347-363
Stewart S.L.,and Zettler L.W.,2002,Symbiotic germination of three semi-aquatic rein orchids(Habenaria repens,H.quinquiseta,H.macroceratitis)from Florida,Aquatic Botany,72(1):25-35
Wu J.,Wang J.F.,Fang L.,Wu H.L.,Zhu M.W.,and Wang R.W.,2013,Domestic research progress of Dendrobium officinale Kimura et Migo,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),48(19):1610-1613(吴建,王建方,方玲,吴华玲,朱明旺,王如伟,2013,国内铁皮石斛研究概况,中国药学杂志,48(19):1610-1613)
Yu N.,Luo D.,Zhang X.,Liu J.,Wang W.,Jin Y.,Dong W.,Liu J.,Liu H.,Yang W.,Zeng L.,Li Q.,He Z,Oldroyd G.E.,and Wang E.,2014,A DELLA protein complex controls the arbuscular mycorrhizal symbiosis in plants,Cell Res.,24(1):130-133
Zhang G.,Liu S.S.,Peng L.,Zhou L.S.,Liu L.L.,Li H.,Hei X.B.,Shen X.,and Guo S.X.,2017,Molecular cloning and characterization of a transcription factor gene DoWRKY3 in Dendrobium officinale,Zhongcaoyao(Chinese Traditional and Herbal Drugs),48(14):2930-2936(张岗,刘思思,彭亮,周丽思,刘亮亮,李欢,黑小斌,沈霞,郭顺星,2017,铁皮石斛转录因子基因DoWRKY3的克隆与分子特性分析,中草药,48(14):2930-2936)
Zhang G.,Zhao M.M.,Zhang D.W.,and Guo S.X.,2013,Reference Gene Selection for Real-Time Quantitative PCR analysis of Dendrobium officinale,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),48(19):1664-1668(张岗,赵明明,张大为,郭顺星,2013,铁皮石斛实时定量PCR内参基因的筛选,中国药学杂志,48(19):1664-1668)
Zhang Z.L.,Ogawa M.,Fleet C.M.,Zentella R,Hu J.,Heo J.O.,Lim J.,Kamiya Y.,Yamaguchi S.,and Sun T.P.,2010,Scarecrow-like 3 promotes gibberellin signaling by antagonizing master growth repressor della in Arabidopsis,Proc.Natl.Acad.Sci.USA,108(5):2160-2165
Brenner S.E.,Chothia C.,and Hubbard T.J.P.,1998,Assessing sequence comparison methods with reliable structurally identified distant evolutionary relationships,Proc.Natl.Acad Sci.USA,95(11):6073-7078
Chen X.M.,Li Y.Y.,and Guo S.X.,2013,Research progress on seed germination of Dendrobium species,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),48(19):1629-1633(陈晓梅,李媛媛,郭顺星,2013,石斛属植物种子萌发的研究进展,中国药学杂志,48(19):1629-1633)
Cenci A.,and Rouard M.,2017,Evolutionary analyses of gras transcription factors in angiosperms,Frontiers in Plant Science,8:273
Di Laurenzio L.,Wysocka-Diller J.,Malamy J.E.,Pysh L.,Helariutta Y.,Freshour G.,Hahn M.G.,Feldmann K.A.,and Benfey P.N.,1996,The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radia organization of the Arabidopsis root,Cell,86(3):423-433
Dong L.L.,Zhao H.S.,Li L.C.,Sun H.Y.,Wang L.L.,and Gao ZM.,2016,Expression characteristics and promoter activity analysis of PeSCL3 gene from Phyllostachys edulis,Reda Yaredai Zhiwu Xuebao(Journal of Tropical and Subtropica Botany),24(3):252-258(董丽莉,赵韩生,李利超,孙化雨王丽丽,高志民,2016,毛竹PeSCL3基因表达特征及其启动子活性研究,热带亚热带植物学报,24(3):252-258)
Gobbato E.,Marsh J.F.,VerniéT.,Wang E.,Maillet F.,Kim J.,Miller J.B.,Sun J.,Bano A.S.,Ratet P.,MysoreK.S.,Deénarie J.,Schultze M.,and Oldroyd G.E.D.,2012,A GRAS-type transcription factor with a specific function in mycorrhizal signaling,Current Biology,22(23):2236-2241
Hirano Y.,Nakagawa M.,Suyama T.,Murase K.,Shirakawa M.,Takayama S.,Sun T.P.,and Hakoshima T.,2017,Structure of the shr-scr heterodimer bound to the bird/idd transcriptional factor jkd,Nat.Plants,3:17010
Hirsch S.,Kim J.,Munoz A.,Heckmann A.B.,Downie J.A.,and Oldroyd G.E.D.,2009,GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula,Plant Cell,21:545-557
KalóP.,Gleason C.,Edwards A.,Marsh J.,Mitra R.M.,Hirsch S.,Jakab J.,Sims S.,Long S.R.,Rogers J.,Kiss G.B.,Downie J.A.,and Oldroyd G.E.D.,2005,Nodulation signaling in legumes requires NSP2,a member of the GR-AS family of transcriptional regulators,Science,308(5729):1786-1789
Kozak M.,1987,An analysis of 5'-noncoding sequences from699 vertebrate messenger RNAs,Nucleic Acids Res.,15(20):8125
Liu S.S.,Chen J.,and Guo S.X.,2015,Review on germination of Orchid seeds,Zhongzi(Seed),34(6):43-50(刘思思,陈娟,郭顺星,2015,兰科植物种子萌发的研究进展,种子,34(6):43-50)
Liu S.S.,Zhang G.,Chen X.M.,Li S.C.,Chen J.,and Guo S.X.,2016,Cloning and expression analysis of gibberellins 3-oxidase gene in Dendrobium officinale,Zhongcaoyao(Chinese Traditional and Herbal Drug),47(6):990-996(刘思思,张岗,陈晓梅,李淑超,陈娟,郭顺星,2016,铁皮石斛赤霉素3-氧化酶基因的克隆及表达分析,中草药,47(6):990-996)
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative pcr and the2-ΔΔctmethod,Methods,25(4):402-408
Llave C.,Xie Z.,Kasschau K.D.,and Carrington J.C.,2002,Cleavage of scarecrow-like m RNA targets directed by a class of Arabidopsis mi RNA,Science,297(5589):2053-2056
Ma H.S.,Liang D.,Shuai P.,Xia X.L.,and Yin W.L.,2010,The salt-and drought-inducible poplar gras protein scl7 confers salt and drought tolerance in Arabidopsis thaliana,J.Exp.Bot.,61(14):4011-4019
Niu Y.L.,Jiang X.M.,and Xu X.Y.,2016,Cloning and VIGSvector construction of GRAS transcription factors family gene SIGLD1 from tomato,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(8):2155-2160(牛义岭,姜秀明,许向阳,2016,番茄GRAS转录因子家族基因SIGLD1的克隆及VIGS载体构建,基因组学与应用生物学,35(8):2155-2160)
Peng J.,Carol P.,Richards D.E.,King K.E.,Cowling R.J.,Murphy G.P.,and Harberd N.P.,1997,The Arabidopsis GAIgene defines a signaling pathway that negatively regulates gibberellin responses,Genes Dev.,11(23):3194-3205
Pimprikar P.,Carbonnel S.,Paries M.,Katzer K.,Klingl V.,Bohmer M.J.,Karl L.,Floss D.S.,Harrison M.J.,Parniske M.,and Gutjahr C.,2016,A ccamk-cyclops-della complex activates transcription of ram1 to regulate arbuscule branching,Current Biology,26(8):987-998
Pysh L.D.,Wysocka-Diller J.W.,Camilleri C.,Bouchez D.,and Benfey P.N.,1999,The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes,The Plant Journal,18(1):111-119
Silverstone A.L.,Ciampaglio C.N.,and Sun T.,1998,The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway,Plant Cell,10(2):155-169
Smit P.,Raedts J.,Portyanko V.,DebelléF.,Gough C.,Bisseling T.,and Geurts R.,2005,NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription,Science,308(5729):1789-1791
Sun X.,Xie Z.,Zhang C.,Mu Q.,Wu W.,Wang B.,and Fang J.,2016,A characterization of grapevine of GRAS domain transcription factor gene family,Functional and Integrative Genomics,16(4):347-363
Stewart S.L.,and Zettler L.W.,2002,Symbiotic germination of three semi-aquatic rein orchids(Habenaria repens,H.quinquiseta,H.macroceratitis)from Florida,Aquatic Botany,72(1):25-35
Wu J.,Wang J.F.,Fang L.,Wu H.L.,Zhu M.W.,and Wang R.W.,2013,Domestic research progress of Dendrobium officinale Kimura et Migo,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),48(19):1610-1613(吴建,王建方,方玲,吴华玲,朱明旺,王如伟,2013,国内铁皮石斛研究概况,中国药学杂志,48(19):1610-1613)
Yu N.,Luo D.,Zhang X.,Liu J.,Wang W.,Jin Y.,Dong W.,Liu J.,Liu H.,Yang W.,Zeng L.,Li Q.,He Z,Oldroyd G.E.,and Wang E.,2014,A DELLA protein complex controls the arbuscular mycorrhizal symbiosis in plants,Cell Res.,24(1):130-133
Zhang G.,Liu S.S.,Peng L.,Zhou L.S.,Liu L.L.,Li H.,Hei X.B.,Shen X.,and Guo S.X.,2017,Molecular cloning and characterization of a transcription factor gene DoWRKY3 in Dendrobium officinale,Zhongcaoyao(Chinese Traditional and Herbal Drugs),48(14):2930-2936(张岗,刘思思,彭亮,周丽思,刘亮亮,李欢,黑小斌,沈霞,郭顺星,2017,铁皮石斛转录因子基因DoWRKY3的克隆与分子特性分析,中草药,48(14):2930-2936)
Zhang G.,Zhao M.M.,Zhang D.W.,and Guo S.X.,2013,Reference Gene Selection for Real-Time Quantitative PCR analysis of Dendrobium officinale,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),48(19):1664-1668(张岗,赵明明,张大为,郭顺星,2013,铁皮石斛实时定量PCR内参基因的筛选,中国药学杂志,48(19):1664-1668)
Zhang Z.L.,Ogawa M.,Fleet C.M.,Zentella R,Hu J.,Heo J.O.,Lim J.,Kamiya Y.,Yamaguchi S.,and Sun T.P.,2010,Scarecrow-like 3 promotes gibberellin signaling by antagonizing master growth repressor della in Arabidopsis,Proc.Natl.Acad.Sci.USA,108(5):2160-2165
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