小黑杨PnGRAS47基因克隆及氮素诱导下的表达模式分析
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摘要
GRAS转录因子是一种植物特有且广泛分布的转录因子,参与调控植物生长发育及在植物响应环境胁迫中发挥重要的功能。本研究通过克隆小黑杨(Populus simonii×P.nigra)Pn GRAS47基因序列,分析其基因及蛋白序列的基本特征;通过实时定量PCR技术检测在不同形态和不同浓度的氮素处理下小黑杨Pn GRAS47基因的组织表达模式。研究结果显示:Pn GRAS47基因全长1 528 bp,编码452个氨基酸。Pn GRAS47蛋白分子质量为50 812.19 Da,理论等电点为5.41,为不稳定蛋白,不存在信号肽,有2段跨膜区域。氨态氮(NH_4~+-N)和硝态氮(NO_3~--N)处理下,小黑杨Pn GRAS47基因呈现组织特异性表达。0.1和10 mmol·L~(-1) NO_3~--N诱导了Pn GRAS47基因在叶和根中的表达,0.1和10 mmol·L~(-1)的NH_4~+-N处理诱导了Pn GRAS47基因在叶和茎中的表达,但抑制了其在根中表达。因此,小黑杨Pn GRAS47基因受不同氮素的诱导,在不同组织中存在差异,GRAS基因可能在小黑杨对氮素的吸收与利用中发挥作用。
GRAS transcription factor is one of the plant-specific transcription factors, widely distributed in plants, involves in the regulation of plant growth and development, and plays an important role in the plant's response to environmental stresses. In this study, we cloned the Pn GRAS47 gene sequence from Populus simonii × P. nigra and analyzed the basic characteristics of its gene and protein sequence. Expression pattern of Pn GRAS47 gene was detected by quantitative real-time PCR under different morphological nitrogens and different nitrogen concentrations. The results show that the full length of Pn GRAS47 gene is 1 528 bp and the gene encodes 452 amino acids. The molecular mass of Pn GRAS47 is 50 812.19 Da and the p I value is 5.41. It is an unstable protein that has no signal peptide and two transmembrane domains. Under the treatments of ammonia nitrogen and nitrate nitrogen, Pn GRAS47 gene showed tissue-specific expressions. 0.1 and 10 mmol·L~(-1) NO_3~--N induced the expression of Pn GRAS47 gene in leaves and roots; 0.1 and 10 mmol·L~(-1) NH_4~+-N induced the expression of Pn GRAS47 gene in leaves and stems, but inhibited that in roots. Therefore, the Pn GRAS47 gene was induced by nitrogen and was differentially induced in various tissues. GRAS may play a role in the absorption and utilization of nitrogen in P. simonii × P. nigra.
GRAS transcription factor is one of the plant-specific transcription factors, widely distributed in plants, involves in the regulation of plant growth and development, and plays an important role in the plant's response to environmental stresses. In this study, we cloned the Pn GRAS47 gene sequence from Populus simonii × P. nigra and analyzed the basic characteristics of its gene and protein sequence. Expression pattern of Pn GRAS47 gene was detected by quantitative real-time PCR under different morphological nitrogens and different nitrogen concentrations. The results show that the full length of Pn GRAS47 gene is 1 528 bp and the gene encodes 452 amino acids. The molecular mass of Pn GRAS47 is 50 812.19 Da and the p I value is 5.41. It is an unstable protein that has no signal peptide and two transmembrane domains. Under the treatments of ammonia nitrogen and nitrate nitrogen, Pn GRAS47 gene showed tissue-specific expressions. 0.1 and 10 mmol·L~(-1) NO_3~--N induced the expression of Pn GRAS47 gene in leaves and roots; 0.1 and 10 mmol·L~(-1) NH_4~+-N induced the expression of Pn GRAS47 gene in leaves and stems, but inhibited that in roots. Therefore, the Pn GRAS47 gene was induced by nitrogen and was differentially induced in various tissues. GRAS may play a role in the absorption and utilization of nitrogen in P. simonii × P. nigra.
引文
Biasini M,Bienert S,Waterhouse A,et al(2014).SWISS-MODEL:modelling protein tertiary and quaternary structure using evolutionary information.Nucl Acids Res,42:W252-W258
Bolle C(2004).The role of GRAS proteins in plant signal transduction and development.Planta,218(5):683-692
Chen YQ,Tai SS,Wang DW,et al(2015).Homology-based analysis of the GRAS gene family in tobacco.Genet Mol Res,14(4):15188-15200
Combet C,Blanchet C,Geourjon C,et al(2000).NPS@:network protein sequence analysis.Trends Biochem Sci,25(3):147-150
Day RB,Shibuya N,Minami E(2003).Identification and characterization of two new members of the GRAS gene family in rice responsive to N-acetylchitooligosaccharide elicitor.Biochim Biophys Acta,1625(3):261-268
Di Laurenzio L,Wysocka-Diller J,Malamy JE,et al(1966).The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.Cell,86:423-433
Fambrini M,Mariotti L,Parlanti S,et al(2015).A GRAS-like gene of sunflower(Helianthus annuus L.)alters the gibberellin content and axillary meristem outgrowth in transgenic Arabidopsis plants.Plant Biol,17(6):1123-1134
Fode B,Siemsen T,Thurow C,et al(2008).The Arabidopsis GRAS protein SCL14 interacts with class II TGAtranscription factors and is essential for the activation of stress-inducible promoters.Plant Cell,20(11):3122-3135
Gasteiger E,Hoogland C,Gattiker A,et al(2005).Protein identification and analysis tools on the Ex PASy server.In:John MW(ed).The Proteomics Protocols Handbook.New York City:Humana Press,571-607
Guex N,Peitsch MC,Schwede T(2009).Automated comparative protein structure modeling with SWISS-MODEL and Swiss-Pdb Viewer:a historical perspective.Electrophoresis,30:S162-S173
Guo H,Jiao Y,Di C,et al(2009).Discovery of Arabidopsis GRAS family genes in response to osmotic and drought stresses.Chin Bull Bot,44(3):290-299(in chinese with English abstract)[郭华军,焦远年,邸超等(2009).拟南芥转录因子GRAS家族基因群响应渗透和干旱胁迫的初步探索.植物学报,44(3):290-299]
Hirsch S,Kim J,Mu?oz A,et al(2009).GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula.Plant Cell,21(2):545-557
Hirsch S,Oldroyd GED(2009).GRAS-domain transcription factors that regulate plant development.Plant Signal Behav,4(8):698-700
Hofmann K,Stoffel W(1993).TMbase:a database of membrane spanning protein segments.Biol Chem,374:166
Huang W,Xian Z,Kang X,et al(2015).Genome-wide identification,phylogeny and expression analysis of GRASgene family in tomato.BMC Plant Biol,15:209
Kopp J,Schwede T(2006).The SWISS-MODEL repository:new features and functionalities.Nucl Acids Res,34:D315-D318
Li Q,Chen J,Di P,et al(2014).Discovery and analysis of GRAS gene family in Isatis indigotica.Eur J Integr Med,6:727-728
Li X,Qian Q,Fu Z,et al(2003).Control of tillering in rice.Nature,422(6932):618-621
Liu X,Widmer A(2014).Genome-wide comparative analysis of the GRAS gene family in Populus,Arabidopsis and rice.Plant Mol Biol Rep,32(6):1129-1145
Lu J,Wang T,Xu Z,et al(2013).Genome-wide analysis of the GRAS gene family in Prunus mume.290(1):3155-3160
Marchler-Bauer A,Bo Y,Han L,et al(2017).CDD/SPAR-CLE:functional classification of proteins via subfamily domain architectures.Nucl Acids Res,45:D200-D203
Peng J,Carol P,Richards DE,et al(1997).The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses.Genes Dev,11(23):3194-3205
Petersen TN,Brunak S,von Heijne G,et al(2011).Signal P4.0:discriminating signal peptides from transmembrane regions.Nat Methods,8(10):785-786
Pysh LD,Wysocka-Diller JW,Camilleri C,et al(1999).The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARE-CROW-LIKE genes.Plant J,18(1):111-119
Silverstone AL,Ciampaglio CN,Sun TP(1998).The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway.Plant Cell,10:155-169
Song XM,Liu TK,Duan WK,et al(2014).Genome-wide analysis of the GRAS gene family in Chinese cabbage(Brassica rapa ssp.pekinensis).Genomics,103(1):135-146
Sun X,Xie Z,Zhang C,et al(2016).A characterization of grapevine of GRAS domain transcription factor gene family.Funct Integr Genomics,16(4):347-363
Tamura K,Peterson D,Peterson N,et al(2011).MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28(10):2731-2739
Thompson JD,Gibson TJ,Plewniak F,et al(1997).The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucl Acids Res,25(24):4876-4882
Tian C,Wan P,Sun S,et al(2004).Genome-wide analysis of the GRAS gene family in rice and Arabidopsis.Plant Mol Biol,54:519-532
Torres-Galea P,Hirtreiter B,Bolle C(2013).Two GRAS proteins,SCARECROW-LIKE21 and PHYTOCHROME ASIGNAL TRANSDUCTION1,function cooperatively in phytochrome A signal transduction.Plant Physiol,161:291-304
Wu ZY,Wu PZ,Chen YP,et al(2015).Genome-wide analysis of the GRAS gene family in physic nut(Jatropha curcas L.).Genet Mol Res,14(4):19211-19224
Yang M,Yang Q,Fu T,et al(2011).Overexpression of the Brassica napus Bn LAS gene in Arabidopsis affects plant development and increases drought tolerance.Plant Cell Rep,30(3):373-388
Zhou L,Yang Z,Zhang F,et al(2013).Expression analysis and cloning of GRAS transcription factor gene from Halostachys caspica.Acta Bot Boreal-Occident Sin,33(6):1091-1097(in chinese with English abstract)[周莲洁,杨中敏,张富春等(2013).新疆盐穗木GRAS转录因子基因克隆及表达分析.西北植物学报,33(6):1091-1097]
Bolle C(2004).The role of GRAS proteins in plant signal transduction and development.Planta,218(5):683-692
Chen YQ,Tai SS,Wang DW,et al(2015).Homology-based analysis of the GRAS gene family in tobacco.Genet Mol Res,14(4):15188-15200
Combet C,Blanchet C,Geourjon C,et al(2000).NPS@:network protein sequence analysis.Trends Biochem Sci,25(3):147-150
Day RB,Shibuya N,Minami E(2003).Identification and characterization of two new members of the GRAS gene family in rice responsive to N-acetylchitooligosaccharide elicitor.Biochim Biophys Acta,1625(3):261-268
Di Laurenzio L,Wysocka-Diller J,Malamy JE,et al(1966).The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root.Cell,86:423-433
Fambrini M,Mariotti L,Parlanti S,et al(2015).A GRAS-like gene of sunflower(Helianthus annuus L.)alters the gibberellin content and axillary meristem outgrowth in transgenic Arabidopsis plants.Plant Biol,17(6):1123-1134
Fode B,Siemsen T,Thurow C,et al(2008).The Arabidopsis GRAS protein SCL14 interacts with class II TGAtranscription factors and is essential for the activation of stress-inducible promoters.Plant Cell,20(11):3122-3135
Gasteiger E,Hoogland C,Gattiker A,et al(2005).Protein identification and analysis tools on the Ex PASy server.In:John MW(ed).The Proteomics Protocols Handbook.New York City:Humana Press,571-607
Guex N,Peitsch MC,Schwede T(2009).Automated comparative protein structure modeling with SWISS-MODEL and Swiss-Pdb Viewer:a historical perspective.Electrophoresis,30:S162-S173
Guo H,Jiao Y,Di C,et al(2009).Discovery of Arabidopsis GRAS family genes in response to osmotic and drought stresses.Chin Bull Bot,44(3):290-299(in chinese with English abstract)[郭华军,焦远年,邸超等(2009).拟南芥转录因子GRAS家族基因群响应渗透和干旱胁迫的初步探索.植物学报,44(3):290-299]
Hirsch S,Kim J,Mu?oz A,et al(2009).GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula.Plant Cell,21(2):545-557
Hirsch S,Oldroyd GED(2009).GRAS-domain transcription factors that regulate plant development.Plant Signal Behav,4(8):698-700
Hofmann K,Stoffel W(1993).TMbase:a database of membrane spanning protein segments.Biol Chem,374:166
Huang W,Xian Z,Kang X,et al(2015).Genome-wide identification,phylogeny and expression analysis of GRASgene family in tomato.BMC Plant Biol,15:209
Kopp J,Schwede T(2006).The SWISS-MODEL repository:new features and functionalities.Nucl Acids Res,34:D315-D318
Li Q,Chen J,Di P,et al(2014).Discovery and analysis of GRAS gene family in Isatis indigotica.Eur J Integr Med,6:727-728
Li X,Qian Q,Fu Z,et al(2003).Control of tillering in rice.Nature,422(6932):618-621
Liu X,Widmer A(2014).Genome-wide comparative analysis of the GRAS gene family in Populus,Arabidopsis and rice.Plant Mol Biol Rep,32(6):1129-1145
Lu J,Wang T,Xu Z,et al(2013).Genome-wide analysis of the GRAS gene family in Prunus mume.290(1):3155-3160
Marchler-Bauer A,Bo Y,Han L,et al(2017).CDD/SPAR-CLE:functional classification of proteins via subfamily domain architectures.Nucl Acids Res,45:D200-D203
Peng J,Carol P,Richards DE,et al(1997).The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses.Genes Dev,11(23):3194-3205
Petersen TN,Brunak S,von Heijne G,et al(2011).Signal P4.0:discriminating signal peptides from transmembrane regions.Nat Methods,8(10):785-786
Pysh LD,Wysocka-Diller JW,Camilleri C,et al(1999).The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARE-CROW-LIKE genes.Plant J,18(1):111-119
Silverstone AL,Ciampaglio CN,Sun TP(1998).The Arabidopsis RGA gene encodes a transcriptional regulator repressing the gibberellin signal transduction pathway.Plant Cell,10:155-169
Song XM,Liu TK,Duan WK,et al(2014).Genome-wide analysis of the GRAS gene family in Chinese cabbage(Brassica rapa ssp.pekinensis).Genomics,103(1):135-146
Sun X,Xie Z,Zhang C,et al(2016).A characterization of grapevine of GRAS domain transcription factor gene family.Funct Integr Genomics,16(4):347-363
Tamura K,Peterson D,Peterson N,et al(2011).MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28(10):2731-2739
Thompson JD,Gibson TJ,Plewniak F,et al(1997).The CLUSTAL_X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tools.Nucl Acids Res,25(24):4876-4882
Tian C,Wan P,Sun S,et al(2004).Genome-wide analysis of the GRAS gene family in rice and Arabidopsis.Plant Mol Biol,54:519-532
Torres-Galea P,Hirtreiter B,Bolle C(2013).Two GRAS proteins,SCARECROW-LIKE21 and PHYTOCHROME ASIGNAL TRANSDUCTION1,function cooperatively in phytochrome A signal transduction.Plant Physiol,161:291-304
Wu ZY,Wu PZ,Chen YP,et al(2015).Genome-wide analysis of the GRAS gene family in physic nut(Jatropha curcas L.).Genet Mol Res,14(4):19211-19224
Yang M,Yang Q,Fu T,et al(2011).Overexpression of the Brassica napus Bn LAS gene in Arabidopsis affects plant development and increases drought tolerance.Plant Cell Rep,30(3):373-388
Zhou L,Yang Z,Zhang F,et al(2013).Expression analysis and cloning of GRAS transcription factor gene from Halostachys caspica.Acta Bot Boreal-Occident Sin,33(6):1091-1097(in chinese with English abstract)[周莲洁,杨中敏,张富春等(2013).新疆盐穗木GRAS转录因子基因克隆及表达分析.西北植物学报,33(6):1091-1097]