菊花CmGATA12基因促进开花的分子机制
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摘要
[目的]本文旨在探究菊花CmGATA12基因的功能,分析其表达特征,并转入拟南芥中对其功能进行初步研究。[方法]从菊花品种‘清露’中克隆获得CmGATA12全长序列,与其他物种进行同源性比对分析,同时利用实时荧光定量PCR分析CmGATA12在菊花不同组织中的表达变化;将CmGATA12基因转入拟南芥中,观察其对拟南芥开花时间和莲座叶片数的影响,并分析CmGATA12转基因拟南芥中相关开花基因的表达变化。[结果]CmGATA12与拟南芥GATA家族的AtGATA13和AtGATA8的遗传距离最近。组织定量分析表明CmGATA12在菊花的茎中表达量最高,在芽中表达量最低。亚细胞定位和转录活性试验表明,CmGATA12定位在细胞核和细胞膜并且具有转录激活活性。统计CmGATA12转基因拟南芥植株的开花时间和莲座叶片数发现,CmGATA12转基因拟南芥植株的开花时间均提前于野生型拟南芥植株,开花时的莲座叶片数均低于野生型拟南芥植株。CmGATA12转基因拟南芥植株相关开花基因的表达中,促进开花的基因AtSPL5和AtGI在CmGATA12转基因植株中较野生型拟南芥中的表达量上调,抑制开花的基因AtSVP、AtFRI、AtTOE1和AtTOE2在CmGATA12转基因植株中较野生型表达量下调。[结论]本研究克隆得到的菊花CmGATA12基因,具有促进拟南芥开花的功能。
[Objectives]The purpose of the current study was to characterize the function of chrysanthemum CmGATA12 gene by analyzing its expression profile in chrysanthemum and heterologous expression in Arabidopsis thaliana to gain more insight into the function of the protein. [Methods]The full-length cDNA of CmGATA12 was cloned from the chrysanthemum variety'Qinglu'and compared with other GATA12 sequences from different species. Simultaneously,real-time quantitative PCR was used to analyze the expression changes of CmGATA12 gene in different tissues of chrysanthemum. The CmGATA12 gene was transfected into A.thaliana,and its effect on flowering time and rosette leaf number in A.thaliana was observed. The expression of flowering related genes in CmGATA12 transgenic A.thaliana was analyzed. [Results]The genetic distance between CmGATA12 and AtGATA13 and AtGATA8 of Arabidopsis GATA family was the closest. Quantitative analysis of CmGATA12 gene in different tissues of chrysanthemum revealed that CmGATA12 gene had the highest expression level in the stem of chrysanthemum and the lowest expression level in bud. Subcellular localization indicated that the CmGATA12 gene was located both on the cell membrane and on the nucleus. The CmGATA12 gene was found to have transcriptional activity in the yeast system. By observing the time taken to flowering and rosette leaf number of CmGATA12 transgenic A.thaliana plants,it was found that the flowering time of CmGATA12 transgenic A.thaliana plants was earlier than that of wild-type plants,and the number of rosette leaves in flowering was lower than that of wild-type plants. Flowering-time related genes involved in flowering promotion(AtSPL5 and AtGI)and flowering inhibition genes(AtSVP,AtFRI,AtTOE1 and AtTOE2)were compared in the transgenic and wild-type plants. The results indicated that flower-promoting genes were up-regulated in the transgenic plants compared to the wild-type plants. Similarly,floral-repression genes were reduced expressed in transgenic plants compared with wild-type plants. [Conclusions]CmGATA12 gene has a role in promoting flowering in the heterologous system.
[Objectives]The purpose of the current study was to characterize the function of chrysanthemum CmGATA12 gene by analyzing its expression profile in chrysanthemum and heterologous expression in Arabidopsis thaliana to gain more insight into the function of the protein. [Methods]The full-length cDNA of CmGATA12 was cloned from the chrysanthemum variety'Qinglu'and compared with other GATA12 sequences from different species. Simultaneously,real-time quantitative PCR was used to analyze the expression changes of CmGATA12 gene in different tissues of chrysanthemum. The CmGATA12 gene was transfected into A.thaliana,and its effect on flowering time and rosette leaf number in A.thaliana was observed. The expression of flowering related genes in CmGATA12 transgenic A.thaliana was analyzed. [Results]The genetic distance between CmGATA12 and AtGATA13 and AtGATA8 of Arabidopsis GATA family was the closest. Quantitative analysis of CmGATA12 gene in different tissues of chrysanthemum revealed that CmGATA12 gene had the highest expression level in the stem of chrysanthemum and the lowest expression level in bud. Subcellular localization indicated that the CmGATA12 gene was located both on the cell membrane and on the nucleus. The CmGATA12 gene was found to have transcriptional activity in the yeast system. By observing the time taken to flowering and rosette leaf number of CmGATA12 transgenic A.thaliana plants,it was found that the flowering time of CmGATA12 transgenic A.thaliana plants was earlier than that of wild-type plants,and the number of rosette leaves in flowering was lower than that of wild-type plants. Flowering-time related genes involved in flowering promotion(AtSPL5 and AtGI)and flowering inhibition genes(AtSVP,AtFRI,AtTOE1 and AtTOE2)were compared in the transgenic and wild-type plants. The results indicated that flower-promoting genes were up-regulated in the transgenic plants compared to the wild-type plants. Similarly,floral-repression genes were reduced expressed in transgenic plants compared with wild-type plants. [Conclusions]CmGATA12 gene has a role in promoting flowering in the heterologous system.
引文
[1] 杨真,李海涛.观赏菊花分类探讨[J].现代园艺,2016(11):81-82.Yang Z,Li H T.Discussion on chrysanthemum classification[J].Modern Gardening,2016(11):81-82(in Chinese).
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[3] 孔小燕.热激启动子驱动CmFTL3调控菊花花期的初步研究与甘菊再生体系的优化[D].南京:南京农业大学,2015:11-13.Kong X Y.The preliminary study about heat shock promoter driving CmFTL3 to regulate chrysanthemum flowering and the optimize of chamomile regeneration system[D].Nanjing:Nanjing Agricultural University,2015:11-13(in Chinese with English abstract).
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[7] Ranftl Q L,Bastakis E,Klermund C,et al.LLM-domain containing B-GATA factors control different aspects of cytokinin-regulated development in Arabidopsis thaliana[J].Plant Physiology,2016,170(4):2295-2311.
[8] Li T,Mu H,Veluchamy S,et al.The GATA-type IVb zinc-finger transcription factor SsNsd1 regulates asexual-sexual development and appressoria formation in Sclerotinia sclerotiorum[J].Molecular Plant Pathology,2018,19(7):1679-1689.
[9] Lu G,Casaretto J A,Ying S,et al.Overexpression of OsGATA12,regulates chlorophyll content,delays plant senescence and improves rice yield under high density planting[J].Plant Molecular Biology,2017,94(112):215-227.
[10] 袁岐,张春利,赵婷婷,等.植物中GATA转录因子的研究进展[J].分子植物育种,2017,15(5):1702-1707.Yuan Q,Zhang C L,Zhao T T,et al.Research advances of GATA transcription factor in plant[J].Molecular Plant Breeding,2017,15(5):1702-1707(in Chinese with English abstract).
[11] Luo X M,Lin W H,Zhu S,et al.Integration of light- and brassinosteroid-signaling pathways by a GATA transcription factor in Arabidopsis[J].Developmental Cell,2010,19(6):872-883.
[12] Behringer C,Bastakis E,Ranftl Q L,et al.Functional diversification within the family of B-GATA transcription factors through the leucine-leucine-methionine domain[J].Plant Physiology,2014,166(1):293-305.
[13] Cockram J,Thiel T,Steuernagel B,et al.Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae[J].PLoS One,2012,7(9):e45307.
[14] Zhao Y,Medrano L,Ohashi K,et al.HANABA TARANU is a GATA transcription factor that regulates shoot apical meristem and flower development in Arabidopsis[J].The Plant Cell,2004,16(10):2586-2600.
[15] Ding L,Yan S,Jiang L,et al.HANABA TARANU regulates the shoot apical meristem and leaf development in cucumber(Cucumis sativus L.)[J].Journal of Experimental Botany,2015,66(22):7075-7087.
[16] Ohnishi A,Wada H,Kobayashi K,et al.Improved photosynthesis in Arabidopsis,roots by activation of GATA transcription factors[J].Photosynthetica,2018,56(1):433-444.
[17] Hayama R,Coupland G.Shedding light on the circadian clock and the photoperiodic control of flowering[J].Current Opinion in Plant Biology,2003,6(1):13-19.
[18] Strayer C,Kay S A.Cloning of the Arabidopsis clock gene TOC1,an autoregulatory response regulator homolog[J].Science,2000,289(80):768-771.
[19] Mouradov A,Cremer F,Coupland G,et al.Control of flowering time:interacting pathways as a basis for diversity[J].The Plant Cell,2002,14(Suppl):S111-S125.
[20] 姜敏,景雅,张宇航,等.转GmGI基因烟草T3株系的鉴定及其农艺性状的统计分析[J].分子植物育种,2016,14(2):524-528.Jiang M,Jing Y,Zhang Y H,et al.Identification of transgenic tobacco T3 strain GmGI transgenic and statistical analysis of its agronomic traits[J].Molecular Plant Breeding,2016,14(2):524-528(in Chinese with English abstract).
[21] Méndez-Vigo B,Martínez-Zapater J M,Alonso-Blanco C,et al.The flowering repressor SVP underlies a novel Arabidopsis thaliana QTL interacting with the genetic background[J].PLoS Genetics,2013,9(1):e1003289.
[22] Guo Y L,Todesco M,Hagmann J,et al.Independent FLC mutations as causes of flowering-time variation in Arabidopsis thaliana and Capsella rubella[J].Genetics,2012,192(2):729-739.
[23] 张新.茉莉酸调控拟南芥和番茄开花时间的分子机制研究[D].杭州:浙江大学,2016:8-10.Zhang X.Research on the molecular mechanism of jasmonic acid regulating the flowering time of Arabidopsis thaliana and tomato[D].Hangzhou:Zhejiang University,2016:8-10(in Chinese with English abstract).
[24] Rao S S,E1-Habbak M H,Havens W M,et al.Overexpression of GmCaM4 in soybean enhances resistance to pathogens and tolerance to salt stress[J].Molecular Plant Pathology,2014,15(2):145-160.
[25] 王楠,向凤宁,李朔.植物膜结合转录因子与胁迫响应[J].生命科学,2016,28(7):799-806.Wang N,Xiang F N,Li S.Advance in plant membrane-bound transcription factors and plant stress response[J].Chinese Bulletin of Life Sciences,2016,28(7):799-806(in Chinese with English abstract).
[26] 王娟,兰海燕.GATA转录因子对植物发育和胁迫响应调控的研究进展[J].植物生理学报,2016,52(12):1785-1794.Wang J,Lan H Y.Advances in the regulation of GATA transcription factors on plant development and stress response[J].Plant Physiology Journal,2016,52(12):1785-1794(in Chinese with English abstract).
[2] 张飞,陈发棣,房伟民,等.菊花花期性状的杂种优势与混合遗传分析[J].南京农业大学学报,2011,34(4):31-36.DOI:10.7685/j.issn.1000-2030.2011.04.006.Zhang F,Chen F D,Fang W M,et al.Heterosis and mixed genetic analysis for florescence-related traits of chrysanthemum[J].Journal of Nanjing Agricultural University,2011,34(4):31-36(in Chinese with English abstract).
[3] 孔小燕.热激启动子驱动CmFTL3调控菊花花期的初步研究与甘菊再生体系的优化[D].南京:南京农业大学,2015:11-13.Kong X Y.The preliminary study about heat shock promoter driving CmFTL3 to regulate chrysanthemum flowering and the optimize of chamomile regeneration system[D].Nanjing:Nanjing Agricultural University,2015:11-13(in Chinese with English abstract).
[4] Bastakis E,Hedtke B,Klermund C,et al.LLM-domain B-GATA transcription factors play multifaceted roles in controlling greening in Arabidopsis[J].The Plant Cell,2018,30(3):582-599.
[5] Hong F C,Hong X S,Ke L,et al.Genome-wide identification,evolution,and expression analysis of GATA transcription factors in apple(Malus× domestica Borkh.)[J].Gene,2017,627(6):460-472.
[6] Richter R,Behringer C,Zourelidou M,et al.Convergence of auxin and gibberellin signaling on the regulation of the GATA transcription factors GNC and GNL in Arabidopsis thaliana[J].Proc Natl Acad Sci USA,2013,110(32):13192-13197.
[7] Ranftl Q L,Bastakis E,Klermund C,et al.LLM-domain containing B-GATA factors control different aspects of cytokinin-regulated development in Arabidopsis thaliana[J].Plant Physiology,2016,170(4):2295-2311.
[8] Li T,Mu H,Veluchamy S,et al.The GATA-type IVb zinc-finger transcription factor SsNsd1 regulates asexual-sexual development and appressoria formation in Sclerotinia sclerotiorum[J].Molecular Plant Pathology,2018,19(7):1679-1689.
[9] Lu G,Casaretto J A,Ying S,et al.Overexpression of OsGATA12,regulates chlorophyll content,delays plant senescence and improves rice yield under high density planting[J].Plant Molecular Biology,2017,94(112):215-227.
[10] 袁岐,张春利,赵婷婷,等.植物中GATA转录因子的研究进展[J].分子植物育种,2017,15(5):1702-1707.Yuan Q,Zhang C L,Zhao T T,et al.Research advances of GATA transcription factor in plant[J].Molecular Plant Breeding,2017,15(5):1702-1707(in Chinese with English abstract).
[11] Luo X M,Lin W H,Zhu S,et al.Integration of light- and brassinosteroid-signaling pathways by a GATA transcription factor in Arabidopsis[J].Developmental Cell,2010,19(6):872-883.
[12] Behringer C,Bastakis E,Ranftl Q L,et al.Functional diversification within the family of B-GATA transcription factors through the leucine-leucine-methionine domain[J].Plant Physiology,2014,166(1):293-305.
[13] Cockram J,Thiel T,Steuernagel B,et al.Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae[J].PLoS One,2012,7(9):e45307.
[14] Zhao Y,Medrano L,Ohashi K,et al.HANABA TARANU is a GATA transcription factor that regulates shoot apical meristem and flower development in Arabidopsis[J].The Plant Cell,2004,16(10):2586-2600.
[15] Ding L,Yan S,Jiang L,et al.HANABA TARANU regulates the shoot apical meristem and leaf development in cucumber(Cucumis sativus L.)[J].Journal of Experimental Botany,2015,66(22):7075-7087.
[16] Ohnishi A,Wada H,Kobayashi K,et al.Improved photosynthesis in Arabidopsis,roots by activation of GATA transcription factors[J].Photosynthetica,2018,56(1):433-444.
[17] Hayama R,Coupland G.Shedding light on the circadian clock and the photoperiodic control of flowering[J].Current Opinion in Plant Biology,2003,6(1):13-19.
[18] Strayer C,Kay S A.Cloning of the Arabidopsis clock gene TOC1,an autoregulatory response regulator homolog[J].Science,2000,289(80):768-771.
[19] Mouradov A,Cremer F,Coupland G,et al.Control of flowering time:interacting pathways as a basis for diversity[J].The Plant Cell,2002,14(Suppl):S111-S125.
[20] 姜敏,景雅,张宇航,等.转GmGI基因烟草T3株系的鉴定及其农艺性状的统计分析[J].分子植物育种,2016,14(2):524-528.Jiang M,Jing Y,Zhang Y H,et al.Identification of transgenic tobacco T3 strain GmGI transgenic and statistical analysis of its agronomic traits[J].Molecular Plant Breeding,2016,14(2):524-528(in Chinese with English abstract).
[21] Méndez-Vigo B,Martínez-Zapater J M,Alonso-Blanco C,et al.The flowering repressor SVP underlies a novel Arabidopsis thaliana QTL interacting with the genetic background[J].PLoS Genetics,2013,9(1):e1003289.
[22] Guo Y L,Todesco M,Hagmann J,et al.Independent FLC mutations as causes of flowering-time variation in Arabidopsis thaliana and Capsella rubella[J].Genetics,2012,192(2):729-739.
[23] 张新.茉莉酸调控拟南芥和番茄开花时间的分子机制研究[D].杭州:浙江大学,2016:8-10.Zhang X.Research on the molecular mechanism of jasmonic acid regulating the flowering time of Arabidopsis thaliana and tomato[D].Hangzhou:Zhejiang University,2016:8-10(in Chinese with English abstract).
[24] Rao S S,E1-Habbak M H,Havens W M,et al.Overexpression of GmCaM4 in soybean enhances resistance to pathogens and tolerance to salt stress[J].Molecular Plant Pathology,2014,15(2):145-160.
[25] 王楠,向凤宁,李朔.植物膜结合转录因子与胁迫响应[J].生命科学,2016,28(7):799-806.Wang N,Xiang F N,Li S.Advance in plant membrane-bound transcription factors and plant stress response[J].Chinese Bulletin of Life Sciences,2016,28(7):799-806(in Chinese with English abstract).
[26] 王娟,兰海燕.GATA转录因子对植物发育和胁迫响应调控的研究进展[J].植物生理学报,2016,52(12):1785-1794.Wang J,Lan H Y.Advances in the regulation of GATA transcription factors on plant development and stress response[J].Plant Physiology Journal,2016,52(12):1785-1794(in Chinese with English abstract).
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