龙眼ELF4同源基因的克隆与功能研究
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摘要
以‘红核子’龙眼(Dimocarpus longan)叶芽为材料,克隆了ELF4同源基因DlELF4-1和DlELF4-2的cDNA全长序列,对序列进行了生物信息学分析,并通过转化拟南芥以及实时荧光定量PCR研究基因功能。蛋白序列比对结果表明,DlELF4-2与AtELF4-L1有较高相似度,而DlELF4-1与AtELF4相似度更高;构建两个基因的植物表达载体转化拟南芥,转基因植株都表现出延迟开花以及不定根生长的现象,表明DlELF4-1和DlELF4-2可能有抑制开花和促进生长素合成功能;DlELF4-1和DlELF4-2在龙眼花芽分化过程的表达模式不同,推测其在龙眼花芽分化过程中有不同的功能。
The full length of c DNA sequences of two ELF4 homolog genes were cloned from Dimocarpus longan‘Red Seed',named DlELF4-1 and DlELF4-2. The functions of these two genes were analyzed by bioinformatics analysis and real-time quantitative PCR. To further study the function of the DlELF4 genes,DlELF4-1 and DlELF4-2 were overexpressed in Arabidopsis thaliana. Sequence analysis and cluster analysis showed that DlELF4-2 was similar to AtELF4-L1,while DlELF4-1 and AtELF4 showed higher similarity. Overexpression of two DlELF4 genes separately in Arabidopsis thaliana resulted in delaying flowering and adventitious roots phenotypes,suggesting that DlELF4 genes may be repressors of flowering and promoting auxin synthesis. However,expression patterns of DlELF4-1 and DlELF4-2 genes were different during bud differentiation of longan,indicating that the two genes may have different functions in longan.
The full length of c DNA sequences of two ELF4 homolog genes were cloned from Dimocarpus longan‘Red Seed',named DlELF4-1 and DlELF4-2. The functions of these two genes were analyzed by bioinformatics analysis and real-time quantitative PCR. To further study the function of the DlELF4 genes,DlELF4-1 and DlELF4-2 were overexpressed in Arabidopsis thaliana. Sequence analysis and cluster analysis showed that DlELF4-2 was similar to AtELF4-L1,while DlELF4-1 and AtELF4 showed higher similarity. Overexpression of two DlELF4 genes separately in Arabidopsis thaliana resulted in delaying flowering and adventitious roots phenotypes,suggesting that DlELF4 genes may be repressors of flowering and promoting auxin synthesis. However,expression patterns of DlELF4-1 and DlELF4-2 genes were different during bud differentiation of longan,indicating that the two genes may have different functions in longan.
引文
Bergonzi S,Albani M C,Ver Loren van Themaat E,Nordstr?m K J,Wang R,Schneeberger K,Moerland P D,Coupland G.2013.Mechanisms of age-dependent response to winter temperature in pe-rennial flowering of Arabis alpine.Science,340:1094-1097.
Boden S A,Weiss D,Ross J J,Davies N W,Trevaskis B,Chandler P M,Swain S M.2014.EARLY FLOWERING 3 regulates flowering in spring barley by mediating gibberellin production and FLOWERING LOCUS T expression.Plant Cell,26(4):1557-1569.
Boerjan W,Cervera M T,Delarue M,Beeckman T,Dewitte W,Bellini C,Caboche M,Onckelen H,Montagu,InzéD.1995.Superroot,a recessive mutation in Arabidopsis,confers auxin overproduction.Plant Cell,7(9):1405-1419.
Celenza J L,Grisafi P L,Fink G R.1995.A pathway for lateral root formation in Arabidopsis thaliana.Genes and Development,9:2131-2142.
Chang Qiang.2006.Study on relationship between off-season floral induction in longan and nutrition of the carbon and nitrogen[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)常强.2006.龙眼反季节成花诱导与碳氮营养关系的研究[硕士论文].福州:福建农林大学.
Chen W W,Qin Q P,Zheng Y P,Wang C,Wang S,Zhou M B,Zhang C,Cui Y Y.2016.Overexpression of Doritaenopsis hybrid EARLYFLOWERING 4-like4 gene,Dh EFL4,postpones flowering in transgenic Arabidopsis.Plant Molecular Biology Reporter,34(1):103-117.
Clough S J,Bent A F.1998.Floral dip:a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana.The Plant Journal,16(6):735-743.
Covington M F,Maloof J N,Straume M,Kay S A,Harmer S L.2008.Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development.Genome Biology,9(8):130.
Doyle M R,Davis S J,Bastow R M,Mc Watters H G,Kozma-Bognár L,Nagy F,Millar A J,Amasino R M.2002.The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana.Nature,419(6902):74-77.
Guan Lei.2008.Characterization and functional analysis of a LEAFY homologous gene[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)官磊.2008.龙眼LEAFY基因克隆与功能研究[硕士论文].福州:福建农林大学.
Huang F N,Fu Z Y,Zeng L H,Michael M B.2017.Isolation and characterization of GI and FKF1 homologous genes in the subtropical fruit tree Dimocarpus longan.Molecular Breeding,37(7):90.
Huang Qiang-wei.1996.The relationship between changes of endogenous hormones and flower bud differentiation with biennial fruiting in longan.Journal of Tropical and Subtropical Botany,2(2):58-62.(in Chinese)黄羌维.1996.龙眼内源激素变化和花芽分化及大小年结果的关系.热带亚热带植物学报,2(2):58-62.
Jia T Q,Wei D F,Meng S,Zeng L H.2014.Identification of regulatory genes implicated in continuous flowering of longan(Dimocarpus longan L.).PLo S ONE,9(12):e114568.
Khanna R,Kikis E A,Quail P H.2003.EARLY FLOWERING 4 functions in phytochrome B-regulated seedling de-etiolation.Plant Physiology,133(4):1530-1538.
Kikis E A,Khanna R,Quail P H.2005.ELF4 is a phytochrome regulated component of a negative feedback loop involving the central oscillator components CCA1 and LHY.The Plant Journal,44(2):300-313.
Kim Y,Lim J,Yeom M,Kim H,Kim J,Wang L,Kim W Y,Somers D E,Nam H G.2013.ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration.Cell Rep,3(3):671-677.
Komeda Y.2004.Genetic regulation of time to flower in Arabidopsis thaliana.Annu Rev Plant Biol,55:521-535.
Li J,Chen X,Xu J,Lan H,Li C,Hu Y,Lu Y.2015.Evolution of DUF1313 family members across plant species and their association with maize photoperiod sensitivity.Genomics,107:199-207.
Marcolino-Gomes J,Nakayama T J,Molinari H B C,Basso M F,Henning L M M,Fuganti-Pagliarini R,Harmon F G,Nepomuceno A L.2017.Functional characterization of a putative Glycine max ELF4 in transgenic Arabidopsis and its role during flowering control.Frontiers in Plant Science,8(618):1-14.
Meng Shan.2013.Promoter cloning of LFY gene and transcriptome sequencing and analysis of grafting new shoots in‘Sijimi’longan[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)孟珊.2013.四季蜜龙眼LFY基因启动子克隆及嫁接新梢转录组测序与分析[硕士论文].福州:福建农林大学.
Mishra P,Panigrahi K C.2015.GIGANTEA-an emerging story.Front Plant Sci,6(8):1-15.
Nusinow D,Helfer A,Hamilton E E,King J J,Imaizumi T,Schultz T F,Farre E M,Kay S A.2011.The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth.Nature,475(7356):398-402.
Peng Ling-tao.2006.Molecular mechanism of flowering time controlling photoperiod pathway in Arabidopsis and rice.Plant Physiology Communication,42(6):1021-1031.(in Chinese)彭凌涛.2006.控制拟南芥和水稻开花时间光周期途径的分子机制.植物生理学通讯,42(6):1021-1031.
Qiu Hong-ye.2016.Preliminary study on the flowering mechanism of longan cv.Sijimi flowering in summer regulated with growth regulators[M.D.Dissertation].Nanning:Guangxi University.(in Chinese)邱宏业.2016.生长调节剂调控四季蜜龙眼夏季成花机理的初步研究[硕士论文].南宁:广西大学.
Sawa M,Kay S A.2011.GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana.Proceedings of the National Academy of Sciences,108(28):11698-11703.
Shim J S,Kubota A,Imaizumi T.2017.Circadian clock and photoperiodic flowering in Arabidopsis:CONSTANS is a hub for signal integration.Plant Physiol,173(1):5-15.
Sukumar P,Maloney G S,Muday G K.2013.Localized induction of the ATP-Binding cassette B19 auxin transporter enhances adventitious root formation in Arabidopsis.Physiologia Plantarum,162:1392-1405.
Wang Chang-chun,Ke Guan-wu.1992.Research on flower buds morphological differentiation.Fujian Journal of Agricultural Sciences,7(1):55-58.(in Chinese)王长春,柯冠武.1992.龙眼花芽形态分化的研究.福建省农科院学报,7(1):55-58.
Winterhagen P,Tiyayon P,Samach A,Hegele M,Wünsche J N.2013.Isolation and characterization of FLOWERING LOCUS T subforms and APETALA1 of the subtropical fruit tree Dimocarpus longan.Plant Physiology and Biochemistry,71:184-5190.
Zhou Qin,Zhang Si-si,Bao Man-zhu,Liu Guo-feng.2017.Advances on molecular mechanism of floral initiation in higher plants.Molecular Plant Breeding,http://kns.cnki.net/kcms/detail/46.1068.S.20171107.1719.004.html.(in Chinese)周琴,张思思,包满珠,刘国锋.2017.高等植物成花诱导的分子机理研究进展.分子植物育种,http://kns.cnki.net/kcms/detail/46.1068.S.20171107.1719.004.html
Boden S A,Weiss D,Ross J J,Davies N W,Trevaskis B,Chandler P M,Swain S M.2014.EARLY FLOWERING 3 regulates flowering in spring barley by mediating gibberellin production and FLOWERING LOCUS T expression.Plant Cell,26(4):1557-1569.
Boerjan W,Cervera M T,Delarue M,Beeckman T,Dewitte W,Bellini C,Caboche M,Onckelen H,Montagu,InzéD.1995.Superroot,a recessive mutation in Arabidopsis,confers auxin overproduction.Plant Cell,7(9):1405-1419.
Celenza J L,Grisafi P L,Fink G R.1995.A pathway for lateral root formation in Arabidopsis thaliana.Genes and Development,9:2131-2142.
Chang Qiang.2006.Study on relationship between off-season floral induction in longan and nutrition of the carbon and nitrogen[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)常强.2006.龙眼反季节成花诱导与碳氮营养关系的研究[硕士论文].福州:福建农林大学.
Chen W W,Qin Q P,Zheng Y P,Wang C,Wang S,Zhou M B,Zhang C,Cui Y Y.2016.Overexpression of Doritaenopsis hybrid EARLYFLOWERING 4-like4 gene,Dh EFL4,postpones flowering in transgenic Arabidopsis.Plant Molecular Biology Reporter,34(1):103-117.
Clough S J,Bent A F.1998.Floral dip:a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana.The Plant Journal,16(6):735-743.
Covington M F,Maloof J N,Straume M,Kay S A,Harmer S L.2008.Global transcriptome analysis reveals circadian regulation of key pathways in plant growth and development.Genome Biology,9(8):130.
Doyle M R,Davis S J,Bastow R M,Mc Watters H G,Kozma-Bognár L,Nagy F,Millar A J,Amasino R M.2002.The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana.Nature,419(6902):74-77.
Guan Lei.2008.Characterization and functional analysis of a LEAFY homologous gene[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)官磊.2008.龙眼LEAFY基因克隆与功能研究[硕士论文].福州:福建农林大学.
Huang F N,Fu Z Y,Zeng L H,Michael M B.2017.Isolation and characterization of GI and FKF1 homologous genes in the subtropical fruit tree Dimocarpus longan.Molecular Breeding,37(7):90.
Huang Qiang-wei.1996.The relationship between changes of endogenous hormones and flower bud differentiation with biennial fruiting in longan.Journal of Tropical and Subtropical Botany,2(2):58-62.(in Chinese)黄羌维.1996.龙眼内源激素变化和花芽分化及大小年结果的关系.热带亚热带植物学报,2(2):58-62.
Jia T Q,Wei D F,Meng S,Zeng L H.2014.Identification of regulatory genes implicated in continuous flowering of longan(Dimocarpus longan L.).PLo S ONE,9(12):e114568.
Khanna R,Kikis E A,Quail P H.2003.EARLY FLOWERING 4 functions in phytochrome B-regulated seedling de-etiolation.Plant Physiology,133(4):1530-1538.
Kikis E A,Khanna R,Quail P H.2005.ELF4 is a phytochrome regulated component of a negative feedback loop involving the central oscillator components CCA1 and LHY.The Plant Journal,44(2):300-313.
Kim Y,Lim J,Yeom M,Kim H,Kim J,Wang L,Kim W Y,Somers D E,Nam H G.2013.ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration.Cell Rep,3(3):671-677.
Komeda Y.2004.Genetic regulation of time to flower in Arabidopsis thaliana.Annu Rev Plant Biol,55:521-535.
Li J,Chen X,Xu J,Lan H,Li C,Hu Y,Lu Y.2015.Evolution of DUF1313 family members across plant species and their association with maize photoperiod sensitivity.Genomics,107:199-207.
Marcolino-Gomes J,Nakayama T J,Molinari H B C,Basso M F,Henning L M M,Fuganti-Pagliarini R,Harmon F G,Nepomuceno A L.2017.Functional characterization of a putative Glycine max ELF4 in transgenic Arabidopsis and its role during flowering control.Frontiers in Plant Science,8(618):1-14.
Meng Shan.2013.Promoter cloning of LFY gene and transcriptome sequencing and analysis of grafting new shoots in‘Sijimi’longan[M.D.Dissertation].Fuzhou:Fujian Agriculture and Forestry University.(in Chinese)孟珊.2013.四季蜜龙眼LFY基因启动子克隆及嫁接新梢转录组测序与分析[硕士论文].福州:福建农林大学.
Mishra P,Panigrahi K C.2015.GIGANTEA-an emerging story.Front Plant Sci,6(8):1-15.
Nusinow D,Helfer A,Hamilton E E,King J J,Imaizumi T,Schultz T F,Farre E M,Kay S A.2011.The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth.Nature,475(7356):398-402.
Peng Ling-tao.2006.Molecular mechanism of flowering time controlling photoperiod pathway in Arabidopsis and rice.Plant Physiology Communication,42(6):1021-1031.(in Chinese)彭凌涛.2006.控制拟南芥和水稻开花时间光周期途径的分子机制.植物生理学通讯,42(6):1021-1031.
Qiu Hong-ye.2016.Preliminary study on the flowering mechanism of longan cv.Sijimi flowering in summer regulated with growth regulators[M.D.Dissertation].Nanning:Guangxi University.(in Chinese)邱宏业.2016.生长调节剂调控四季蜜龙眼夏季成花机理的初步研究[硕士论文].南宁:广西大学.
Sawa M,Kay S A.2011.GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana.Proceedings of the National Academy of Sciences,108(28):11698-11703.
Shim J S,Kubota A,Imaizumi T.2017.Circadian clock and photoperiodic flowering in Arabidopsis:CONSTANS is a hub for signal integration.Plant Physiol,173(1):5-15.
Sukumar P,Maloney G S,Muday G K.2013.Localized induction of the ATP-Binding cassette B19 auxin transporter enhances adventitious root formation in Arabidopsis.Physiologia Plantarum,162:1392-1405.
Wang Chang-chun,Ke Guan-wu.1992.Research on flower buds morphological differentiation.Fujian Journal of Agricultural Sciences,7(1):55-58.(in Chinese)王长春,柯冠武.1992.龙眼花芽形态分化的研究.福建省农科院学报,7(1):55-58.
Winterhagen P,Tiyayon P,Samach A,Hegele M,Wünsche J N.2013.Isolation and characterization of FLOWERING LOCUS T subforms and APETALA1 of the subtropical fruit tree Dimocarpus longan.Plant Physiology and Biochemistry,71:184-5190.
Zhou Qin,Zhang Si-si,Bao Man-zhu,Liu Guo-feng.2017.Advances on molecular mechanism of floral initiation in higher plants.Molecular Plant Breeding,http://kns.cnki.net/kcms/detail/46.1068.S.20171107.1719.004.html.(in Chinese)周琴,张思思,包满珠,刘国锋.2017.高等植物成花诱导的分子机理研究进展.分子植物育种,http://kns.cnki.net/kcms/detail/46.1068.S.20171107.1719.004.html