葡萄VvRGL基因应答GA调控无核果实发育的研究
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摘要
DELLA蛋白是GA信号传导途径的核心作用元件,属于GRAS核转录调节因子家族。该研究以‘魏可’葡萄品种为试材,通过基因克隆、启动子分析、染色体定位、基因蛋白结构及系统进化分析,鉴定VvRGL1和VvRGL2精确序列,预测其潜在功能,并采用qRT-PCR技术检测VvRGL基因应答GA在果皮、果肉及种子(区)的时空表达特征。结果表明:(1)VvRGL1及VvRGL2的染色体定位为Chr14和Chr7,开放阅读框(ORF)为2 007bp和1 815bp,编码氨基酸数量为668和604个;二者均含有GRAS保守结构域,但却不具备DELLA结构域,属于GRAS转录因子大家族成员,而不属于DELLA亚家族;基因结构分析表明,VvRGL1的DNA序列有1个内含子,2个外显子,而VvRGL2的DNA序列无内含子,有1个外显子,基因结构高度保守;进化分析表明,VvRGL1与拟南芥和柑桔的亲缘关系较近,而VvRGL2与草莓和杨树的亲缘关系较近。(2)2个基因的启动子均含有响应赤霉素和胚乳发育相关的作用元件,表明它们可能参与响应GA信号传导和种子胚乳发育过程。(3)qRT-PCR结果显示,外源GA处理均不同程度降低了2个基因在葡萄果皮和种子区的表达,但却上调了其在幼果期果肉中的表达,表明在果皮与种子(区)中,GA可能通过抑制葡萄VvRGL1/RGL2基因的表达参与调控葡萄无核果实的发育。本研究结果为进一步阐明VvRGL在赤霉素信号传导及葡萄无核果实发育机理中的作用机制提供重要依据。
DELLA protein is a core component of GA signaling pathway,and belongs to the GRAS nuclear transcription regulator family.In the present study,we used the grapevine cv.‘Wink'as the test material to identify the precise sequences and predict the potential functions of VvRGL1 and VvRGL2 through gene cloning,promoter analysis,chromosomal location,gene protein structure and phylogenetic analysis.And the qRT-PCR method was used to detect the temporal and spatial expression of VvRGLgenes in the grape berry peel,berry flesh and seed(or seed area)induced by exogenous GA.Result showed that:(1)the chromosomal localization of VvRGL1/RGL2 were Chr14 and Chr7,the open reading frame(ORF)were 2007 bp and 1 815 bp,and the number of amino acid were 668 and 604,respectively.Both of VvRGL1 andVvRGL2 contained a GRAS conserved domain,but did not have a DELLA domain,and were members of the GRAS transcription factor gene family,but not the DELLA subfamily.Gene structure analysis result showed that their gene structures were highly conserved with the DNA sequence of VvRGL1 was only one intron and two exons,while the DNA sequence of VvRGL2 had no intron and only one exon.Phylogenetic analysis showed that VvRGL1 was closely related to Arabidopsis and citrus,while VvRGL2 was closely related to strawberry and poplar.(2)The promoters of two genes both contained elements that were responsible for GA and endosperm development,suggesting that they might be involved in response to GA signaling and endosperm development.(3)The results of qRT-PCR showed that exogenous GA treatment reduced the expression of two genes in grape peel and seed area,but up-regulated their expression in the flesh of young fruit,suggesting that in the peel and seed areas,GA might participate in the regulation of grape seedless fruit development by inhibiting the expression of VvRGL1/RGL2.This study provides an important basis for further explore the function of VvRGLin GA signaling pathway and in seedless fruit development of grapevine.
DELLA protein is a core component of GA signaling pathway,and belongs to the GRAS nuclear transcription regulator family.In the present study,we used the grapevine cv.‘Wink'as the test material to identify the precise sequences and predict the potential functions of VvRGL1 and VvRGL2 through gene cloning,promoter analysis,chromosomal location,gene protein structure and phylogenetic analysis.And the qRT-PCR method was used to detect the temporal and spatial expression of VvRGLgenes in the grape berry peel,berry flesh and seed(or seed area)induced by exogenous GA.Result showed that:(1)the chromosomal localization of VvRGL1/RGL2 were Chr14 and Chr7,the open reading frame(ORF)were 2007 bp and 1 815 bp,and the number of amino acid were 668 and 604,respectively.Both of VvRGL1 andVvRGL2 contained a GRAS conserved domain,but did not have a DELLA domain,and were members of the GRAS transcription factor gene family,but not the DELLA subfamily.Gene structure analysis result showed that their gene structures were highly conserved with the DNA sequence of VvRGL1 was only one intron and two exons,while the DNA sequence of VvRGL2 had no intron and only one exon.Phylogenetic analysis showed that VvRGL1 was closely related to Arabidopsis and citrus,while VvRGL2 was closely related to strawberry and poplar.(2)The promoters of two genes both contained elements that were responsible for GA and endosperm development,suggesting that they might be involved in response to GA signaling and endosperm development.(3)The results of qRT-PCR showed that exogenous GA treatment reduced the expression of two genes in grape peel and seed area,but up-regulated their expression in the flesh of young fruit,suggesting that in the peel and seed areas,GA might participate in the regulation of grape seedless fruit development by inhibiting the expression of VvRGL1/RGL2.This study provides an important basis for further explore the function of VvRGLin GA signaling pathway and in seedless fruit development of grapevine.
引文
[1]WANG M Q,SUN X,WANG C,et al.Characterization of miR061and its target genes in grapevine responding to exogenous gibberellic acid[J].Functional and Integrative Genomics,2017,17(5):537-549.
[2]HAN J,FANG J G,WANG C,et al.Grapevine microRNAs responsive to exogenous gibberellin[J].BMC Genomics,2014,15(1):111.
[3]张梦燕,孙军利,赵宝龙.外源ALA对克瑞森无核葡萄叶片光合特性及果实品质的影响[J].西北植物学报,2018,38(3):493-500.ZHNAG M Y,SUN J L,ZHAO B L.Effect of 5-aminolevulinicacid(ALA)on leaf photosynthesis and fruit quality of Crimson seedless grapevine[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(3):493-500.
[4]张文颖,王晨,汤崴,等.果树果实胚败育研究进展[J].分子植物育种,2018,16(12):4043-4054.ZHANG W Y,WANG C,TANG W,et al.Advances study on embryo abortion of fruit trees[J].Molecular Plant Breeding,2018,16(12):4043-4054.
[5]WANG X C,ZHAO M Z,WU W M,et al.Comparative transcriptome analysis of berry-sizing effects of gibberellin(GA3)on seedless Vitis vinifera L.[J].Genes and Genomics,2017,39(5):493-507.
[6]CHENG C X,XU X Z,SINGER S D,et al.Effect of GA3treatment on seed development and seed-related gene expression in grape[J].PLoS One,2013,8(11):e80044.
[7]KANNO Y,OIKAWA T,CHIBA Y,et al.AtSWEET13and AtSWEET14 regulate gibberellin-mediated physiological processes[J].Nature Communications,2016,7:13245.
[8]王文然,樊秀彩,张文颖,等.果树赤霉素代谢与信号途径研究进展[J].生物技术通报,2017,33(11):1-7.WANG W R,FAN X C,ZHANG W Y,et al.Study progress on gibberellin metabolism and signaling transduction pathway in fruits trees[J].Biotechnology Bulletin,2017,33(11):1-7.
[9]周莲洁,张富春,王艳.GRAS家族基因在植物生长、代谢及逆境胁迫中的功能研究进展[J].植物生理学报,2013,49(9):855-860.ZHOU L J,ZHANG F C,WANG Y.Research progress on the functional mechanism of GRAS family genes in plant growth,metabolism and stress[J].Plant Physiology Journal,2013,49(9):855-860.
[10]ZHAO B,LI H T,LI J J,et al.Brassica napus DS-3,encoding a DELLA protein,negatively regulates stem elongation through gibberellin signaling pathway[J].Theoretical and Applied Genetics,2017,130(4):727-741.
[11]LI W J,ZHANG J X,SUN H Y,et al.FveRGA1,encoding a DELLA protein,negatively regulates runner production in Fragaria vesca[J].Planta,2018,247(4):941-951.
[12]GALLEGO-BARTOLOME J,MINGUET E G,MARIN J A,et al.Transcriptional diversification and functional conservation between DELLA proteins in Arabidopsis[J].Molecular Biology and Evolution,2010,27(6):1 247-1 256.
[13]DAI C,XUE H W.Rice early flowering1,a CKI,phosphorylates DELLA protein SLR1to negatively regulate gibberellin signalling[J].The Embo Journal,2010,29(11):1 916-1 927.
[14]张文颖,王晨,朱旭东,等.葡萄全基因组DELLA蛋白基因家族鉴定及其应答外源赤霉素调控葡萄果实发育的特征[J].中国农业科学,2018,51(16):3 130-3 146.ZHANG W Y,WANG C,ZHU X D,et al.Genome-wide identification and expression of DELLA protein gene family during the development of grape berry induced by exogenous GA[J].Scientia Agricultura Sinica,2018,51(16):3 130-3146.
[15]LEE S,CHENG H,KING K E,et al.Gibberellin regulates Arabidopsis seed germination via RGL2,a GAI/RGA-like gene whose expression is up-regulated following imbibition[J].Genes&Development,2002,16(5):646-658.
[16]WEN C K,CHANG C.Arabidopsis RGL1encodes a negative regulator of gibberellin responses[J].Plant Cell,2002,14(1):87-100.
[17]SUZUKI H,PARK S H,OKUBO K,et al.Differential expression and affinities of Arabidopsis gibberellin receptors can explain variation in phenotypes of multiple knockout mutants[J].The Plant Journal,2009,60(1):48-55.
[18]张彦苹,王晨,于华平,等.适于葡萄不同组织RNA提取方法的筛选[J].西北农业学报,2010,19(11):135-140.ZHANG Y P,WANG C,YU H P,et al.Screening of RNA extraction methods for various grapevine organs and tissues[J].Acta Agriculturae Boreali-Occidentalis Sinica,2010,19(11):135-140.
[19]FAN S,ZHANG D,XING L B,et al.Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus[J].Molecular Genetics and Genomics,2017,292(4):755-771.
[20]LIU B,De STORME N,GEELEN D.Gibberellin induces diploid pollen formation by interfering with meiotic cytokinesis[J].Plant Physiology,2017,173(1):338-353.
[21]SUN X,JONES W T,RIKKERINK E H.GRAS proteins:the versatile roles of intrinsically disordered proteins in plant signalling[J].Biochemical Journal,2012,442(1):1-12.
[22]LEE M H,KIM B,SONG S K,et al.Large-scale analysis of the GRAS gene family in Arabidopsis thaliana[J].Plant Molecular Biology,2008,67(6):659-670.
[23]VAN De V K,RUELENS P,GEUTEN K,et al.Exploiting DELLA signaling in cereals[J].Trends in Plant Science,2017,22(10):880-893.
[24]CAI H,CHEN Y,ZHANG M,et al.A novel GRAS transcription factor,ZmGRAS20,regulates starch biosynthesis in rice endosperm[J].Physiology and Molecular Biology of Plants,2017,23(1):1-12.
[25]GRIMPLET J,AGUDELO-ROMERO P,TEIXEIRA R T,et al.Structural and functional analysis of the GRAS gene family in grapevine indicates a role of GRAS proteins in the control of development and stress responses[J].Frontiers in Plant Science,2016,7(e39547):353.
[26]TYLER L,THOMAS S G,HU J H,et al.DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis[J].Plant Physiology,2004,135(2):1 008-1 019.
[27]WANG Y J,DENG D X.Molecular basis and evolutionary pattern of GA-GID1-DELLA regulatory module[J].Molecular Genetics and Genomics,2014,289(1):1-9.
[28]杨光,曹雪,房经贵,等.‘藤稔’葡萄VvGAI基因的克隆、亚细胞定位及时空表达分析[J].园艺学报,2011,38(10):1 883-1 892.YANG G,CAO X,FANG J G,et al.Cloning,subcellular localization and spatiotemporal expression of a VvGAI gene from grapevine‘Fujiminori’[J].Acta Horticulturae Sinica,2011,38(10):1 883-1 892.
[2]HAN J,FANG J G,WANG C,et al.Grapevine microRNAs responsive to exogenous gibberellin[J].BMC Genomics,2014,15(1):111.
[3]张梦燕,孙军利,赵宝龙.外源ALA对克瑞森无核葡萄叶片光合特性及果实品质的影响[J].西北植物学报,2018,38(3):493-500.ZHNAG M Y,SUN J L,ZHAO B L.Effect of 5-aminolevulinicacid(ALA)on leaf photosynthesis and fruit quality of Crimson seedless grapevine[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(3):493-500.
[4]张文颖,王晨,汤崴,等.果树果实胚败育研究进展[J].分子植物育种,2018,16(12):4043-4054.ZHANG W Y,WANG C,TANG W,et al.Advances study on embryo abortion of fruit trees[J].Molecular Plant Breeding,2018,16(12):4043-4054.
[5]WANG X C,ZHAO M Z,WU W M,et al.Comparative transcriptome analysis of berry-sizing effects of gibberellin(GA3)on seedless Vitis vinifera L.[J].Genes and Genomics,2017,39(5):493-507.
[6]CHENG C X,XU X Z,SINGER S D,et al.Effect of GA3treatment on seed development and seed-related gene expression in grape[J].PLoS One,2013,8(11):e80044.
[7]KANNO Y,OIKAWA T,CHIBA Y,et al.AtSWEET13and AtSWEET14 regulate gibberellin-mediated physiological processes[J].Nature Communications,2016,7:13245.
[8]王文然,樊秀彩,张文颖,等.果树赤霉素代谢与信号途径研究进展[J].生物技术通报,2017,33(11):1-7.WANG W R,FAN X C,ZHANG W Y,et al.Study progress on gibberellin metabolism and signaling transduction pathway in fruits trees[J].Biotechnology Bulletin,2017,33(11):1-7.
[9]周莲洁,张富春,王艳.GRAS家族基因在植物生长、代谢及逆境胁迫中的功能研究进展[J].植物生理学报,2013,49(9):855-860.ZHOU L J,ZHANG F C,WANG Y.Research progress on the functional mechanism of GRAS family genes in plant growth,metabolism and stress[J].Plant Physiology Journal,2013,49(9):855-860.
[10]ZHAO B,LI H T,LI J J,et al.Brassica napus DS-3,encoding a DELLA protein,negatively regulates stem elongation through gibberellin signaling pathway[J].Theoretical and Applied Genetics,2017,130(4):727-741.
[11]LI W J,ZHANG J X,SUN H Y,et al.FveRGA1,encoding a DELLA protein,negatively regulates runner production in Fragaria vesca[J].Planta,2018,247(4):941-951.
[12]GALLEGO-BARTOLOME J,MINGUET E G,MARIN J A,et al.Transcriptional diversification and functional conservation between DELLA proteins in Arabidopsis[J].Molecular Biology and Evolution,2010,27(6):1 247-1 256.
[13]DAI C,XUE H W.Rice early flowering1,a CKI,phosphorylates DELLA protein SLR1to negatively regulate gibberellin signalling[J].The Embo Journal,2010,29(11):1 916-1 927.
[14]张文颖,王晨,朱旭东,等.葡萄全基因组DELLA蛋白基因家族鉴定及其应答外源赤霉素调控葡萄果实发育的特征[J].中国农业科学,2018,51(16):3 130-3 146.ZHANG W Y,WANG C,ZHU X D,et al.Genome-wide identification and expression of DELLA protein gene family during the development of grape berry induced by exogenous GA[J].Scientia Agricultura Sinica,2018,51(16):3 130-3146.
[15]LEE S,CHENG H,KING K E,et al.Gibberellin regulates Arabidopsis seed germination via RGL2,a GAI/RGA-like gene whose expression is up-regulated following imbibition[J].Genes&Development,2002,16(5):646-658.
[16]WEN C K,CHANG C.Arabidopsis RGL1encodes a negative regulator of gibberellin responses[J].Plant Cell,2002,14(1):87-100.
[17]SUZUKI H,PARK S H,OKUBO K,et al.Differential expression and affinities of Arabidopsis gibberellin receptors can explain variation in phenotypes of multiple knockout mutants[J].The Plant Journal,2009,60(1):48-55.
[18]张彦苹,王晨,于华平,等.适于葡萄不同组织RNA提取方法的筛选[J].西北农业学报,2010,19(11):135-140.ZHANG Y P,WANG C,YU H P,et al.Screening of RNA extraction methods for various grapevine organs and tissues[J].Acta Agriculturae Boreali-Occidentalis Sinica,2010,19(11):135-140.
[19]FAN S,ZHANG D,XING L B,et al.Phylogenetic analysis of IDD gene family and characterization of its expression in response to flower induction in Malus[J].Molecular Genetics and Genomics,2017,292(4):755-771.
[20]LIU B,De STORME N,GEELEN D.Gibberellin induces diploid pollen formation by interfering with meiotic cytokinesis[J].Plant Physiology,2017,173(1):338-353.
[21]SUN X,JONES W T,RIKKERINK E H.GRAS proteins:the versatile roles of intrinsically disordered proteins in plant signalling[J].Biochemical Journal,2012,442(1):1-12.
[22]LEE M H,KIM B,SONG S K,et al.Large-scale analysis of the GRAS gene family in Arabidopsis thaliana[J].Plant Molecular Biology,2008,67(6):659-670.
[23]VAN De V K,RUELENS P,GEUTEN K,et al.Exploiting DELLA signaling in cereals[J].Trends in Plant Science,2017,22(10):880-893.
[24]CAI H,CHEN Y,ZHANG M,et al.A novel GRAS transcription factor,ZmGRAS20,regulates starch biosynthesis in rice endosperm[J].Physiology and Molecular Biology of Plants,2017,23(1):1-12.
[25]GRIMPLET J,AGUDELO-ROMERO P,TEIXEIRA R T,et al.Structural and functional analysis of the GRAS gene family in grapevine indicates a role of GRAS proteins in the control of development and stress responses[J].Frontiers in Plant Science,2016,7(e39547):353.
[26]TYLER L,THOMAS S G,HU J H,et al.DELLA proteins and gibberellin-regulated seed germination and floral development in Arabidopsis[J].Plant Physiology,2004,135(2):1 008-1 019.
[27]WANG Y J,DENG D X.Molecular basis and evolutionary pattern of GA-GID1-DELLA regulatory module[J].Molecular Genetics and Genomics,2014,289(1):1-9.
[28]杨光,曹雪,房经贵,等.‘藤稔’葡萄VvGAI基因的克隆、亚细胞定位及时空表达分析[J].园艺学报,2011,38(10):1 883-1 892.YANG G,CAO X,FANG J G,et al.Cloning,subcellular localization and spatiotemporal expression of a VvGAI gene from grapevine‘Fujiminori’[J].Acta Horticulturae Sinica,2011,38(10):1 883-1 892.