葡萄VvAGL11和VvAGL15基因的鉴定及其在赤霉素诱导葡萄无核果实发育过程中的作用
|
摘要
[目的]本文旨在研究VvAGL11和VvAGL15在应答赤霉素(GA3)信号诱导葡萄无核果实发育过程中的作用。[方法]以‘巨峰’葡萄为试材,鉴定了VvAGL11和VvAGL15的c DNA全长序列,并通过分析基因的启动子顺式作用元件预测其潜在功能,同时运用RT-qPCR方法检测对照和GA3处理组葡萄VvAGL11和VvAGL15的时空表达水平。[结果]GA3处理能够诱导葡萄果实无核化,使葡萄果粒和果穗显著伸长,穗轴增粗。同时,鉴定到VvAGL11(MG581423)和VvAGL15(MG581424)的c DNA全长序列。VvAGL11和VvAGL15蛋白序列与可可和棉花的亲缘关系较近;二者均含有MADS-MEF2-like和K-box家族保守结构域,属于MADS-box转录因子家族;其主要二级结构元件为α-螺旋和无规则卷曲,且主要定位于细胞核中,蛋白结构具有保守性。启动子顺式作用元件分析显示:二者均含有响应赤霉素和胚乳发育相关的motifs。RT-qPCR分析显示,在果实硬核期和转色期的种子中,VvAGL11和VvAGL15的表达量较高,而GA3处理能够显著抑制其在种子中的表达。[结论]鉴定到MADS-box基因家族中的2个成员,分别为VvAGL11和VvAGL15。GA3处理可抑制VvAGL11和VvAGL15在种子区的表达,影响种胚的正常发育,从而形成无核果实。
[Objectives]The paper aimed to study the role of VvAGL11 and VvAGL15 in development process of seedless grape berry induced by gibberellin. [Methods]Full-length of c DNA of VvAGL11 and VvAGL15 was obtained from‘Kyoho'. Potential function of VvAGL11 and VvAGL15 was predicted by analyzing its promoter motif elements. Spatio-temporal expression level of VvAGL11 and VvAGL15 in grape berry from control and GA3 treatment groups was detected by RT-qPCR. [Results]Seedless grape berries were induced by GA3 treatment which can significantly elongate grape berry and clusters and thickening spike stalk. At the same time,c DNA full-length of VvAGL11( MG581423) and VvAGL15( MG581424) was identified. Both VvAGL11 and VvAGL15 contained conserved domains of MADS-MEF2-like and K-box,belonging to the MADS-box transcription factor family. The main secondary structural elements were α-helix and random coil,and these two genes were mainly located in the nucleus. The protein structure was conservative. Promoter cis-acting element analysis showed that both of them contained the gibberellin-responsive elements and the elements required for endosperm expression. RT-qPCR results showed that VvAGL11 and VvAGL15 expressed highly in grape stone hardening stage and veraison stage. GA3 treatment could significantly down-regulate the expression of them only in the seed region.[Conclusions]VvAGL11 and VvAGL15 were identified as members of the MADS-box gene family. GA3 can inhibit the development of seed by down-regulating the expression of VvAGL11 and VvAGL15.
[Objectives]The paper aimed to study the role of VvAGL11 and VvAGL15 in development process of seedless grape berry induced by gibberellin. [Methods]Full-length of c DNA of VvAGL11 and VvAGL15 was obtained from‘Kyoho'. Potential function of VvAGL11 and VvAGL15 was predicted by analyzing its promoter motif elements. Spatio-temporal expression level of VvAGL11 and VvAGL15 in grape berry from control and GA3 treatment groups was detected by RT-qPCR. [Results]Seedless grape berries were induced by GA3 treatment which can significantly elongate grape berry and clusters and thickening spike stalk. At the same time,c DNA full-length of VvAGL11( MG581423) and VvAGL15( MG581424) was identified. Both VvAGL11 and VvAGL15 contained conserved domains of MADS-MEF2-like and K-box,belonging to the MADS-box transcription factor family. The main secondary structural elements were α-helix and random coil,and these two genes were mainly located in the nucleus. The protein structure was conservative. Promoter cis-acting element analysis showed that both of them contained the gibberellin-responsive elements and the elements required for endosperm expression. RT-qPCR results showed that VvAGL11 and VvAGL15 expressed highly in grape stone hardening stage and veraison stage. GA3 treatment could significantly down-regulate the expression of them only in the seed region.[Conclusions]VvAGL11 and VvAGL15 were identified as members of the MADS-box gene family. GA3 can inhibit the development of seed by down-regulating the expression of VvAGL11 and VvAGL15.
引文
[1]陈俊伟,谢鸣,吴江.遗传工程无籽果实原理及其研究进展[J].浙江农业学报,2003,15(6):365-371.Chen J W,Xie M,Wu J. The principle of breeding of seedless fruit by genetic engineering and its study advance[J]. Acta Agriculturae Zhejiangensis,2003,15(6):365-371(in Chinese with English abstract).
[2] Yao J L,Dong Y H,Bret A M. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor[J]. Proc Natl Acad Sci USA,2001,98(3):1306-1311.
[3] Singh D P,Jermakow A W,Swain S M. Gibberellins are required for seed development and pollen tube growth in Arabidopsis[J]. Plant Cell,2002,14(12):3133-3147.
[4]崔梦杰,王晨,张文颖,等.无核葡萄研究进展[J].植物生理学报,2017,53(3):317-330.Cui M J,Wang C,Zhang W Y,et al. Progress in the study of seedless grapes[J]. Plant Physiology Journal,2017,53(3):317-330(in Chinese with English abstract).
[5]孙其宝,俞飞飞,孙俊.葡萄无核化研究进展[J].安徽农业科学,2004,32(2):360-362.Sun Q B,Yu F F,Sun J. Progress in the study of grapevine berry seedless[J]. Journal of Anhui Agricultural Sciences,2004,32(2):360-362(in Chinese with English abstract).
[6]肖祥希,李明,邱栋梁.果实无核机理研究进展[J].经济林研究,2009,27(2):104-110.Xiao X X,Li M,Qiu D L. Research progress of seedless mechanism of fruits[J]. Nonwood Forest Research,2009,27(2):104-110(in Chinese with English abstract).
[7] Alvarezbuylla E R,Liljegren S J,Pelaz S,et al. MADS-box gene evolution beyond flowers:expression in pollen,endosperm,guard cells,roots and trichomes[J]. Plant Journal for Cell&Molecular Biology,2010,24(4):457-466.
[8] Battaglia R,Brambilla V,Colombo L,et al. Functional analysis of MADS-box genes controlling ovule development in Arabidopsis using the ethanolinducible alc gene-expression system[J]. Mechanisms of Development,2006,123(4):267-276.
[9] Becker A,Theissen G. The major clades of MADS-box genes and their role in the development and evolution of flowering plants[J]. Molecular Phylogenetics&Evolution,2003,29(3):464-489.
[10] Huang B,Routaboul J M,Liu M,et al. Overexpression of the class D MADS-box gene Sl-AGL11 impacts fleshy tissue differentiation and structure in tomato fruits[J]. Journal of Experimental Botany,2017,68(17):4869-4884.
[11] Ocarez N,Mejía N. Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[J]. Plant Cell Reports,2016,35(1):239.
[12] Wang H,Caruso L V,Downie A B,et al. The Embryo MADS domain protein AGAMOUS-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism[J]. Plant Cell,2004,16(5):1206-1219.
[13] Zheng Y,Perry S E. Global identification of targets of the Arabidopsis MADS domain protein AGAMOUS-Like 15[J]. The Plant Cell,2009,21(9):2563.
[14]王文举,马治国.赤霉素和链霉素及促生灵促进巨峰葡萄果粒增大和无核的试验[J].落叶果树,2003,35(4):43-44.Wang W J,Ma Z G. Test of gibberellin,streptomycin and tomatotone on promoting Kyoho grape berry enlargement and seedless[J]. Deciduous Fruits,2003,35(4):43-44(in Chinese with English abstract).
[15]刘佳,刘晓,陈建.四川地区赤霉素和链霉素处理对巨峰葡萄无核化及果实发育的影响[J].西南农业学报,2011,24(1):220-224.Liu J,Liu X,Chen J. Effect of GA3and SM treatment on inducing seedless and berry growth of Kyoho grape in Sichuan area[J]. Southwest China Journal of Agricultural Sciences,2011,24(1):220-224(in Chinese with English abstract).
[16]陈发河,蔡慧农,冯作山,等.葡萄浆果发育过程中激素水平的变化[J].植物生理与分子生物学学报,2002,28(5):391-395.Chen F H,Cai H N,Feng Z S,et al. Changes of hormone levels during the development of grape berries[J]. Journal of Plant Physiology and Molecular Biology,2002,28(5):391-395(in Chinese with English abstract).
[17]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 Borealioccidentalis Sinica,2010,19(11):135-140(in Chinese with English abstract).
[18] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[19] Riechmann J L,Meyerowitz E M. Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1,AP3,PI,and AG is independent of their DNA-binding specificity[J]. Molecular Biology of the Cell,1997,8(7):1243-1259.
[20] Folter S,Shchennikova A V,Franken J,et al. A BsisterMADS-box gene involved in ovule and seed development in petunia and Arabidopsis[J].The Plant Journal,2006,47(6):934-946.
[21] Deng W,Chen G,Peng F,et al. Transparent testa16 plays multiple roles in plant development and is involved in lipid synthesis and embryo development in canola[J]. Plant Physiology,2012,160(2):978-989.
[22] Xu W,Fiume E,Coen O,et al. Endosperm and nucellus develop antagonistically in Arabidopsis seeds[J]. The Plant Cell,2016,28(6):1343-1360.
[23] Yang Z,Li C,Wang Y,et al. GhAGL15s,preferentially expressed during somatic embryogenesis,promote embryogenic callus formation in cotton(Gossypium hirsutum L.)[J]. Molecular Genetics&Genomics,2014,289(5):873-883.
[24]董庆龙,冀志蕊,迟福梅,等.苹果MADS-box转录因子的生物信息学及其在不同组织中的表达[J].中国农业科学,2014,47(6):1151-1161.Dong Q L,Ji Z R,Chi F M,et al. Bioinformatics of the MADS-Box transcription factor and their expression in different apple tissues[J]. Scientia Agricultura Sinica,2014,47(6):1151-1161(in Chinese with English abstract).
[25]蔡英卿.龙眼体胚发生过程中SERK等胚性相关基因的克隆与表达分析[D].福州:福建农林大学,2011.Cai Y Q. Cloning and epression of SERK and sme other somatic embryogenesis-related genes during somatic embryogenesis in Dimocarpus longan Lour[D]. Fuzhou:Fujian Agriculture and Forestry University,2011(in Chinese with English abstract).
[26] ParenicovaL,De S F,Kieffer M,et al. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis:new openings to the MADS world[J]. The Plant Cell,2003,15(7):1538-1551.
[27]王飞.葡萄无核品种及其杂种胚败育机理与胚挽救技术研究[D].杨凌:西北农林科技大学,2002.Wang F. Mechanism of embryo abortion in stonespermic seedless grape and embryo rescue with seedless grape[D]. Yangling:Northwest A&F University,2002(in Chinese with English abstract).
[2] Yao J L,Dong Y H,Bret A M. Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor[J]. Proc Natl Acad Sci USA,2001,98(3):1306-1311.
[3] Singh D P,Jermakow A W,Swain S M. Gibberellins are required for seed development and pollen tube growth in Arabidopsis[J]. Plant Cell,2002,14(12):3133-3147.
[4]崔梦杰,王晨,张文颖,等.无核葡萄研究进展[J].植物生理学报,2017,53(3):317-330.Cui M J,Wang C,Zhang W Y,et al. Progress in the study of seedless grapes[J]. Plant Physiology Journal,2017,53(3):317-330(in Chinese with English abstract).
[5]孙其宝,俞飞飞,孙俊.葡萄无核化研究进展[J].安徽农业科学,2004,32(2):360-362.Sun Q B,Yu F F,Sun J. Progress in the study of grapevine berry seedless[J]. Journal of Anhui Agricultural Sciences,2004,32(2):360-362(in Chinese with English abstract).
[6]肖祥希,李明,邱栋梁.果实无核机理研究进展[J].经济林研究,2009,27(2):104-110.Xiao X X,Li M,Qiu D L. Research progress of seedless mechanism of fruits[J]. Nonwood Forest Research,2009,27(2):104-110(in Chinese with English abstract).
[7] Alvarezbuylla E R,Liljegren S J,Pelaz S,et al. MADS-box gene evolution beyond flowers:expression in pollen,endosperm,guard cells,roots and trichomes[J]. Plant Journal for Cell&Molecular Biology,2010,24(4):457-466.
[8] Battaglia R,Brambilla V,Colombo L,et al. Functional analysis of MADS-box genes controlling ovule development in Arabidopsis using the ethanolinducible alc gene-expression system[J]. Mechanisms of Development,2006,123(4):267-276.
[9] Becker A,Theissen G. The major clades of MADS-box genes and their role in the development and evolution of flowering plants[J]. Molecular Phylogenetics&Evolution,2003,29(3):464-489.
[10] Huang B,Routaboul J M,Liu M,et al. Overexpression of the class D MADS-box gene Sl-AGL11 impacts fleshy tissue differentiation and structure in tomato fruits[J]. Journal of Experimental Botany,2017,68(17):4869-4884.
[11] Ocarez N,Mejía N. Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[J]. Plant Cell Reports,2016,35(1):239.
[12] Wang H,Caruso L V,Downie A B,et al. The Embryo MADS domain protein AGAMOUS-Like 15 directly regulates expression of a gene encoding an enzyme involved in gibberellin metabolism[J]. Plant Cell,2004,16(5):1206-1219.
[13] Zheng Y,Perry S E. Global identification of targets of the Arabidopsis MADS domain protein AGAMOUS-Like 15[J]. The Plant Cell,2009,21(9):2563.
[14]王文举,马治国.赤霉素和链霉素及促生灵促进巨峰葡萄果粒增大和无核的试验[J].落叶果树,2003,35(4):43-44.Wang W J,Ma Z G. Test of gibberellin,streptomycin and tomatotone on promoting Kyoho grape berry enlargement and seedless[J]. Deciduous Fruits,2003,35(4):43-44(in Chinese with English abstract).
[15]刘佳,刘晓,陈建.四川地区赤霉素和链霉素处理对巨峰葡萄无核化及果实发育的影响[J].西南农业学报,2011,24(1):220-224.Liu J,Liu X,Chen J. Effect of GA3and SM treatment on inducing seedless and berry growth of Kyoho grape in Sichuan area[J]. Southwest China Journal of Agricultural Sciences,2011,24(1):220-224(in Chinese with English abstract).
[16]陈发河,蔡慧农,冯作山,等.葡萄浆果发育过程中激素水平的变化[J].植物生理与分子生物学学报,2002,28(5):391-395.Chen F H,Cai H N,Feng Z S,et al. Changes of hormone levels during the development of grape berries[J]. Journal of Plant Physiology and Molecular Biology,2002,28(5):391-395(in Chinese with English abstract).
[17]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 Borealioccidentalis Sinica,2010,19(11):135-140(in Chinese with English abstract).
[18] Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J]. Methods,2001,25(4):402-408.
[19] Riechmann J L,Meyerowitz E M. Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1,AP3,PI,and AG is independent of their DNA-binding specificity[J]. Molecular Biology of the Cell,1997,8(7):1243-1259.
[20] Folter S,Shchennikova A V,Franken J,et al. A BsisterMADS-box gene involved in ovule and seed development in petunia and Arabidopsis[J].The Plant Journal,2006,47(6):934-946.
[21] Deng W,Chen G,Peng F,et al. Transparent testa16 plays multiple roles in plant development and is involved in lipid synthesis and embryo development in canola[J]. Plant Physiology,2012,160(2):978-989.
[22] Xu W,Fiume E,Coen O,et al. Endosperm and nucellus develop antagonistically in Arabidopsis seeds[J]. The Plant Cell,2016,28(6):1343-1360.
[23] Yang Z,Li C,Wang Y,et al. GhAGL15s,preferentially expressed during somatic embryogenesis,promote embryogenic callus formation in cotton(Gossypium hirsutum L.)[J]. Molecular Genetics&Genomics,2014,289(5):873-883.
[24]董庆龙,冀志蕊,迟福梅,等.苹果MADS-box转录因子的生物信息学及其在不同组织中的表达[J].中国农业科学,2014,47(6):1151-1161.Dong Q L,Ji Z R,Chi F M,et al. Bioinformatics of the MADS-Box transcription factor and their expression in different apple tissues[J]. Scientia Agricultura Sinica,2014,47(6):1151-1161(in Chinese with English abstract).
[25]蔡英卿.龙眼体胚发生过程中SERK等胚性相关基因的克隆与表达分析[D].福州:福建农林大学,2011.Cai Y Q. Cloning and epression of SERK and sme other somatic embryogenesis-related genes during somatic embryogenesis in Dimocarpus longan Lour[D]. Fuzhou:Fujian Agriculture and Forestry University,2011(in Chinese with English abstract).
[26] ParenicovaL,De S F,Kieffer M,et al. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis:new openings to the MADS world[J]. The Plant Cell,2003,15(7):1538-1551.
[27]王飞.葡萄无核品种及其杂种胚败育机理与胚挽救技术研究[D].杨凌:西北农林科技大学,2002.Wang F. Mechanism of embryo abortion in stonespermic seedless grape and embryo rescue with seedless grape[D]. Yangling:Northwest A&F University,2002(in Chinese with English abstract).