大叶落地生根(Kalanchoe daigremontiana)KdSOC1基因的克隆及自激活检测
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摘要
大叶落地生根(Kalanchoe daigremontiana)为景天科多年生肉质草本植物,是研究无性繁殖的模式植物。SOC1 (Suppressor of overexpression of constans 1)基因是植物成花过程中MADS-box基因家族中的关键转录因子。为深入研究大叶落地生根KdSOC1基因的功能,于大叶落地生根中克隆KdSOC1基因,进行生物信息学分析,构建酵母双杂交诱饵表达载体pGBKT7-KdSOC1,并采用LiAc法将重组质粒pGBKT7-KdSOC1转化至Y2HGold酵母菌株中,通过营养缺陷型培养基培养进行自激活检测。结果表明,成功克隆了KdSOC1基因,该基因开放阅读框共有684 bp。生物信息学分析结果显示,Kd SOC1蛋白共有227个氨基酸残基,分子量为25.88 kD,大叶落地生根SOC1蛋白与牡丹的亲缘关系最为接近。双杂交诱饵表达载体pGBKT7-KdSOC1构建成功,并在酵母细胞中正常表达,表达产物对报告基因无自激活作用。本研究为筛选大叶落地生根中与其互作的蛋白及后续实验提供了帮助。
Kalanchoe daigremontiana is a perennial succulent herb of sedum, which is a model plant for the study of asexual reproduction. The SOC1(Suppressor of overexpression of constans 1) gene is a key transcription factor in the MADS-box gene family during plant flowering. In order to further study the function of KdSOC1 of Kalanchoe daigremontiana, KdSOC1 gene was cloned from Kalanchoe daigremontiana for bioinformatics analysis, and yeast two-hybrid bait expression vector pGBKT7-KdSOC1 was constructed. Recombinant plasmid pGBKT7-KdSOC1 was transformed into Y2 HGold yeast strain by Li Ac method, and self-activation assays were performed by auxotrophic medium culture. The results showed that the KdSOC1 gene was successfully cloned, and the open reading frame of the gene was 684 bp. Bioinformatics analysis showed that KdSOC1 protein had 227 amino acid residues and the molecular weight was 25.88 kD. The SOC1 protein of Kalanchoe daigremontiana had the highest homologous characteristics with peony. The two-hybrid bait expression vector pGBKT7-KdSOC1 was successfully constructed and expressed in yeast cells. The expression product had no self-activation effect on the reporter gene.This study would be helpful for screening proteins interacting in the Kalanchoe daigremontiana and conducting subsequent experiments.
Kalanchoe daigremontiana is a perennial succulent herb of sedum, which is a model plant for the study of asexual reproduction. The SOC1(Suppressor of overexpression of constans 1) gene is a key transcription factor in the MADS-box gene family during plant flowering. In order to further study the function of KdSOC1 of Kalanchoe daigremontiana, KdSOC1 gene was cloned from Kalanchoe daigremontiana for bioinformatics analysis, and yeast two-hybrid bait expression vector pGBKT7-KdSOC1 was constructed. Recombinant plasmid pGBKT7-KdSOC1 was transformed into Y2 HGold yeast strain by Li Ac method, and self-activation assays were performed by auxotrophic medium culture. The results showed that the KdSOC1 gene was successfully cloned, and the open reading frame of the gene was 684 bp. Bioinformatics analysis showed that KdSOC1 protein had 227 amino acid residues and the molecular weight was 25.88 kD. The SOC1 protein of Kalanchoe daigremontiana had the highest homologous characteristics with peony. The two-hybrid bait expression vector pGBKT7-KdSOC1 was successfully constructed and expressed in yeast cells. The expression product had no self-activation effect on the reporter gene.This study would be helpful for screening proteins interacting in the Kalanchoe daigremontiana and conducting subsequent experiments.
引文
Borner R.,Kampmann G.,Chandler J.,Gleissner R.,Wisman E.,Apel K.,and Melzer S.,2000,A MADS domain gene involved in the transition to flowering in Arabidopsis,Plant J.,24(5):591-599
Chen Z.,Wang L.,Chen J.,Liu C.,Zeng H.,and Wang H.,2017,Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana,Sci.Rep.,7(1):5629
Cseke L.J.,Zheng J.,and Podila G.K.,2003,Characterization of PTM5 in Aspen trees:a MADS-box gene expressed during woody vascular development,Gene,318(1):55-67
Cui B.,Wang J.Q.,Song C.X.,Jiang S.H.,Liang F.,Yuan X.Y.,and Ma J.,2016,Cloning and expression analysis of SOC1gene from Phalaenopsis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):548-553(崔波,王洁琼,宋彩霞,蒋素华,梁芳,袁秀云,马杰,2016,蝴蝶兰SOC1基因的克隆及表达分析,分子植物育种,15(3):548-553)
Dan S.N.,Hu Y.,He X.H.,Zhang Y.,An Z.Y.,Xu C.,Luo C.,and Huang G.X.,2015,Cloning and expression analysins of FcSOC1 homologous gene from Fortunella crassifolia Swingle,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(12):2651-2659(旦帅男,胡颖,何新华,张奕,安振宇,徐趁,罗聪,黄桂香,2015,金柑FcSOC1同源基因的克隆及表达分析,基因组学与应用生物学,34(12):2651-2659)
Fields S.,and Song O.,1989,A novel genetic system to detect protein protein interactions,Nature,340(6230):245-246
Fu J.X.,Yang L.W.,Jie S.,Dai Y.,and Dai S.L.,2013,Functional study of SOC1 homologous genes,ClSOC1-a and ClSOC1-b,In:Editorial Committee of the Chinese Society for Horticultural Science,eds.,Advances in Ornamental Horticulture of China,Beijing,China,pp.204-209(付建新,杨立文,亓帅,戴翼,戴思兰,2013,甘菊SOC1同源基因ClSOC1-a和ClSOC1-b功能研究,见:中国园艺学会编委,编著,中国观赏园艺研究进展,中国,北京,pp.204-209)
Heuer S.,Hansen S.,Bantin J.,Brettschneider R.,Kranz E.,Lorz H.,and Dresselhaus T.,2001,The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development,in egg cells,and early embryogenesis,Plant Physiol.,127(1):33-45
Lee H.,Suh S.S.,Park E.,Cho E.,Ji H.A.,Kim S.G.,Jong S.L.,Young M.K.,and Ilha L.,2000,The AGAMOUS-LIKE 20MADS domain protein integrates floral inductive pathways in Arabidopsis,Genes Dev.,14(18):2366-2376
Lee J.,and Lee I.,2010,Regulation and function of SOC1,a flowering pathway integrator,J.Exp.Bot.,61(9):2247-2254
Lee J.,Oh M.,Park H.,and Lee I.,2008,SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy,Plant J.,55(5):832-843
Lee S.,Kim J.,Han J.J.,Han M.J.,and An G.,2004,Functional analyses of the flowering time gene OsMADS50,the putative suppressor of overexpression of co1/AGAMOUS-like 20(SOC1/AGL20)ortholog in rice,Plant J.,38(5):754-764
Liu C.J.,Wang S.L.,Xue J.Q.,Zhu F.Y.,Ren X.X.,Li M.Y.,and Zhang X.X.,2014,Molecular cloning and expression analysis of the flowering-regulating transcription factor PrSOC1gene in tree peony,Yuanyi Xuebao(Acta Horticulturae Sinica),41(11):2259-2267(刘传娇,王顺利,薛璟祺,朱富勇,任秀霞,李名扬,张秀新,2014,牡丹开花调控转录因子基因Pr SOC1的克隆与表达分析,园艺学报,41(11):2259-2267)
Liu C.L.,2016,Cloning and function analysis of KdSOC1 in Kalanchoe daigremontiana,Thesis for M.S.,Beijing Forestry University,Supervisor:Zeng H.M.,pp.15-27(刘成兰,2016,大叶落地生根KdSOC1基因的克隆与功能的初步鉴定,硕士学位论文,北京林业大学,导师:曾会明,pp.15-27)
Liu F.F.,Li H.Y.,Wang S.,and Liu G.F.,2011,Cloning and periodic expression of BpSOC1 gene in Betula platyphylla,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),39(4):1-4(刘菲菲,李慧玉,王姗,刘桂丰,2011,白桦BpSOC1基因的克隆及时序表达分析,东北林业大学学报,39(4):1-4)
Melzer S.,Lens F.,Gennen J.,Vanneste S.,Rohde A.,and Beeckman T.,2008,Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana,Nat.Genet.,40(12):1489-1492
Moon J.,Suh S.S.,Lee H.,Choi K.R.,Hong C.B.,Paek N.C.,Kim S.G.,and Lee I.,2003,The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis,Plant J.,35(5):613-623
Mouhu K.,Kurokura T.,Koskela E.A.,Albert V.A.,Elomaa P.,and Hytonen T.,2013,The Fragaria vesca homolog of SUP-PRESSOR OF OVEREXPRESSION OF CONSTANS1 represses flowering and promotes vegetative growth,Plant Cell,25(9):3296-3310
Onouchi H.,Igeno M.I.,Périlleux C.,Graves K.,and Coupland G.,2000,Mutagenesis of plants overexpressing CONSTANSdemonstrates novel interactions among Arabidopsis flowering-time genes,Plant Cell,12(6):885-900
Riechmann J.L.,and Meyerowitz E.M.,1997a,Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1,AP3,PI,and AG is independent of their DNA-binding specificity,Mol.Biol.Cell,8(7):1243-1259
Riechmann J.L.,and Meyerowitz E.M.,1997b,MADS domain proteins in plant development,Biol.Chem.,378(10):1079-1101
Seo E.,Lee H.,Jin J.,Park H.,Kim J.,Noh Y.S.,and Lee I.,2009,Crosstalk between cold response and flowering in Arabidopsis is mediated through the flowering-time gene SOC1 and its upstream negative regulator,Plant Cell,21(10):3185
Shi Y.C.,Yang Y.Y.,and Liu W.Q.,2014,Cloning and expression of SOC1 gene in tobacco,Zhongguo Yancao Xuebao(Acta Tabacaria Sinica),20(2):99-103(石永春,杨永银,刘卫群,2014,烟草SOC1基因的克隆和表达分析,中国烟草学报,20(2):99-103)
Shitsukawa N.,Ikari C.,Mitsuya T.,Sakiyama T.,Ishikawa A.,Takumi S.,and Murai K.,2007,Wheat SOC1 functions independently of WAP1/VRN1,an integrator of vernalization and photoperiod flowering promotion pathways,Physiologia Plantarum,130(4):627-636
Tadege M.,Sheldon C.C.,Helliwell C.A.,Upadhyaya N.M.,Dennis E.S.,and Peacock W.J.,2003,Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice,Plant Biotechnol.J.,1(5):361-369
Wu D.,Wu J.H.,and Zhang Z.Z.,2017,Cloning and sub-cellular localization of NtSOC1 gene from Narcissus tazetta var.Chinensis,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(10):1889-1895(武丹,吴菁华,张志忠,2017,中国水仙NtSOC1基因克隆及亚细胞定位,西北植物学报,37(10):1889-1895)
Zhong X.,Dai X.,Xu J.,Wu H.,Liu B.,and Li H.,2012,Cloning and expression analysis of GmGAL1,SOC1 homolog gene in soybean,Mol.Biol.Rep.,39(6):6967-6974
Wu J.Y.,Yan P.,Shen W.T.,and Zhou P.,2010,Construction and autoactivation identification of bait vector containing papaya eIF4E and eIFiso4E gene inyeast two hybrid system,Redai Shengwu Xuebao(Journal of Tropical Organisms),1(1):50-54(吴金燕,言普,沈文涛,周鹏,2010,番木瓜eIF4E和eIFiso4E酵母双杂交诱饵表达载体的构建及自激活检测,热带生物学报,1(1):50-54)
Chen Z.,Wang L.,Chen J.,Liu C.,Zeng H.,and Wang H.,2017,Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana,Sci.Rep.,7(1):5629
Cseke L.J.,Zheng J.,and Podila G.K.,2003,Characterization of PTM5 in Aspen trees:a MADS-box gene expressed during woody vascular development,Gene,318(1):55-67
Cui B.,Wang J.Q.,Song C.X.,Jiang S.H.,Liang F.,Yuan X.Y.,and Ma J.,2016,Cloning and expression analysis of SOC1gene from Phalaenopsis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(3):548-553(崔波,王洁琼,宋彩霞,蒋素华,梁芳,袁秀云,马杰,2016,蝴蝶兰SOC1基因的克隆及表达分析,分子植物育种,15(3):548-553)
Dan S.N.,Hu Y.,He X.H.,Zhang Y.,An Z.Y.,Xu C.,Luo C.,and Huang G.X.,2015,Cloning and expression analysins of FcSOC1 homologous gene from Fortunella crassifolia Swingle,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(12):2651-2659(旦帅男,胡颖,何新华,张奕,安振宇,徐趁,罗聪,黄桂香,2015,金柑FcSOC1同源基因的克隆及表达分析,基因组学与应用生物学,34(12):2651-2659)
Fields S.,and Song O.,1989,A novel genetic system to detect protein protein interactions,Nature,340(6230):245-246
Fu J.X.,Yang L.W.,Jie S.,Dai Y.,and Dai S.L.,2013,Functional study of SOC1 homologous genes,ClSOC1-a and ClSOC1-b,In:Editorial Committee of the Chinese Society for Horticultural Science,eds.,Advances in Ornamental Horticulture of China,Beijing,China,pp.204-209(付建新,杨立文,亓帅,戴翼,戴思兰,2013,甘菊SOC1同源基因ClSOC1-a和ClSOC1-b功能研究,见:中国园艺学会编委,编著,中国观赏园艺研究进展,中国,北京,pp.204-209)
Heuer S.,Hansen S.,Bantin J.,Brettschneider R.,Kranz E.,Lorz H.,and Dresselhaus T.,2001,The maize MADS box gene ZmMADS3 affects node number and spikelet development and is co-expressed with ZmMADS1 during flower development,in egg cells,and early embryogenesis,Plant Physiol.,127(1):33-45
Lee H.,Suh S.S.,Park E.,Cho E.,Ji H.A.,Kim S.G.,Jong S.L.,Young M.K.,and Ilha L.,2000,The AGAMOUS-LIKE 20MADS domain protein integrates floral inductive pathways in Arabidopsis,Genes Dev.,14(18):2366-2376
Lee J.,and Lee I.,2010,Regulation and function of SOC1,a flowering pathway integrator,J.Exp.Bot.,61(9):2247-2254
Lee J.,Oh M.,Park H.,and Lee I.,2008,SOC1 translocated to the nucleus by interaction with AGL24 directly regulates leafy,Plant J.,55(5):832-843
Lee S.,Kim J.,Han J.J.,Han M.J.,and An G.,2004,Functional analyses of the flowering time gene OsMADS50,the putative suppressor of overexpression of co1/AGAMOUS-like 20(SOC1/AGL20)ortholog in rice,Plant J.,38(5):754-764
Liu C.J.,Wang S.L.,Xue J.Q.,Zhu F.Y.,Ren X.X.,Li M.Y.,and Zhang X.X.,2014,Molecular cloning and expression analysis of the flowering-regulating transcription factor PrSOC1gene in tree peony,Yuanyi Xuebao(Acta Horticulturae Sinica),41(11):2259-2267(刘传娇,王顺利,薛璟祺,朱富勇,任秀霞,李名扬,张秀新,2014,牡丹开花调控转录因子基因Pr SOC1的克隆与表达分析,园艺学报,41(11):2259-2267)
Liu C.L.,2016,Cloning and function analysis of KdSOC1 in Kalanchoe daigremontiana,Thesis for M.S.,Beijing Forestry University,Supervisor:Zeng H.M.,pp.15-27(刘成兰,2016,大叶落地生根KdSOC1基因的克隆与功能的初步鉴定,硕士学位论文,北京林业大学,导师:曾会明,pp.15-27)
Liu F.F.,Li H.Y.,Wang S.,and Liu G.F.,2011,Cloning and periodic expression of BpSOC1 gene in Betula platyphylla,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),39(4):1-4(刘菲菲,李慧玉,王姗,刘桂丰,2011,白桦BpSOC1基因的克隆及时序表达分析,东北林业大学学报,39(4):1-4)
Melzer S.,Lens F.,Gennen J.,Vanneste S.,Rohde A.,and Beeckman T.,2008,Flowering-time genes modulate meristem determinacy and growth form in Arabidopsis thaliana,Nat.Genet.,40(12):1489-1492
Moon J.,Suh S.S.,Lee H.,Choi K.R.,Hong C.B.,Paek N.C.,Kim S.G.,and Lee I.,2003,The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis,Plant J.,35(5):613-623
Mouhu K.,Kurokura T.,Koskela E.A.,Albert V.A.,Elomaa P.,and Hytonen T.,2013,The Fragaria vesca homolog of SUP-PRESSOR OF OVEREXPRESSION OF CONSTANS1 represses flowering and promotes vegetative growth,Plant Cell,25(9):3296-3310
Onouchi H.,Igeno M.I.,Périlleux C.,Graves K.,and Coupland G.,2000,Mutagenesis of plants overexpressing CONSTANSdemonstrates novel interactions among Arabidopsis flowering-time genes,Plant Cell,12(6):885-900
Riechmann J.L.,and Meyerowitz E.M.,1997a,Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1,AP3,PI,and AG is independent of their DNA-binding specificity,Mol.Biol.Cell,8(7):1243-1259
Riechmann J.L.,and Meyerowitz E.M.,1997b,MADS domain proteins in plant development,Biol.Chem.,378(10):1079-1101
Seo E.,Lee H.,Jin J.,Park H.,Kim J.,Noh Y.S.,and Lee I.,2009,Crosstalk between cold response and flowering in Arabidopsis is mediated through the flowering-time gene SOC1 and its upstream negative regulator,Plant Cell,21(10):3185
Shi Y.C.,Yang Y.Y.,and Liu W.Q.,2014,Cloning and expression of SOC1 gene in tobacco,Zhongguo Yancao Xuebao(Acta Tabacaria Sinica),20(2):99-103(石永春,杨永银,刘卫群,2014,烟草SOC1基因的克隆和表达分析,中国烟草学报,20(2):99-103)
Shitsukawa N.,Ikari C.,Mitsuya T.,Sakiyama T.,Ishikawa A.,Takumi S.,and Murai K.,2007,Wheat SOC1 functions independently of WAP1/VRN1,an integrator of vernalization and photoperiod flowering promotion pathways,Physiologia Plantarum,130(4):627-636
Tadege M.,Sheldon C.C.,Helliwell C.A.,Upadhyaya N.M.,Dennis E.S.,and Peacock W.J.,2003,Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice,Plant Biotechnol.J.,1(5):361-369
Wu D.,Wu J.H.,and Zhang Z.Z.,2017,Cloning and sub-cellular localization of NtSOC1 gene from Narcissus tazetta var.Chinensis,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(10):1889-1895(武丹,吴菁华,张志忠,2017,中国水仙NtSOC1基因克隆及亚细胞定位,西北植物学报,37(10):1889-1895)
Zhong X.,Dai X.,Xu J.,Wu H.,Liu B.,and Li H.,2012,Cloning and expression analysis of GmGAL1,SOC1 homolog gene in soybean,Mol.Biol.Rep.,39(6):6967-6974
Wu J.Y.,Yan P.,Shen W.T.,and Zhou P.,2010,Construction and autoactivation identification of bait vector containing papaya eIF4E and eIFiso4E gene inyeast two hybrid system,Redai Shengwu Xuebao(Journal of Tropical Organisms),1(1):50-54(吴金燕,言普,沈文涛,周鹏,2010,番木瓜eIF4E和eIFiso4E酵母双杂交诱饵表达载体的构建及自激活检测,热带生物学报,1(1):50-54)