草莓FaBBX15基因克隆与功能分析
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摘要
植物BBX蛋白作为转录因子参与植物多种生命活动。为了研究草莓中BBX家族成员的基因功能,在前期分析转录组数据得到一个响应不同光质处理的草莓BBX家族成员。通过同源克隆的方法从‘红颜’草莓中分离克隆得到一个BBX蛋白编码c DNA序列,并对其进行生物信息学分析、基因表达分析、亚细胞定位与酵母转录激活分析。结果表明:克隆得到一条1 403 bp的cDNA序列,含有编码蛋白长度为447个氨基酸的CDS序列,编码蛋白与拟南芥(Arabidopsis thaliana) AtBBX15蛋白同源,命名为FaBBX15。蛋白质预测分子量49 831.42 Da。FaBBX15蛋白质含有1个B-box保守结构域和1个CCT保守结构域,属于BBX蛋白家族第3亚家族成员。定量PCR分析表明FaBBX15基因表达受到红光与蓝光抑制。FaBBX15蛋白质在烟草表皮亚细胞中定位于细胞核,在酵母细胞中具有转录激活能力,具有典型的转录因子特征。本研究结果可为进一步揭示FaBBX15蛋白在光信号转导中的的基因功能及分子机理提供理论参考。
As transcript factors, BBX proteins participate in various physiologically processes of plant. In order to investigate the gene function of BBXs in strawberry, a member of BBX family in strawberry responding to different light quality treatments was obtained by analyzing transcriptome data in advance. A BBX protein encoding cDNA sequence was isolated and cloned from 'Benihhope' strawberry by homologous cloning, and its bioinformatics analysis, gene expression analysis, subcellular localization and yeast transcription activation analysis were carried out. The results showed that a 1 403 bp c DNA sequence was cloned, containing a CDS sequence with 447 amino acids. The coding protein was homologous to Arabidopsis thaliana AtBBX15 protein and named FaBBX15.The predicted molecular weight was 49 831.42 Da. Fa BBX15 protein contained one B-box conserved domain and one CCT conserved domain and belonged to the third subfamily of BBX protein family. Quantitative PCR analysis indicated that FaBBX15 gene expression was inhibited by red and blue light. FaBBX15 protein was located in the nucleus of tobacco epidermal subcells and has transcriptional activation ability in yeast cells with typical transcription factor characteristics. The result of this study might provide a theoretical reference for further revealing the gene function and molecular mechanism of Fa BBX15 protein in light signal transduction.
As transcript factors, BBX proteins participate in various physiologically processes of plant. In order to investigate the gene function of BBXs in strawberry, a member of BBX family in strawberry responding to different light quality treatments was obtained by analyzing transcriptome data in advance. A BBX protein encoding cDNA sequence was isolated and cloned from 'Benihhope' strawberry by homologous cloning, and its bioinformatics analysis, gene expression analysis, subcellular localization and yeast transcription activation analysis were carried out. The results showed that a 1 403 bp c DNA sequence was cloned, containing a CDS sequence with 447 amino acids. The coding protein was homologous to Arabidopsis thaliana AtBBX15 protein and named FaBBX15.The predicted molecular weight was 49 831.42 Da. Fa BBX15 protein contained one B-box conserved domain and one CCT conserved domain and belonged to the third subfamily of BBX protein family. Quantitative PCR analysis indicated that FaBBX15 gene expression was inhibited by red and blue light. FaBBX15 protein was located in the nucleus of tobacco epidermal subcells and has transcriptional activation ability in yeast cells with typical transcription factor characteristics. The result of this study might provide a theoretical reference for further revealing the gene function and molecular mechanism of Fa BBX15 protein in light signal transduction.
引文
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Chen Q.,Yu H.,Tang H.,and Wang X.,2012,Identification and expression analysis of genes involved in anthocyanin and proanthocyanidin biosynthesis in the fruit of blackberry,Sci.Hortic.,141:61-68
Chu Z.,Wang X.,Li Y.,Yu H.,Li J.,Lu Y.,Li H.,and Ouyang B.,2016,Genomic organization,phylogenetic and expression analysis of the b-box gene family in tomato,Front.Plant Sci.,7:1552
Gangappa S.N.,and Botto J.F.,2014,The BBX family of plant transcription factors,Trends Plant Sci.,19(7):460-470
Griffiths S.,Dunford R.P.,Coupland G.,and Laurie D.A.,2003,The evolution of constans-like gene families in barley,rice,and Arabidopsis,Plant Physiol.,131(4):1855-1867
Hoecker U.,2017,The activities of the E3 ubiquitin ligase COP1/SPA,a key repressor in light signaling,Curr.Opin.Plant Biol.,37:63-69
Huang J.,Zhao X.,Weng X.,Wang L.,and Xie W.,2012,The rice B-box zinc finger gene family:genomic identification,characterization,expression profiling and diurnal analysis,PLoS One,7(10):e48242
Khanna R.,Kronmiller B.,Maszle D.R.,Coupland G.,Holm M.,Mizuno T.,and Wu S.H.,2009,The Arabidopsis b-box zinc finger family,Plant Cell,21(11):3416-3420
Kurokura T.,Samad S.,Koskela E.,Mouhu K.,and Hytonen T.,2017,Fragaria vesca constans controls photoperiodic flow ering and vegetative development,J.Exp.Bot.,68(17):4839-4850
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Methods,25(4):402-408
Min J.H.,Chung J.S.,Lee K.H.,and Kim C.S.,2015,The constans-like 4 transcription factor,At COL4,positively regulates abiotic stress tolerance through an abscisic acid-dependent manner in Arabidopsis,J.Integr.Plant Biol.,57(3):313-324
Putterill J.,Robson F.,Lee K.,Simon R.,and Coupland G.,1995,The constans gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors,Cell,80(6):847-857
Soitamo A.J.,Piippo M.,Allahverdiyeva Y.,Battchikova N.,and Aro E.M.,2008,Light has a specific role in modulating Arabidopsis gene expression at low temperature,BMC Plant Biol.,8(1):13
Tiwari S.B.,Shen Y.,Chang H.C.,Hou Y.,Harris A.,Ma S.F.,McPartland M.,Hymus G.J.,Adam L.,Marion C.,Belachew A.,Repetti P.P.,Reuber T.L.,and Ratcliffe O.J.,2010,The flowering time regulator constans is recruited to the flowering locust promoter via a unique cis-element,New Phytol.,187(1):57-66
Valverde F.,Mouradov A.,Soppe W.,Ravenscroft D.,Samach A.,and Coupland G.,2004,Photoreceptor regulation of CON-STANS protein in photoperiodic flowering,Science,303(5660):1003-1006
Wang H.,Zhang Z.,Li H.,Zhao X.,Liu X.,Ortiz M.,Lin C.T.,and Liu B.,2013,Constans-like 7 regulates branching and shade avoidance response in Arabidopsis,J.Exp.Bot.,64(4):1017-1024
Xu D.,Jiang Y.,Li J.,Holm M.,and Deng X.W.,2017,The b-box domain protein BBX21 promotes photo morphogenesis,Plant Physiol.,17:1305
Xue D.L.,Zeng S.H.,and Wang Y.,2015,Molecular cloning and expression analysis of Flowering Locus T in Lycium ruthenicum Murr.and L.barbarum L.,Jiyinzuxue Yu Yingy ong Shengwuxue(Genomics and Applied Biology),34(3):565-570(薛定磊,曾少华,王瑛,2015,黑果枸杞和宁夏枸杞FT(Flowering Locus T)基因的克隆及表达分析,基因组学与应用生物学,34(3):565-570)
Yang Y.,Ma C.,Xu Y.,Wei Q.,Imtiaz M.,Lan H.,Gao S.,Cheng L.,Wang M.,Fei F.,Hong B.,and Gao J.,2014,A zinc finger protein regulates flowering time and abiotic stress tolerance in chrysanthemum by modulating gibberellin biosynthesis,Plant Cell,26(5):2038-2054
Yano M.,Katayose Y.,Ashikari M.,Yamanouchi,U.,Monna L.,Fuse T.,Baba T.,Yamamoto K.,Umehara Y.,Nagamura Y.,and Sasaki T.,2000,Hd1,a major photoperiod sensitivity quantitative trait locus in rice,is closely related to the Arabidopsis flowering time gene constans,Plant Cell,12(12):2473-2483
Ye Y.T.,Lou K.F.,Wang X.R.,Liu Y.Q.,Chen Q.,and Tang H.R.,2016,Genome-wide identification and expression investigation of the b-box gene family in Fragaria vesca,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):827-834(叶云天,娄可飞,王熙然,刘勇强,陈清,汤浩茹,2016,森林草莓B-box基因家族全基因组序列鉴定与表达分析,分子植物育种,14(4):827-834)
Zhang Y.T.,Jiang L.Y.,Ye Y.T.,Feng C.,Sun B.,Wang X.R.,and Tang H.R.,2017,Cloning and expression analysis of FaCOP1 gene from Fragaria×ananassa'Toyonaka',Yuanyi Xuebao(Acta Horticulturae Sinica),44(3):547-556(张云婷,江雷雨,叶云天,冯琛,孙勃,王小蓉,汤浩茹,2017,草莓FaCOP1的克隆及其表达特异性分析,园艺学报,44(3):547-556)
Zhang Z.,Ji R.,Li H.,Zhao T.,Liu J.,Lin C.,and Liu B.,2014,Constans-like 7(COL7)is involved in phytochrome B(phyB)-mediated light-quality regulation of auxin homeostasis,Mol.Plant,7(9):1429-1440
Zou Z.Y.,Wang R.H.,Wang R.L.,Yang S.,and Yang Y.J.,2018,Genome-wide identification,phylogenetic analysis,and expression profiling of the BBX family genes in pear,J.Hortic.Sci.Biotechnol.,93(1):37-50
Chen Q.,Yu H.,Tang H.,and Wang X.,2012,Identification and expression analysis of genes involved in anthocyanin and proanthocyanidin biosynthesis in the fruit of blackberry,Sci.Hortic.,141:61-68
Chu Z.,Wang X.,Li Y.,Yu H.,Li J.,Lu Y.,Li H.,and Ouyang B.,2016,Genomic organization,phylogenetic and expression analysis of the b-box gene family in tomato,Front.Plant Sci.,7:1552
Gangappa S.N.,and Botto J.F.,2014,The BBX family of plant transcription factors,Trends Plant Sci.,19(7):460-470
Griffiths S.,Dunford R.P.,Coupland G.,and Laurie D.A.,2003,The evolution of constans-like gene families in barley,rice,and Arabidopsis,Plant Physiol.,131(4):1855-1867
Hoecker U.,2017,The activities of the E3 ubiquitin ligase COP1/SPA,a key repressor in light signaling,Curr.Opin.Plant Biol.,37:63-69
Huang J.,Zhao X.,Weng X.,Wang L.,and Xie W.,2012,The rice B-box zinc finger gene family:genomic identification,characterization,expression profiling and diurnal analysis,PLoS One,7(10):e48242
Khanna R.,Kronmiller B.,Maszle D.R.,Coupland G.,Holm M.,Mizuno T.,and Wu S.H.,2009,The Arabidopsis b-box zinc finger family,Plant Cell,21(11):3416-3420
Kurokura T.,Samad S.,Koskela E.,Mouhu K.,and Hytonen T.,2017,Fragaria vesca constans controls photoperiodic flow ering and vegetative development,J.Exp.Bot.,68(17):4839-4850
Livak K.J.,and Schmittgen T.D.,2001,Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod,Methods,25(4):402-408
Min J.H.,Chung J.S.,Lee K.H.,and Kim C.S.,2015,The constans-like 4 transcription factor,At COL4,positively regulates abiotic stress tolerance through an abscisic acid-dependent manner in Arabidopsis,J.Integr.Plant Biol.,57(3):313-324
Putterill J.,Robson F.,Lee K.,Simon R.,and Coupland G.,1995,The constans gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zinc finger transcription factors,Cell,80(6):847-857
Soitamo A.J.,Piippo M.,Allahverdiyeva Y.,Battchikova N.,and Aro E.M.,2008,Light has a specific role in modulating Arabidopsis gene expression at low temperature,BMC Plant Biol.,8(1):13
Tiwari S.B.,Shen Y.,Chang H.C.,Hou Y.,Harris A.,Ma S.F.,McPartland M.,Hymus G.J.,Adam L.,Marion C.,Belachew A.,Repetti P.P.,Reuber T.L.,and Ratcliffe O.J.,2010,The flowering time regulator constans is recruited to the flowering locust promoter via a unique cis-element,New Phytol.,187(1):57-66
Valverde F.,Mouradov A.,Soppe W.,Ravenscroft D.,Samach A.,and Coupland G.,2004,Photoreceptor regulation of CON-STANS protein in photoperiodic flowering,Science,303(5660):1003-1006
Wang H.,Zhang Z.,Li H.,Zhao X.,Liu X.,Ortiz M.,Lin C.T.,and Liu B.,2013,Constans-like 7 regulates branching and shade avoidance response in Arabidopsis,J.Exp.Bot.,64(4):1017-1024
Xu D.,Jiang Y.,Li J.,Holm M.,and Deng X.W.,2017,The b-box domain protein BBX21 promotes photo morphogenesis,Plant Physiol.,17:1305
Xue D.L.,Zeng S.H.,and Wang Y.,2015,Molecular cloning and expression analysis of Flowering Locus T in Lycium ruthenicum Murr.and L.barbarum L.,Jiyinzuxue Yu Yingy ong Shengwuxue(Genomics and Applied Biology),34(3):565-570(薛定磊,曾少华,王瑛,2015,黑果枸杞和宁夏枸杞FT(Flowering Locus T)基因的克隆及表达分析,基因组学与应用生物学,34(3):565-570)
Yang Y.,Ma C.,Xu Y.,Wei Q.,Imtiaz M.,Lan H.,Gao S.,Cheng L.,Wang M.,Fei F.,Hong B.,and Gao J.,2014,A zinc finger protein regulates flowering time and abiotic stress tolerance in chrysanthemum by modulating gibberellin biosynthesis,Plant Cell,26(5):2038-2054
Yano M.,Katayose Y.,Ashikari M.,Yamanouchi,U.,Monna L.,Fuse T.,Baba T.,Yamamoto K.,Umehara Y.,Nagamura Y.,and Sasaki T.,2000,Hd1,a major photoperiod sensitivity quantitative trait locus in rice,is closely related to the Arabidopsis flowering time gene constans,Plant Cell,12(12):2473-2483
Ye Y.T.,Lou K.F.,Wang X.R.,Liu Y.Q.,Chen Q.,and Tang H.R.,2016,Genome-wide identification and expression investigation of the b-box gene family in Fragaria vesca,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(4):827-834(叶云天,娄可飞,王熙然,刘勇强,陈清,汤浩茹,2016,森林草莓B-box基因家族全基因组序列鉴定与表达分析,分子植物育种,14(4):827-834)
Zhang Y.T.,Jiang L.Y.,Ye Y.T.,Feng C.,Sun B.,Wang X.R.,and Tang H.R.,2017,Cloning and expression analysis of FaCOP1 gene from Fragaria×ananassa'Toyonaka',Yuanyi Xuebao(Acta Horticulturae Sinica),44(3):547-556(张云婷,江雷雨,叶云天,冯琛,孙勃,王小蓉,汤浩茹,2017,草莓FaCOP1的克隆及其表达特异性分析,园艺学报,44(3):547-556)
Zhang Z.,Ji R.,Li H.,Zhao T.,Liu J.,Lin C.,and Liu B.,2014,Constans-like 7(COL7)is involved in phytochrome B(phyB)-mediated light-quality regulation of auxin homeostasis,Mol.Plant,7(9):1429-1440
Zou Z.Y.,Wang R.H.,Wang R.L.,Yang S.,and Yang Y.J.,2018,Genome-wide identification,phylogenetic analysis,and expression profiling of the BBX family genes in pear,J.Hortic.Sci.Biotechnol.,93(1):37-50