海岛棉纤维均一化酵母双杂交文库的构建与GbTCP5互作蛋白的筛选
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摘要
为探究转录因子在海岛棉纤维中的生物学功能,以海岛棉8个时期的纤维为研究对象,利用SMART技术合成ds-cDNA,经DSN进行均一化处理,构建以pGADT7为载体的海岛棉纤维均一化酵母cDNA文库,利用酵母双杂交系统筛选与海岛棉GbTCP5互作的蛋白。根据克隆得到的GbTCP5基因的CDS区设计含有NcoⅠ、EcoRI酶切位点的引物,构建诱饵载体pGBKT7-GbTCP5。经自激活和毒性检测后共转化酵母菌株Y2HGold,筛选与GbTCP5互作的蛋白。实时荧光定量PCR显示,GbTCP5基因在纤维发育起始期和伸长期的表达量较高,且在花瓣和花萼中的表达量高于其他组织。文库的质量检测显示,构建的均一化cDNA文库库容为9.15×10~7 cfu·mL~(-1),重组率为100%,插入片段大小在500～2 000 bp之间。诱饵载体pGBKT7-GbTCP5通过酵母双杂交系统多次筛选、回转验证,最终确定了4个互作的蛋白,分别是GbTCP20、GbTCP42、GbTCP45和GbAHP1蛋白。海岛棉纤维均一化cDNA文库的构建和筛选,为进一步研究海岛棉TCP转录因子及其互作蛋白在纤维发育时期中的作用机制奠定了基础。
In order to study explore the biological function of transcription factors in Gossypium barbadense fibers, taking the fibers of the Gossypium barbadense for 8 periods as the research object, and reverse-synthesized ds-cDNA by SMART technology. Uniform processing with DSN, and a homogenization yeast cDNA library of sea-island cotton fiber with pGADT7 as a vector was constructed, and the protein interacting with GbTCP5 of Gossypium barbadense was screened by yeast two-hybrid system. The primers containing NcoⅠ and EcoRI restriction sites were designed based on the CDS region of the cloned GbTCP5 gene to construct the bait vector pGBKT7-GbTCP5. Co-transformation of yeast strain Y2 HGold after self-activation and toxicity detection, screening for GbTCPS interacting proteins. Real-time PCR showed that the expression of GbTCP5 gene was higher at the beginning and elongation of fiber development, and higher in petals and calyx than other tissues. Through the quality test of the library, the constructed homologous cDNA library has a library capacity of 9.15×10~7 cfu·mL~(-1), the recombination rate is 100%, and the insert size is between 500～2000 bp. The bait vector pGBKT7-GbTCP5 was screened and rotated by yeast two-hybrid system, and finally confirmed four interacting proteins, namely GbTCP20, GbTCP42, GbTCP45 and GbAHP1 proteins. The construction and screening of the homogenized cDNA library of the island cotton fiber laid a foundation for further study of the mechanism of the interaction between the Gossypium barbadense cotton transcription factor and its interaction protein during fiber development.
In order to study explore the biological function of transcription factors in Gossypium barbadense fibers, taking the fibers of the Gossypium barbadense for 8 periods as the research object, and reverse-synthesized ds-cDNA by SMART technology. Uniform processing with DSN, and a homogenization yeast cDNA library of sea-island cotton fiber with pGADT7 as a vector was constructed, and the protein interacting with GbTCP5 of Gossypium barbadense was screened by yeast two-hybrid system. The primers containing NcoⅠ and EcoRI restriction sites were designed based on the CDS region of the cloned GbTCP5 gene to construct the bait vector pGBKT7-GbTCP5. Co-transformation of yeast strain Y2 HGold after self-activation and toxicity detection, screening for GbTCPS interacting proteins. Real-time PCR showed that the expression of GbTCP5 gene was higher at the beginning and elongation of fiber development, and higher in petals and calyx than other tissues. Through the quality test of the library, the constructed homologous cDNA library has a library capacity of 9.15×10~7 cfu·mL~(-1), the recombination rate is 100%, and the insert size is between 500～2000 bp. The bait vector pGBKT7-GbTCP5 was screened and rotated by yeast two-hybrid system, and finally confirmed four interacting proteins, namely GbTCP20, GbTCP42, GbTCP45 and GbAHP1 proteins. The construction and screening of the homogenized cDNA library of the island cotton fiber laid a foundation for further study of the mechanism of the interaction between the Gossypium barbadense cotton transcription factor and its interaction protein during fiber development.
引文
[1] Danisman S.TCP Transcription factors at the interface between environmental challenges and the plant’s growth responses[J].Frontiers in Plant Science,2016,7(406):1930
[2] 贺超,肖小军,章智,彭琦,黄英金,廖江林.高温胁迫下水稻灌浆初期籽粒的全长cDNA文库构建及其分析[J].核农学报,2015,29(8):1437-1443
[3] Tran T,Chi Ⅴ T Q,Abdullah M,Omar H,Mostafa N,Huynh K,Suhaimi N.Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda[J].Molecular Biology Reports,2011,38(1):177-182
[4] Fields S,Song O K.A novel genetic system to detect protein-protein interactions[J].Nature,1989,340(6230):245
[5] Cubas P,Lauter N,Doebley J,Coen E.The TCP domain:A motif found in proteins regulating plant growth and development[J].Plant Journal,2010,18(2):215-222
[6] Kosugi S,Ohashi Y.PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene[J].Plant Cell,1997,9(9):1607-1619
[7] Martíntrillo M,Cubas P.TCP genes:A family snapshot ten years later[J].Trends in Plant Science,2010,15(1):31-39
[8] Hervé C,Dabos P,Bardet C,Jauneau A,Auriac M C,Ramboer A,Lacout F,Tremousaygue D.In vivo interference with AtTCP20 function induces severe plant growth alterations and deregulates the expression of many genes important for development[J].Plant Physiology,2009,149(3):1462-1477
[9] Palatnik J F,Allen E,Wu X,Schommer C,Schwab R,Carrington J C,Weigel D.Control of leaf morphogenesis by microRNAs[J].Nature,2003,425(6955):257
[10] Pagnussat G C,Yu H J,Ngo Q A,Rajani S,Mayalagu S,Johnson C S,Capron A,Xie L F,Ye D,Sundaresan Ⅴ.Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis[J].Development,2005,132(3):603-614
[11] Takeda T,Amano K,Ohto M,Nakamura K,Sato S,Kato T,Tabata S,Ueguchi C.RNA interference of the Arabidopsis putative transcription factor TCP16 gene results in abortion of early pollen development[J].Plant Molecular Biology,2006,61(1/2):165-177
[12] Prunedapaz J L,Breton G,Para A,Kay S A.A functional genomics approach reveals CHE as a component of the arabidopsis circadian clock[J].Science,2009,323(5920):1481-1485
[13] Zheng K,Ni Z,Qu Y,Cai Y,Yang Z,Sun G,Chen Q.Genome-wide identification and expression analyses of TCP transcription factor genes in Gossypium barbadense[J].Scientific Reports,2018,8(1):14526
[14] 邱迎风,倪志勇,刘娜,陈全家,曲延英.陆地棉GhMYB9基因克隆及结合特性分析[J].核农学报,2017,31(3):440-446
[15] Hao J,Tu L,Hu H,Tan J,Deng F,Tang W,Nie Y,Zhang X.GbTCP,a cotton TCP transcription factor,confers fibre elongation and root hair development by a complex regulating system[J].Journal of Experimental Botany,2012,63(17):6267-6281
[16] Wang M Y,Xia G X.The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation[J].Plant Physiology,2013,162(3):1669-1680
[17] 郑凯,倪志勇,曲延英,王怡,蔡永生,陈全家.海岛棉GbTCP15基因的克隆与表达分析[J].西北植物学报,2016,36(10):1925-1932
[18] 陈炜,赵建文,张智俊,林新春,郭小勤.竹类植物中CYP85A1基因的克隆及表达分析[J].核农学报,2019,33(4):653-663
[19] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408
[20] T?htiharju S,Rijpkema A S,Vetterli A,Albert V A,Teeri T H,Elomaa P.Evolution and diversification of the CYC/TB1 Gene family in Asteraceae-a comparative study in Gerbera (Mutisieae) and Sunflower (Heliantheae)[J].Molecular Biology Evolution,2012,29(4):1155-1166
[21] Danisman S,Dijk A D J Ⅴ,Bimbo A,Wal F V D,Hennig L,Folter S D,Angenent G C,Immink R G H.Analysis of functional redundancies within the Arabidopsis TCP transcription factor family[J].Journal of Experimental Botany,2013,64(18):5673-5685
[22] Kieffer M,Master Ⅴ,Waites R,Davies B.TCP14 and TCP15 affect internode length and leaf shape in Arabidopsis[J].Plant Journal for Cell & Molecular Biology,2011,68(1):147-158
[23] Li Z,Li B,Shen W H,Huang H,Dong A.TCP transcription factors interact with AS2 in the repression of class-Ⅰ KNOX genes in Arabidopsis thaliana[J].Plant Journal,2012,1:99-107
[24] Navaud O,Dabos P,Carnus E,Tremousaygue D,Hervé C.TCP transcription factors predate the emergence of land plants[J].Journal of Molecular Evolution,2007,65(1):23-33
[25] Reeves P A,Olmstead R G.Evolution of the TCP gene family in Asteridae:Cladistic and network approaches to understanding regulatory gene family diversification and its impact on morphological evolution[J].Molecular Biology and Evolution,2003,20(12):1997-2009
[26] K?lsch A,Gleissberg S.Diversification of CYCLOIDEA -like TCP genes in the basal eudicot families fumariaceae and Papaveraceae s.str[J].Plant Biology,2010,8(5):680-687
[27] Citerne H L,Luo D,Pennington R T,Coen E,Cronk Q C B.A Phylogenomic investigation of CYCLOIDEA-Like TCP genes in the Leguminosae[J].Plant Physiology,2003,131(3):1042-1053
[28] Li S.The Arabidopsis thaliana TCP transcription factors:A broadening horizon beyond development[J].Plant Signaling & Behavior,2015,10(7):e1044192
[29] Yao X,Ma H,Wang J,Zhang D.Genome-wide comparative analysis and expression pattern of TCP gene families in Arabidopsis thaliana and Oryza sativa[J].Journal of Integrative Plant Biology,2007,49(6):885-897
[30] Ma X D,Ma J C,Fan D,Li C F,Jiang Y Z,Luo K M.Genome-wide identification of TCP family transcription factors from Populus euphratica and their involvement in leaf Shape Regulation[J].Scientific Reports,2016,6:32795
[31] Parapunova V,Busscher M,Busscher-Lange J,Lammers M,Karlova R,Bovy A G,Angenent G C,de Maage R A.Identification,cloning and characterization of the tomato TCP transcription factor family[J].The BMC Plant Biology,2014,14(1):157
[32] Shi P,Guy K M,Wu W,Fang B,Yang J,Zhang M,Hu Z.Genome-wide identification and expression analysis of the ClTCP transcription factors in Citrullus lanatus[J].BMC Plant Biology,2016,16(1):85
[33] De Paolo S,Gaudio L,Aceto S.Analysis of the TCP genes expressed in the inflorescence of the orchid Orchis italica[J].Scientific Reports,2015,5(1):16265
[34] Francis A,Dhaka N,Bakshi M,Jung K H,Sharma M K,Sharma R.Corrigendum:Comparative phylogenomic analysis provides insights into TCP gene functions in Sorghum[J].Scientific Reports,2017,6(1):38488
[35] Ma J,Wang Q,Sun R,Xie F,Jones D C,Zhang B.Genome-wide identification and expression analysis of TCP transcription factors in Gossypium raimondii[J].Scientific Reports,2014,4:6645
[36] Ma J,Fang L,Wang Q,Wang K,Jones D C,Zhang B.Comprehensive analysis of TCP transcription factors and their expression during cotton (Gossypium arboreum) fiber early development[J].Scientific Reports,2016,6(1):21535
[37] Li W,Li D D,Han L H,Tao M,Hu Q Q,Wu W Y,Zhang J B,Li X B,Huang G Q.Genome-wide identification and characterization of TCP transcription factor genes in upland cotton (Gossypium hirsutum)[J].Scientific Reports,2017,7(1):10118
[38] Tran C D,Chu H D,Nguyen K H,Watanabe Y,La H Ⅴ,Tran K D,Tran L S P.Genome-wide identification of the TCP transcription factor family in Chickpea (Cicer arietinum L.) and their transcriptional responses to dehydration and exogenous abscisic acid treatments[J].Journal of Plant Growth Regulation,2018,37(4):1286-1299
[39] Guo Z H,Shu W S,Cheng H Y,Wang G M,Qi K J,Zhang S L,Gu C.Expression analysis of TCP genes in peach reveals an involvement of PpTCP.A2 in ethylene biosynthesis during fruit ripening[J].Plant Molecular Biology Reporter,2018,36(4):588-595
[40] Danisman S,van der wal F,Dhondt S,Waites R,de Folter S,Bimbo A van Dijk A D,Muino J M,Cutri L,Dornelas M C,Angenent G C,Immink R G.Arabidopsis class Ⅰ and class Ⅱ TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically[J].Plant Physiology,2012,159(4):1511-1523
[41] Aguilar-Martínez J A,Neelima S.Analysis of the role of Arabidopsis class ITCP genes AtTCP7,AtTCP8,AtTCP22,and AtTCP23 in leaf development[J].Frontiers in Plant Science,2013,4(1):406
[2] 贺超,肖小军,章智,彭琦,黄英金,廖江林.高温胁迫下水稻灌浆初期籽粒的全长cDNA文库构建及其分析[J].核农学报,2015,29(8):1437-1443
[3] Tran T,Chi Ⅴ T Q,Abdullah M,Omar H,Mostafa N,Huynh K,Suhaimi N.Construction of cDNA library and preliminary analysis of expressed sequence tags from green microalga Ankistrodesmus convolutus Corda[J].Molecular Biology Reports,2011,38(1):177-182
[4] Fields S,Song O K.A novel genetic system to detect protein-protein interactions[J].Nature,1989,340(6230):245
[5] Cubas P,Lauter N,Doebley J,Coen E.The TCP domain:A motif found in proteins regulating plant growth and development[J].Plant Journal,2010,18(2):215-222
[6] Kosugi S,Ohashi Y.PCF1 and PCF2 specifically bind to cis elements in the rice proliferating cell nuclear antigen gene[J].Plant Cell,1997,9(9):1607-1619
[7] Martíntrillo M,Cubas P.TCP genes:A family snapshot ten years later[J].Trends in Plant Science,2010,15(1):31-39
[8] Hervé C,Dabos P,Bardet C,Jauneau A,Auriac M C,Ramboer A,Lacout F,Tremousaygue D.In vivo interference with AtTCP20 function induces severe plant growth alterations and deregulates the expression of many genes important for development[J].Plant Physiology,2009,149(3):1462-1477
[9] Palatnik J F,Allen E,Wu X,Schommer C,Schwab R,Carrington J C,Weigel D.Control of leaf morphogenesis by microRNAs[J].Nature,2003,425(6955):257
[10] Pagnussat G C,Yu H J,Ngo Q A,Rajani S,Mayalagu S,Johnson C S,Capron A,Xie L F,Ye D,Sundaresan Ⅴ.Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis[J].Development,2005,132(3):603-614
[11] Takeda T,Amano K,Ohto M,Nakamura K,Sato S,Kato T,Tabata S,Ueguchi C.RNA interference of the Arabidopsis putative transcription factor TCP16 gene results in abortion of early pollen development[J].Plant Molecular Biology,2006,61(1/2):165-177
[12] Prunedapaz J L,Breton G,Para A,Kay S A.A functional genomics approach reveals CHE as a component of the arabidopsis circadian clock[J].Science,2009,323(5920):1481-1485
[13] Zheng K,Ni Z,Qu Y,Cai Y,Yang Z,Sun G,Chen Q.Genome-wide identification and expression analyses of TCP transcription factor genes in Gossypium barbadense[J].Scientific Reports,2018,8(1):14526
[14] 邱迎风,倪志勇,刘娜,陈全家,曲延英.陆地棉GhMYB9基因克隆及结合特性分析[J].核农学报,2017,31(3):440-446
[15] Hao J,Tu L,Hu H,Tan J,Deng F,Tang W,Nie Y,Zhang X.GbTCP,a cotton TCP transcription factor,confers fibre elongation and root hair development by a complex regulating system[J].Journal of Experimental Botany,2012,63(17):6267-6281
[16] Wang M Y,Xia G X.The cotton transcription factor TCP14 functions in auxin-mediated epidermal cell differentiation and elongation[J].Plant Physiology,2013,162(3):1669-1680
[17] 郑凯,倪志勇,曲延英,王怡,蔡永生,陈全家.海岛棉GbTCP15基因的克隆与表达分析[J].西北植物学报,2016,36(10):1925-1932
[18] 陈炜,赵建文,张智俊,林新春,郭小勤.竹类植物中CYP85A1基因的克隆及表达分析[J].核农学报,2019,33(4):653-663
[19] Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408
[20] T?htiharju S,Rijpkema A S,Vetterli A,Albert V A,Teeri T H,Elomaa P.Evolution and diversification of the CYC/TB1 Gene family in Asteraceae-a comparative study in Gerbera (Mutisieae) and Sunflower (Heliantheae)[J].Molecular Biology Evolution,2012,29(4):1155-1166
[21] Danisman S,Dijk A D J Ⅴ,Bimbo A,Wal F V D,Hennig L,Folter S D,Angenent G C,Immink R G H.Analysis of functional redundancies within the Arabidopsis TCP transcription factor family[J].Journal of Experimental Botany,2013,64(18):5673-5685
[22] Kieffer M,Master Ⅴ,Waites R,Davies B.TCP14 and TCP15 affect internode length and leaf shape in Arabidopsis[J].Plant Journal for Cell & Molecular Biology,2011,68(1):147-158
[23] Li Z,Li B,Shen W H,Huang H,Dong A.TCP transcription factors interact with AS2 in the repression of class-Ⅰ KNOX genes in Arabidopsis thaliana[J].Plant Journal,2012,1:99-107
[24] Navaud O,Dabos P,Carnus E,Tremousaygue D,Hervé C.TCP transcription factors predate the emergence of land plants[J].Journal of Molecular Evolution,2007,65(1):23-33
[25] Reeves P A,Olmstead R G.Evolution of the TCP gene family in Asteridae:Cladistic and network approaches to understanding regulatory gene family diversification and its impact on morphological evolution[J].Molecular Biology and Evolution,2003,20(12):1997-2009
[26] K?lsch A,Gleissberg S.Diversification of CYCLOIDEA -like TCP genes in the basal eudicot families fumariaceae and Papaveraceae s.str[J].Plant Biology,2010,8(5):680-687
[27] Citerne H L,Luo D,Pennington R T,Coen E,Cronk Q C B.A Phylogenomic investigation of CYCLOIDEA-Like TCP genes in the Leguminosae[J].Plant Physiology,2003,131(3):1042-1053
[28] Li S.The Arabidopsis thaliana TCP transcription factors:A broadening horizon beyond development[J].Plant Signaling & Behavior,2015,10(7):e1044192
[29] Yao X,Ma H,Wang J,Zhang D.Genome-wide comparative analysis and expression pattern of TCP gene families in Arabidopsis thaliana and Oryza sativa[J].Journal of Integrative Plant Biology,2007,49(6):885-897
[30] Ma X D,Ma J C,Fan D,Li C F,Jiang Y Z,Luo K M.Genome-wide identification of TCP family transcription factors from Populus euphratica and their involvement in leaf Shape Regulation[J].Scientific Reports,2016,6:32795
[31] Parapunova V,Busscher M,Busscher-Lange J,Lammers M,Karlova R,Bovy A G,Angenent G C,de Maage R A.Identification,cloning and characterization of the tomato TCP transcription factor family[J].The BMC Plant Biology,2014,14(1):157
[32] Shi P,Guy K M,Wu W,Fang B,Yang J,Zhang M,Hu Z.Genome-wide identification and expression analysis of the ClTCP transcription factors in Citrullus lanatus[J].BMC Plant Biology,2016,16(1):85
[33] De Paolo S,Gaudio L,Aceto S.Analysis of the TCP genes expressed in the inflorescence of the orchid Orchis italica[J].Scientific Reports,2015,5(1):16265
[34] Francis A,Dhaka N,Bakshi M,Jung K H,Sharma M K,Sharma R.Corrigendum:Comparative phylogenomic analysis provides insights into TCP gene functions in Sorghum[J].Scientific Reports,2017,6(1):38488
[35] Ma J,Wang Q,Sun R,Xie F,Jones D C,Zhang B.Genome-wide identification and expression analysis of TCP transcription factors in Gossypium raimondii[J].Scientific Reports,2014,4:6645
[36] Ma J,Fang L,Wang Q,Wang K,Jones D C,Zhang B.Comprehensive analysis of TCP transcription factors and their expression during cotton (Gossypium arboreum) fiber early development[J].Scientific Reports,2016,6(1):21535
[37] Li W,Li D D,Han L H,Tao M,Hu Q Q,Wu W Y,Zhang J B,Li X B,Huang G Q.Genome-wide identification and characterization of TCP transcription factor genes in upland cotton (Gossypium hirsutum)[J].Scientific Reports,2017,7(1):10118
[38] Tran C D,Chu H D,Nguyen K H,Watanabe Y,La H Ⅴ,Tran K D,Tran L S P.Genome-wide identification of the TCP transcription factor family in Chickpea (Cicer arietinum L.) and their transcriptional responses to dehydration and exogenous abscisic acid treatments[J].Journal of Plant Growth Regulation,2018,37(4):1286-1299
[39] Guo Z H,Shu W S,Cheng H Y,Wang G M,Qi K J,Zhang S L,Gu C.Expression analysis of TCP genes in peach reveals an involvement of PpTCP.A2 in ethylene biosynthesis during fruit ripening[J].Plant Molecular Biology Reporter,2018,36(4):588-595
[40] Danisman S,van der wal F,Dhondt S,Waites R,de Folter S,Bimbo A van Dijk A D,Muino J M,Cutri L,Dornelas M C,Angenent G C,Immink R G.Arabidopsis class Ⅰ and class Ⅱ TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically[J].Plant Physiology,2012,159(4):1511-1523
[41] Aguilar-Martínez J A,Neelima S.Analysis of the role of Arabidopsis class ITCP genes AtTCP7,AtTCP8,AtTCP22,and AtTCP23 in leaf development[J].Frontiers in Plant Science,2013,4(1):406