GhMYB4基因过量表达载体的构建及拟南芥转化
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摘要
作为广泛参与植物体内的多种生理生化反应的MYB转录因子是植物中最大的转录因子家族之一,为阐明GhMYB4基因在棉纤维发育过程中的功能,本研究构建GhMYB4基因过量表达载体,以pGEM-T Easy-GhMYB4质粒为模板进行PCR扩增,成功将该基因构建到植物表达载体pEGAD中,并利用冻融法转入农杆菌GV3101菌株,通过农杆菌浸染法转化拟南芥植株,经过含草甘膦成分的除草剂筛选和PCR检测初步证明已获得转GhMYB4基因的拟南芥。为后期探讨转化棉花GhMYB4基因的生物学功能和棉花纤维发育机制提供理论参考。
MYB transcription factors, which are involved in many physiological and biochemical reactions in plants, are one of the largest transcription factors in plants. In order to elucidate the function of GhMYB4 gene in cotton fiber development. In this study, the overexpression vector of GhMYB4 gene was constructed using pGEM-T Easy-GhMYB4 plasmid as template for PCR amplification. The gene was successfully constructed into the plant expression vector p EGAD. Then, it was transformed into Agrobacterium tumefaciens GV3101 strain by freezing and thawing method, and transformed into Arabidopsis thaliana plant by Agrobacterium tumefaciens soaking method. After herbicide containing glyphosate screening and PCR detection, it was preliminarily proved that the Arabidopsis thaliana transformed with GhMYB4 was obtained. It would provide a theoretical reference for the later study on the biological function and cotton fiber development mechanism of transformed cotton GhMYB4 gene.
MYB transcription factors, which are involved in many physiological and biochemical reactions in plants, are one of the largest transcription factors in plants. In order to elucidate the function of GhMYB4 gene in cotton fiber development. In this study, the overexpression vector of GhMYB4 gene was constructed using pGEM-T Easy-GhMYB4 plasmid as template for PCR amplification. The gene was successfully constructed into the plant expression vector p EGAD. Then, it was transformed into Agrobacterium tumefaciens GV3101 strain by freezing and thawing method, and transformed into Arabidopsis thaliana plant by Agrobacterium tumefaciens soaking method. After herbicide containing glyphosate screening and PCR detection, it was preliminarily proved that the Arabidopsis thaliana transformed with GhMYB4 was obtained. It would provide a theoretical reference for the later study on the biological function and cotton fiber development mechanism of transformed cotton GhMYB4 gene.
引文
Du X.M.,Pan J.J.,and Wang R.H.,2000,Differentiation and development of cotton fiber cells,Mianhua Xuebao(Acta Gossypii Sinia),12(4):212-217(杜雄明,潘家驹,汪若海,2000,棉纤维细胞分化和发育,棉花学报,12(4):212-217)
Froidure S.,Roby D.,and Rivas S.,2010,Expression of the Arabidopsis transcription factor AtMYB30 is post-transcriptionally regulated,Plant Physiol.Biochem.,48(8):735-739
Guan X.Y.,Pang M.X.,Nah G.,Shi X.L.,Ye W.X.,Stell D.M.,and Chen Z.J.,2014,mi R828 and mi R858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development,Nat.Commun.,5(1):3050-3064
Hülskamp M.,Miséra S.,and Jürgens A.G.,1994,Genetic dissection of trichome cell development in Arabidopsis,Cell,76(3):555-566
Machado A.,Wu Y.,Yang Y.,Llewellyn D.J.,and Dennis E.S.,2009,The MYB transcription factor GhMYB25 regulates early fibre and trichome development,Plant J.,59(1):52-62
Niu Y.L.,Jiang X.M.,and Xu X.Y.,2016,Advances in the study of MYB gene family of plant transcription factor,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(8):2050-2059(牛义岭,姜秀明,许向阳,2016,植物转录因子MYB基因家族的研究进展,分子植物育种,14(8):2050-2059)
Poethig S.,1989,Genetic mosaics and cell lineage analysis in plants,Trends in Genetics,5(8):273-277
Qiu Y.F.,2016,The expression pattern analysis of three MYBtranscription factors and functional verification of GhMYB-4a,Xinjiang Agricultural University,Supervisor:Qu Y.Y.,pp.34(邱迎风,2016,三个棉花MYB转录因子的表达特性及GhMYB4a的功能验证,新疆农业大学,导师:曲延英,pp.34)
Rosinski J.A.,and Atchley W.R.,1998,Molecular evolution of the MYB family of transcription factors:evidence for polyphyletic origin,Journal of Molecular Evolution,46(1):74-83
Schwab R.,Ossowski S.,Riester M.,Warthmann N.,and Weigel D.,2006,Highly specific gene silencing by artificial microRNAs in Arabidopsis,Plant Cell,18(5):1121-1133
Smith L.G.,and Hake S.,1992,The initiation and determination of leaves,Plant Cell,4(9):1017-1027
Stracke R.,Werber M.,and Weisshaar B.,2001,The R2R3-MYBgene family in Arabidopsis thaliana,Curr.Opin.Plant Biol.,4(5):447-456
Walford S.A.,Wu Y.,Llewellyn D.J.,and Dennis E.S.,2011,GhMYB25-like:a key factor in early cotton fibre development,Plant J.,65(5):785-797
Wang L.W.,Chen H.Z.,and Zhang X.X.,2015,Interaction between MYB protein and target DNA binding sites in plants,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(4):946-950(王丽文,陈红忠,张欣欣,2015,植物MYB蛋白与相应靶DNA结合位点间的相互作用,分子植物育种,13(4):946-950)
Wang N.H.,Yu W.W.,Wu M.,Feng M.Q.,Li L.X.,Feng P.W.,Jing H.,Huang S.L.,Zhang J.F.,and Yu S.X.,2014,Cloning,expression analysis and functional identification of GhMYB0gene in cotton,Zuowu Xuebao(Acta Agronomica Sinica),40(9):1540-1548(王诺菡,于霁雯,吴嫚,马启锋,裴文锋,李海晶,黄双领,张金发,喻树迅,2014,棉花GhMYB0基因的克隆、表达分析及功能鉴定,作物学报,40(9):1540-1548)
Wang S.,Wang J.W.,Yu N.,Li C.H.,Luo B.,Gou J.Y.,Wang L.J.,and Chen X.Y.,2004,Control of plant trichome development by a cotton fiber MYB gene,Plant Cell,16(9):2323-2334
Yang W.J.,Du H.,Fang F.,Yang W.S.,Wu Y.M.,and Tang Y.X.,2008,Cloning and characterization of two new MYB transcription factor genes from soybean,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),41(4):961-970(杨文杰,杜海,方芳,杨婉身,吴燕民,唐益雄,2008,大豆两个MYB转录因子基因的克隆及表达分析,中国农业科学,41(4):961-970)
Zhang H.,Tang W.K.,Tan X.,Gong L.L.,and Li X.B.,2007,Progresses in the study of gene regulation of cotton fiber development,Zhiwu Xuebao(Chinese Bulletin of Botany),24(2):127-133(张辉,汤文开,谭新,龚路路,李学宝,2007,棉纤维发育及其相关基因表达调控研究进展,植物学报,24(2):127-133)
Zhao K.M.,and Bartley L.E.,2014,Comparative genomic analysis of the R2R3 MYB secondary cell wall regulators of Arabidopsis,Poplar,Rice,Maize,and Switchgrass,BMC Plant Biol.,14:135
Froidure S.,Roby D.,and Rivas S.,2010,Expression of the Arabidopsis transcription factor AtMYB30 is post-transcriptionally regulated,Plant Physiol.Biochem.,48(8):735-739
Guan X.Y.,Pang M.X.,Nah G.,Shi X.L.,Ye W.X.,Stell D.M.,and Chen Z.J.,2014,mi R828 and mi R858 regulate homoeologous MYB2 gene functions in Arabidopsis trichome and cotton fibre development,Nat.Commun.,5(1):3050-3064
Hülskamp M.,Miséra S.,and Jürgens A.G.,1994,Genetic dissection of trichome cell development in Arabidopsis,Cell,76(3):555-566
Machado A.,Wu Y.,Yang Y.,Llewellyn D.J.,and Dennis E.S.,2009,The MYB transcription factor GhMYB25 regulates early fibre and trichome development,Plant J.,59(1):52-62
Niu Y.L.,Jiang X.M.,and Xu X.Y.,2016,Advances in the study of MYB gene family of plant transcription factor,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(8):2050-2059(牛义岭,姜秀明,许向阳,2016,植物转录因子MYB基因家族的研究进展,分子植物育种,14(8):2050-2059)
Poethig S.,1989,Genetic mosaics and cell lineage analysis in plants,Trends in Genetics,5(8):273-277
Qiu Y.F.,2016,The expression pattern analysis of three MYBtranscription factors and functional verification of GhMYB-4a,Xinjiang Agricultural University,Supervisor:Qu Y.Y.,pp.34(邱迎风,2016,三个棉花MYB转录因子的表达特性及GhMYB4a的功能验证,新疆农业大学,导师:曲延英,pp.34)
Rosinski J.A.,and Atchley W.R.,1998,Molecular evolution of the MYB family of transcription factors:evidence for polyphyletic origin,Journal of Molecular Evolution,46(1):74-83
Schwab R.,Ossowski S.,Riester M.,Warthmann N.,and Weigel D.,2006,Highly specific gene silencing by artificial microRNAs in Arabidopsis,Plant Cell,18(5):1121-1133
Smith L.G.,and Hake S.,1992,The initiation and determination of leaves,Plant Cell,4(9):1017-1027
Stracke R.,Werber M.,and Weisshaar B.,2001,The R2R3-MYBgene family in Arabidopsis thaliana,Curr.Opin.Plant Biol.,4(5):447-456
Walford S.A.,Wu Y.,Llewellyn D.J.,and Dennis E.S.,2011,GhMYB25-like:a key factor in early cotton fibre development,Plant J.,65(5):785-797
Wang L.W.,Chen H.Z.,and Zhang X.X.,2015,Interaction between MYB protein and target DNA binding sites in plants,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),13(4):946-950(王丽文,陈红忠,张欣欣,2015,植物MYB蛋白与相应靶DNA结合位点间的相互作用,分子植物育种,13(4):946-950)
Wang N.H.,Yu W.W.,Wu M.,Feng M.Q.,Li L.X.,Feng P.W.,Jing H.,Huang S.L.,Zhang J.F.,and Yu S.X.,2014,Cloning,expression analysis and functional identification of GhMYB0gene in cotton,Zuowu Xuebao(Acta Agronomica Sinica),40(9):1540-1548(王诺菡,于霁雯,吴嫚,马启锋,裴文锋,李海晶,黄双领,张金发,喻树迅,2014,棉花GhMYB0基因的克隆、表达分析及功能鉴定,作物学报,40(9):1540-1548)
Wang S.,Wang J.W.,Yu N.,Li C.H.,Luo B.,Gou J.Y.,Wang L.J.,and Chen X.Y.,2004,Control of plant trichome development by a cotton fiber MYB gene,Plant Cell,16(9):2323-2334
Yang W.J.,Du H.,Fang F.,Yang W.S.,Wu Y.M.,and Tang Y.X.,2008,Cloning and characterization of two new MYB transcription factor genes from soybean,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),41(4):961-970(杨文杰,杜海,方芳,杨婉身,吴燕民,唐益雄,2008,大豆两个MYB转录因子基因的克隆及表达分析,中国农业科学,41(4):961-970)
Zhang H.,Tang W.K.,Tan X.,Gong L.L.,and Li X.B.,2007,Progresses in the study of gene regulation of cotton fiber development,Zhiwu Xuebao(Chinese Bulletin of Botany),24(2):127-133(张辉,汤文开,谭新,龚路路,李学宝,2007,棉纤维发育及其相关基因表达调控研究进展,植物学报,24(2):127-133)
Zhao K.M.,and Bartley L.E.,2014,Comparative genomic analysis of the R2R3 MYB secondary cell wall regulators of Arabidopsis,Poplar,Rice,Maize,and Switchgrass,BMC Plant Biol.,14:135