马铃薯R2R3-MYB转录因子的克隆及植物表达载体的构建
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摘要
MYB转录因子家族是植物转录因子中最大的家族之一,广泛参与植物初生和次生代谢调控、细胞形态的建成、环境胁迫的应答等。前期转录组测序表明抗晚疫病的马铃薯材料加湘1号接种晚疫病菌后其MYB基因家族中的一个R2R3-MYB转录因子PGSC0003DMG400011243的表达量显著上调。为了探讨R2R3-MYB转录因子PGSC0003DMG400011243在马铃薯晚疫病抗病过程中发挥的作用。本研究通过PCR扩增了加湘1号中的该基因,并通过XcmⅠ酶切、连接将其装载到pCXSN载体上,构建了该基因的超表达载体pCXSN-JX-R2R3,并转化到农杆菌GV3101中。该研究为R2R3-MYB转录因子的基因转化及后续的功能分析提供了理论指导。
The MYB transcription factor family is one of the largest family of plant transcription factors which widely involved in the regulation of primary and secondary metabolism, cell morphogenesis, response to environmental stresses and so on. In previous RNA-Seq analysis, the expression of a R2 R3-MYB transcription factor PGSC0003 DMG400011243 from the MYB gene family increased significantly after potato material Jiaxiang No.1 with resistance to late blight was inoculated with Phytophthora infestans. In order to investigate the role of R2 R3-MYB transcription factor PGSC0003 DMG400011243 in the resistance of potato late blight. The gene was amplified by PCR from Jiaxiang No.1 DNA and cloned into plasmid pCXSN by Xcm Ⅰ enzyme digestion and linkage. The overexpression vector pCXSN-JX-R2 R3 was constructed and transformed into Agrobacterium GV3101. The study might lay the foundation for the gene transformation of R2 R3-MYB transcription factor and further functional analysis.
The MYB transcription factor family is one of the largest family of plant transcription factors which widely involved in the regulation of primary and secondary metabolism, cell morphogenesis, response to environmental stresses and so on. In previous RNA-Seq analysis, the expression of a R2 R3-MYB transcription factor PGSC0003 DMG400011243 from the MYB gene family increased significantly after potato material Jiaxiang No.1 with resistance to late blight was inoculated with Phytophthora infestans. In order to investigate the role of R2 R3-MYB transcription factor PGSC0003 DMG400011243 in the resistance of potato late blight. The gene was amplified by PCR from Jiaxiang No.1 DNA and cloned into plasmid pCXSN by Xcm Ⅰ enzyme digestion and linkage. The overexpression vector pCXSN-JX-R2 R3 was constructed and transformed into Agrobacterium GV3101. The study might lay the foundation for the gene transformation of R2 R3-MYB transcription factor and further functional analysis.
引文
Bedon F.,Bomal C.,Caron S.,Levasseur C.,Boyle B.,Mansfield S.D.,Schmidt A.,Gershenzon J.,Grima-Pettenati J.,Seguin A.,and MacKay J.,2010,Subgroup 4 R2R3-MYBs in conifer trees:gene family expansion and contribution to the isoprenoid and flavonoid-oriented responses,J.Exp.Bot.,61(14):3847-3864
Chen S.C.,Peng S.Q.,Huang G.X.,Wu K.X.,Fu X.H.,and Chen Z.Q.,2003,Association of decreased expression of a Myb transcription factor with the TPD(tapping panel dryness)syndrome in Hevea brasiliensis,Plant Mol.Biol.,51(1):51-58
Chen Y.H.,Yang X.Y.,He K.,Liu M.H.,Li J.G.,Gao Z.F.,Lin Z.Q.,Zhang Y.F.,Wang X.X.,Qiu X.M.,Shen Y.P.,Zhang L.,Deng X.H.,Luo J.C.,Deng X.W.,Chen Z.L.,Gu H.Y.,and Qu L.J.,2006,The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family,Plant Mol.Biol.,60(1):107-124
Doyle J.J.,and Doyle J.L.,1987,A rapid DNA isolation procedure for small quantities of leaf tissue,Phytochem.Bull.,19(1):11-15
Du H.,Yang W.J.,Liu L.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.,2008,Cloning and functional identification of the two MYB transcription factors GmMYBJ6 and GmMYBJ7in soybean,Zuowu Xuebao(Acta Agronomica Sinica),34(7):1179-1187(杜海,杨文杰,刘蕾,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,大豆MYB转录因子基因GmMYBJ6和GmMYBJ7的克隆及表达分析,作物学报,34(7):1179-1187)
Dubos C.,Le G.J.,Baudry A.,Huep G.,Lanet E.,Debeaujon I.,Routaboul J.M.,Alboresi A.,Weisshaar B.,and Lepiniec L.,2008,MYBL2 is a new regulator of flavonoid biosynthesisin Arabidopsis thaliana,Plant J.,55(6):940-953
Fry W.,2008,Phytophthora infestans:the plant(and R gene)destroyer,Mol.Plant Pathol.,9(3):385-402
Hoeren F.U.,Dolferus R.,Wu Y.,Peacock W.J.,and Dennis E.S.,1998,Evidence for a role for At MYB2 in the induction of the Arabidopsis alcohol dehydrogenase gene(ADH1)by low oxygen,Genetics,149(2):479-490
Huang Y.J.,Deng R.Y.,Gao F.,Luo X.P.,and Wu Q.,2015,Cloning and expression analysis of transcription factor gene FtMYB21 from tartary buckwheat under abiotic stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9):1939-1945(黄云吉,邓仁榆,高飞,雒晓鹏,吴琦,2015,苦荞转录因子基因FtMYB21的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学,34(9):1939-1945)
Huang Z.J.,Huang R.F.,and Huang D.F.,2004,ERF transcription factor and their roles in plant defense responses,Zhiwu Bingli Xuebao(Acta Phytopathologica Sinica),34(3):193-198(黄泽军,黄荣峰,黄大昉,2004,ERF转录因子及其在植物防卫反应中的作用,植物病理学报,34(3):193-198)
Koes R.,Verweij W.,and Quattrocchio F.,2005,Flavonoids:a colorful model for the regulation and evolution of biochemical pathways,Trends Plant Sci.,10(5):236-242
Lacombe E.,Doorsselaere J.V.,Boerjan W.,Boudet A.M.,and Grima-Pettenati J.,2000,Characterization of cis-elements required for vascular expression of the cinnamoyl coa reductase gene and for protein-DNA complex formation,Plant J.,23(5):663-676
Lea U.S.,Slimestad R.,Smedvig P.,and Lillo C.,2007,Nitrogen deficiency enhances expression of specific MYB and bHLHtranscription factors and accumulation of end products in the flavonoid pathway,Planta,225(5):1245-1253
Lee M.M.,and Schiefelbein J.,2002,Cell pattern in the Arabidopsis root epidermis determined by lateral inhibition with feedback,Plant Cell,14(3):611-618
Legay S.,Lacombe E.,Goicoechea M.,Brière C.,Sèguin A.,Mackay J.,and Grima-Pettenati J.,2007,Molecular characterization of EgM YB1,a putative transcriptional repressor of the lignin biosynthetic pathway,Plant Sci.,173(5):542-549
Li W.J.,Qin J.H.,Gu J.M.,Qiu Z.J.,Zhou J.,Xie K.Y.,and Lu X.P.,2015,Developing potato as a staple food in China based on world potato development,Zhongguo Shiwu Yu Yingyang(Food and Nutrition in China),21(7):5-9(李文娟,秦军红,谷建苗,仇志军,周俊,谢开云,卢肖平,2015,从世界马铃薯产业发展谈中国马铃薯的主粮化,中国食物与营养,21(7):5-9)
Mu H.M.,Du X.J.,Zhang X.S.,Zhang M.,and Cao X.,2015,Study on plants MYB transcription factors regulate biologi-cal synthesis of phenylpropanoid metabolism,Beifang Yuanyi(Northern Horticulture),(24):171-174(穆红梅,杜秀菊,张秀省,张敏,曹兴,2015,植物MYB转录因子调控苯丙烷类生物合成研究,北方园艺,(24):171-174)
Patzlaff A.,Newman L.J.,Dubos C.,Whetten R.W.,Smith C.,McInnis S.,Bevan M.W.,Sederoff R.R.,and Campbell M.M.,2003,Characterisation of Pt MYB1,an R2R3-MYBfrom pine xylem,Plant Mol.Biol.,53(4):597-608
Uimari A.,and Strommer J.,1997,Myb26:a MYB-like protein of pea flowers with affinity for promoters of phenylpropanoid genes,Plant J.,12(6):1273-1284
Yang X.Y.,Li J.G.,Pei M.,Gu H.,Chen Z.L.,and Qu L.J.,2007,Over-expression of a flower-specific transcription factor gene AtMYB24 causes aberrant another development,Plant Cell Rep.,26(2):219-228
Zhong R.,Lee C.H.,Richardson E.A.,and Ye Z.H.,2007,The MYB46 transcription factor is a direct target of SND1 and regulates secondary wall biosynthesis in Arabidopsis,Plant Cell,19(9):2776-2792
Zhou J.,Lee C.,Zhong R.,and Ye Z.H.,2009,MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis,Plant Cell,21(1):248-266
Zhu X.Y.,Cui C.,Zhou H.J.,Wu Y.C.,and Wang Z.Z.,2015,Cloning and expression analysis of the transcription factor SmMYB7 from Salvia miltiorrhiza bunge,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(2):365-372(朱小亚,崔璀,周宏骏,武玉翠,王喆之,2015,丹参转录因子SmMYB7的克隆及表达模式分析,基因组学与应用生物学,34(2):365-372)
Chen S.C.,Peng S.Q.,Huang G.X.,Wu K.X.,Fu X.H.,and Chen Z.Q.,2003,Association of decreased expression of a Myb transcription factor with the TPD(tapping panel dryness)syndrome in Hevea brasiliensis,Plant Mol.Biol.,51(1):51-58
Chen Y.H.,Yang X.Y.,He K.,Liu M.H.,Li J.G.,Gao Z.F.,Lin Z.Q.,Zhang Y.F.,Wang X.X.,Qiu X.M.,Shen Y.P.,Zhang L.,Deng X.H.,Luo J.C.,Deng X.W.,Chen Z.L.,Gu H.Y.,and Qu L.J.,2006,The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family,Plant Mol.Biol.,60(1):107-124
Doyle J.J.,and Doyle J.L.,1987,A rapid DNA isolation procedure for small quantities of leaf tissue,Phytochem.Bull.,19(1):11-15
Du H.,Yang W.J.,Liu L.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.,2008,Cloning and functional identification of the two MYB transcription factors GmMYBJ6 and GmMYBJ7in soybean,Zuowu Xuebao(Acta Agronomica Sinica),34(7):1179-1187(杜海,杨文杰,刘蕾,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,大豆MYB转录因子基因GmMYBJ6和GmMYBJ7的克隆及表达分析,作物学报,34(7):1179-1187)
Dubos C.,Le G.J.,Baudry A.,Huep G.,Lanet E.,Debeaujon I.,Routaboul J.M.,Alboresi A.,Weisshaar B.,and Lepiniec L.,2008,MYBL2 is a new regulator of flavonoid biosynthesisin Arabidopsis thaliana,Plant J.,55(6):940-953
Fry W.,2008,Phytophthora infestans:the plant(and R gene)destroyer,Mol.Plant Pathol.,9(3):385-402
Hoeren F.U.,Dolferus R.,Wu Y.,Peacock W.J.,and Dennis E.S.,1998,Evidence for a role for At MYB2 in the induction of the Arabidopsis alcohol dehydrogenase gene(ADH1)by low oxygen,Genetics,149(2):479-490
Huang Y.J.,Deng R.Y.,Gao F.,Luo X.P.,and Wu Q.,2015,Cloning and expression analysis of transcription factor gene FtMYB21 from tartary buckwheat under abiotic stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(9):1939-1945(黄云吉,邓仁榆,高飞,雒晓鹏,吴琦,2015,苦荞转录因子基因FtMYB21的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学,34(9):1939-1945)
Huang Z.J.,Huang R.F.,and Huang D.F.,2004,ERF transcription factor and their roles in plant defense responses,Zhiwu Bingli Xuebao(Acta Phytopathologica Sinica),34(3):193-198(黄泽军,黄荣峰,黄大昉,2004,ERF转录因子及其在植物防卫反应中的作用,植物病理学报,34(3):193-198)
Koes R.,Verweij W.,and Quattrocchio F.,2005,Flavonoids:a colorful model for the regulation and evolution of biochemical pathways,Trends Plant Sci.,10(5):236-242
Lacombe E.,Doorsselaere J.V.,Boerjan W.,Boudet A.M.,and Grima-Pettenati J.,2000,Characterization of cis-elements required for vascular expression of the cinnamoyl coa reductase gene and for protein-DNA complex formation,Plant J.,23(5):663-676
Lea U.S.,Slimestad R.,Smedvig P.,and Lillo C.,2007,Nitrogen deficiency enhances expression of specific MYB and bHLHtranscription factors and accumulation of end products in the flavonoid pathway,Planta,225(5):1245-1253
Lee M.M.,and Schiefelbein J.,2002,Cell pattern in the Arabidopsis root epidermis determined by lateral inhibition with feedback,Plant Cell,14(3):611-618
Legay S.,Lacombe E.,Goicoechea M.,Brière C.,Sèguin A.,Mackay J.,and Grima-Pettenati J.,2007,Molecular characterization of EgM YB1,a putative transcriptional repressor of the lignin biosynthetic pathway,Plant Sci.,173(5):542-549
Li W.J.,Qin J.H.,Gu J.M.,Qiu Z.J.,Zhou J.,Xie K.Y.,and Lu X.P.,2015,Developing potato as a staple food in China based on world potato development,Zhongguo Shiwu Yu Yingyang(Food and Nutrition in China),21(7):5-9(李文娟,秦军红,谷建苗,仇志军,周俊,谢开云,卢肖平,2015,从世界马铃薯产业发展谈中国马铃薯的主粮化,中国食物与营养,21(7):5-9)
Mu H.M.,Du X.J.,Zhang X.S.,Zhang M.,and Cao X.,2015,Study on plants MYB transcription factors regulate biologi-cal synthesis of phenylpropanoid metabolism,Beifang Yuanyi(Northern Horticulture),(24):171-174(穆红梅,杜秀菊,张秀省,张敏,曹兴,2015,植物MYB转录因子调控苯丙烷类生物合成研究,北方园艺,(24):171-174)
Patzlaff A.,Newman L.J.,Dubos C.,Whetten R.W.,Smith C.,McInnis S.,Bevan M.W.,Sederoff R.R.,and Campbell M.M.,2003,Characterisation of Pt MYB1,an R2R3-MYBfrom pine xylem,Plant Mol.Biol.,53(4):597-608
Uimari A.,and Strommer J.,1997,Myb26:a MYB-like protein of pea flowers with affinity for promoters of phenylpropanoid genes,Plant J.,12(6):1273-1284
Yang X.Y.,Li J.G.,Pei M.,Gu H.,Chen Z.L.,and Qu L.J.,2007,Over-expression of a flower-specific transcription factor gene AtMYB24 causes aberrant another development,Plant Cell Rep.,26(2):219-228
Zhong R.,Lee C.H.,Richardson E.A.,and Ye Z.H.,2007,The MYB46 transcription factor is a direct target of SND1 and regulates secondary wall biosynthesis in Arabidopsis,Plant Cell,19(9):2776-2792
Zhou J.,Lee C.,Zhong R.,and Ye Z.H.,2009,MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis,Plant Cell,21(1):248-266
Zhu X.Y.,Cui C.,Zhou H.J.,Wu Y.C.,and Wang Z.Z.,2015,Cloning and expression analysis of the transcription factor SmMYB7 from Salvia miltiorrhiza bunge,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(2):365-372(朱小亚,崔璀,周宏骏,武玉翠,王喆之,2015,丹参转录因子SmMYB7的克隆及表达模式分析,基因组学与应用生物学,34(2):365-372)