芒果MinMYBPA基因的克隆及载体的构建
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摘要
MYB (Myeloblastosis)是植物最大的转录因子家族之一,广泛参与植物代谢调控。为了研究其在芒果果皮中花青苷合成的作用机理,本研究从芒果果皮中克隆得到一个与花青苷合成相关的基因,将其命名为MinMYBPA,其cDNA全长序列为1 081 bp,开放阅读框为843 bp,编码280个氨基酸,蛋白质分子量为32.09 k D,等电点为9.01,对MinMYBPA基因编码的蛋白进行系统发育分析,发现其与克莱门柚具有较近的亲缘关系。本研究为深入了解其在芒果花青素合成和积累的分子机理,进一步构建了pTRV2-MinMYBPA沉默表达和pGreenⅡ62-SK-MinMYBPA过表达载体,为MinMYBPA的功能鉴定提供参考依据。
MYB(Myeloblastosis) is one of the largest transcriptional factor families in plants and is widely involved in the regulation of plant metabolism. In order to study the mechanism of anthocyanin synthesis in mango peel, a gene related to anthocyanin synthesis was cloned from mango peel. It was named MinMYBPA and the full-length c DNA sequence was 1 081 bp. Its open reading frame was 843 bp, encoding 280 amino acids. The molecular weight of MinMYBPA protein was 32.09 kD and the isoelectric point was 9.01. Phylogenetic analysis of the protein encoded by MinMYBPA gene showed that it was closely related to Clement pomelo. In order to understand the molecular mechanism of anthocyanin synthesis and accumulation of MinMYBPA in mango, the silencing expression vector of pTRV2-MinMYBPA and the overexpression vector of pGreenⅡ 62-SK-MinMYBPA were constructed, which laid a theoretical foundation for the functional identification of MinMYBPA.
MYB(Myeloblastosis) is one of the largest transcriptional factor families in plants and is widely involved in the regulation of plant metabolism. In order to study the mechanism of anthocyanin synthesis in mango peel, a gene related to anthocyanin synthesis was cloned from mango peel. It was named MinMYBPA and the full-length c DNA sequence was 1 081 bp. Its open reading frame was 843 bp, encoding 280 amino acids. The molecular weight of MinMYBPA protein was 32.09 kD and the isoelectric point was 9.01. Phylogenetic analysis of the protein encoded by MinMYBPA gene showed that it was closely related to Clement pomelo. In order to understand the molecular mechanism of anthocyanin synthesis and accumulation of MinMYBPA in mango, the silencing expression vector of pTRV2-MinMYBPA and the overexpression vector of pGreenⅡ 62-SK-MinMYBPA were constructed, which laid a theoretical foundation for the functional identification of MinMYBPA.
引文
Albert N.W.,Davies K.M.,Lewis D.H.,Zhang H.,Montefiori M.,Brendolise C.,Boase M.R.,Ngo H.,Jameson P.E.,and Schwinn K.E.,2014,A conserved network of transcriptional activators and repressors regulates anthocyanin pigmentation,Plant Cell,26(3):962-980
Cedroni M.L.,Cronn R.C.,Adams K.L.,Wilkins T.A.,and Wendel J.F.,2003,Evoluti on and expression of MYB genes in diploid and polyploid cotton,Plant Mol.Biol.,51(3):313-325
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
Du H.,Yang S.S.,Liang Z.,Feng B.R.,Liu L.,Huang Y.B.,and Tang Y.X.,2012,Genome-wide analysis of the MYB transcription factor super-family in soybean,BMC Plant Biol.,12(1):106
Dubos C.,Stracke R.,Grotewold E.,Weisshaar B.,Martin C.,and Lepiniec L.,2010,MYB transcription factors in Araidopsis,Trends Plant Sci.,15(10):573-581
Feng S.Q.,Wang Y.L.,Yang S.,Xu Y.T.,and Chen X.S.,2010,Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor Py MYB10,Planta,232(1):245-255
Fournier-Level A.,Lacombe T.,Le Cunff L.,Boursiquot J.M.,and This P.,2010,Evolution of the Vv MybA gene family,the major determinant of berry colour in cultivated grapevine(Vitis vinifera L.),Heredity,104(4):351-362
Hichri I.,Barrieu F.,Bogs J.,Kappel C.,Delrot S.,and Lauvergeat V.,2011,Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway,J.Exp.Bot.,62(8):2465-2483
Jaakola L.,2013,New insights into the regulation of anthocyanin bio-synthesis in fruits,Trends Plant Sci.,18(9):477-483
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
Lin-Wang K.,Micheletti D.,Palmer J.,Volz R.,Lozano L.,Espley R.,Hellens R.P.,ChagnèD.,Rowan D.D.,Troggio M.,Iglesias I.,and Allan A.C.,2011,High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex,Plant Cell Environ.,34(7):1176-1190
Luo X.B.,2011,Nutritional value,health function and edible method of mango,Zhongguo Shiwu Yu Yingyang(Chinese Food and Nutrition),17(7):77-79(罗学兵,2011,芒果的营养价值、保健功能及食用方法,中国食物与营养,17(7):77-79)
Matus J.T.,Aquea F.,and Arce-Johnson P.,2008,Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related cladesand conserved gene structure organization across Vitis and Arabidopsis genomes,BMC Plant Biol.,8(1):83
Palapol Y.,Ketsa S.,Lin-Wang K.,Ferguson I.B.,and Allan A.C.,2009,A MYB transcription factor regulates anthocyanin biosynthesis in mangosteen(Garcinia mangostana L.)fruit during ripening,Planta,229(6):1323-1334
Petroni K.,and Tonelli C.,2011,Recent advances on the regulation of anthocyanin synthesis in reproductive organs,Plant Sci.,181(3):219-229
Rabinowicz P.D.,Braun E.L.,Wolfe A.D.,Bowen B.,and Grotewold E.,1999,Maize R2R3 Myb genes:Sequence analysis reveals amplification in the higher plants,Genetics,153(1):427-444
Ravaglia D.,Espley R.V.,Henry-Kirk R.A.,Andreotti C.,Ziosi V.,Hellens R.P.,Costa G.,and Allan A.C.,2013,Transcriptional regulation of flavonoid biosynthesis in nectarine(Prunus persica)by a set of R2R3 MYB transcription factors,BMCPlant Biol.,13(1):68
Shen X.J.,Zhao K.,Liu L.L.,Zhang K.C.,Yuan H.Z.,Liao X.,Wang Q.,Guo X.W.,Li F.,and Li T.H.,2014,A role for PacMYBA in ABA-regulated anthocyanin biosynthesis in red-colored sweet cherry cv.Hong Deng(Prunus avium L.),Plant Cell Physiol.,55(5):862-880
Stracke R.,Werber M.,and Weisshaar B.,2001,The R2R3-MYB gene family in Arabidopsis thaliana,Curr.Opin.Plant Biol.,4(5):447-456
Cedroni M.L.,Cronn R.C.,Adams K.L.,Wilkins T.A.,and Wendel J.F.,2003,Evoluti on and expression of MYB genes in diploid and polyploid cotton,Plant Mol.Biol.,51(3):313-325
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
Du H.,Yang S.S.,Liang Z.,Feng B.R.,Liu L.,Huang Y.B.,and Tang Y.X.,2012,Genome-wide analysis of the MYB transcription factor super-family in soybean,BMC Plant Biol.,12(1):106
Dubos C.,Stracke R.,Grotewold E.,Weisshaar B.,Martin C.,and Lepiniec L.,2010,MYB transcription factors in Araidopsis,Trends Plant Sci.,15(10):573-581
Feng S.Q.,Wang Y.L.,Yang S.,Xu Y.T.,and Chen X.S.,2010,Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor Py MYB10,Planta,232(1):245-255
Fournier-Level A.,Lacombe T.,Le Cunff L.,Boursiquot J.M.,and This P.,2010,Evolution of the Vv MybA gene family,the major determinant of berry colour in cultivated grapevine(Vitis vinifera L.),Heredity,104(4):351-362
Hichri I.,Barrieu F.,Bogs J.,Kappel C.,Delrot S.,and Lauvergeat V.,2011,Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway,J.Exp.Bot.,62(8):2465-2483
Jaakola L.,2013,New insights into the regulation of anthocyanin bio-synthesis in fruits,Trends Plant Sci.,18(9):477-483
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
Lin-Wang K.,Micheletti D.,Palmer J.,Volz R.,Lozano L.,Espley R.,Hellens R.P.,ChagnèD.,Rowan D.D.,Troggio M.,Iglesias I.,and Allan A.C.,2011,High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex,Plant Cell Environ.,34(7):1176-1190
Luo X.B.,2011,Nutritional value,health function and edible method of mango,Zhongguo Shiwu Yu Yingyang(Chinese Food and Nutrition),17(7):77-79(罗学兵,2011,芒果的营养价值、保健功能及食用方法,中国食物与营养,17(7):77-79)
Matus J.T.,Aquea F.,and Arce-Johnson P.,2008,Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related cladesand conserved gene structure organization across Vitis and Arabidopsis genomes,BMC Plant Biol.,8(1):83
Palapol Y.,Ketsa S.,Lin-Wang K.,Ferguson I.B.,and Allan A.C.,2009,A MYB transcription factor regulates anthocyanin biosynthesis in mangosteen(Garcinia mangostana L.)fruit during ripening,Planta,229(6):1323-1334
Petroni K.,and Tonelli C.,2011,Recent advances on the regulation of anthocyanin synthesis in reproductive organs,Plant Sci.,181(3):219-229
Rabinowicz P.D.,Braun E.L.,Wolfe A.D.,Bowen B.,and Grotewold E.,1999,Maize R2R3 Myb genes:Sequence analysis reveals amplification in the higher plants,Genetics,153(1):427-444
Ravaglia D.,Espley R.V.,Henry-Kirk R.A.,Andreotti C.,Ziosi V.,Hellens R.P.,Costa G.,and Allan A.C.,2013,Transcriptional regulation of flavonoid biosynthesis in nectarine(Prunus persica)by a set of R2R3 MYB transcription factors,BMCPlant Biol.,13(1):68
Shen X.J.,Zhao K.,Liu L.L.,Zhang K.C.,Yuan H.Z.,Liao X.,Wang Q.,Guo X.W.,Li F.,and Li T.H.,2014,A role for PacMYBA in ABA-regulated anthocyanin biosynthesis in red-colored sweet cherry cv.Hong Deng(Prunus avium L.),Plant Cell Physiol.,55(5):862-880
Stracke R.,Werber M.,and Weisshaar B.,2001,The R2R3-MYB gene family in Arabidopsis thaliana,Curr.Opin.Plant Biol.,4(5):447-456