Cloning and expression analysis of the FvNCED3 gene and its promoter from ash(Fraxinus velutina)
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摘要
The 9-cis-epoxycarotenoid dioxygenase(NCED)gene is rate-limiting in abscisic acid(ABA) biosynthesis.In this study, an NCED gene, designated FvNCED3(KY008746), was cloned from velvet ash(Fraxinus velutina Torr.) with a RACE method. The full length c DNA of FvNCED3 encodes a 573-amino acid polypeptide.Sequencing analysis showed that the FvNCED3 protein was highly homologous to other NCED proteins. The expression patterns of FvNCED3 in different ash organs were analyzed by real-time PCR which revealed that FvNCED3 expression levels were highest in leaves and lowest in roots. The gene expression patterns of FvNCED3 under abiotic stress indicated that its expression increased under drought, salt and ABA stress and decreased due to high and low temperatures. There were no obvious changes under ultraviolet light. The 1094-bp upstream sequence 5' flank regulation region of the FvNCED3 gene was also cloned from ash using the Genome Walking method. To assess the activity of the FvNCED3 promoter, a p FvNCED3 p::GUS plant expression vector was constructed for tobacco transformation. GUS expression of the FvNCED3 GUS enzyme activity was detected in almost all transgenic tobacco tissues, especially in the young leaves,stigma, anther, ovule and ovary. After treating the transgenic tobacco with NaCl and placing it under drought stress, GUS staining of tobacco leaves increased compared with that under normal growth conditions. This result indicates that gene expression driven by the FvNCED3 promoter can be induced by salt and drought stress.
The 9-cis-epoxycarotenoid dioxygenase(NCED)gene is rate-limiting in abscisic acid(ABA) biosynthesis.In this study, an NCED gene, designated FvNCED3(KY008746), was cloned from velvet ash(Fraxinus velutina Torr.) with a RACE method. The full length c DNA of FvNCED3 encodes a 573-amino acid polypeptide.Sequencing analysis showed that the FvNCED3 protein was highly homologous to other NCED proteins. The expression patterns of FvNCED3 in different ash organs were analyzed by real-time PCR which revealed that FvNCED3 expression levels were highest in leaves and lowest in roots. The gene expression patterns of FvNCED3 under abiotic stress indicated that its expression increased under drought, salt and ABA stress and decreased due to high and low temperatures. There were no obvious changes under ultraviolet light. The 1094-bp upstream sequence 5' flank regulation region of the FvNCED3 gene was also cloned from ash using the Genome Walking method. To assess the activity of the FvNCED3 promoter, a p FvNCED3 p::GUS plant expression vector was constructed for tobacco transformation. GUS expression of the FvNCED3 GUS enzyme activity was detected in almost all transgenic tobacco tissues, especially in the young leaves,stigma, anther, ovule and ovary. After treating the transgenic tobacco with NaCl and placing it under drought stress, GUS staining of tobacco leaves increased compared with that under normal growth conditions. This result indicates that gene expression driven by the FvNCED3 promoter can be induced by salt and drought stress.
The 9-cis-epoxycarotenoid dioxygenase(NCED)gene is rate-limiting in abscisic acid(ABA) biosynthesis.In this study, an NCED gene, designated FvNCED3(KY008746), was cloned from velvet ash(Fraxinus velutina Torr.) with a RACE method. The full length c DNA of FvNCED3 encodes a 573-amino acid polypeptide.Sequencing analysis showed that the FvNCED3 protein was highly homologous to other NCED proteins. The expression patterns of FvNCED3 in different ash organs were analyzed by real-time PCR which revealed that FvNCED3 expression levels were highest in leaves and lowest in roots. The gene expression patterns of FvNCED3 under abiotic stress indicated that its expression increased under drought, salt and ABA stress and decreased due to high and low temperatures. There were no obvious changes under ultraviolet light. The 1094-bp upstream sequence 5' flank regulation region of the FvNCED3 gene was also cloned from ash using the Genome Walking method. To assess the activity of the FvNCED3 promoter, a p FvNCED3 p::GUS plant expression vector was constructed for tobacco transformation. GUS expression of the FvNCED3 GUS enzyme activity was detected in almost all transgenic tobacco tissues, especially in the young leaves,stigma, anther, ovule and ovary. After treating the transgenic tobacco with NaCl and placing it under drought stress, GUS staining of tobacco leaves increased compared with that under normal growth conditions. This result indicates that gene expression driven by the FvNCED3 promoter can be induced by salt and drought stress.
引文
Bang SW,Park SH,Jeong JS,Kim YS,Jung H,Ha SH,Kim JK(2013)Characterization of the inducible OsNCED3 promoter in stress different transgenic rice organs and over three homozygous generations.Planta 237(1):211-224
Carol P,Lorenzo A,Champa SG(2004)Targeting transgene expression in research,agricultural,and environmental applications:promoters used in plant transformation.Vitro Cell Dev Biol Plant 40(1):1-22
Chaves MM,Flexas J,Pinheiro C(2009)Photosynthesis under drought and salt stress:regulation mechanisms from whole plant to cell.Ann Bot 103(4):551-560
Chernys JT,Zeevaart JAD(2000)Characterization of the 9-cisepoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado.Plant Physiol124(1):343-353
Cutler SR,Rodriguez PL,Finkelstein RR,Abrams SR(2010)Abscisic acid:emergence of a core signaling network.Annu Rev Plant Biol 61:651-679
Farifteh J,Farshad A,George RJ(2006)Assessing salt-affected soils using remote sensing,solute modeling,and geophysics.Geoderma 130(3-4):191-206
Gong W,Shen YP,Ma LG,Pan Y,Du YL,Wang DH,Yang JY,Hu LD,Liu XF,Dong CX(2004)Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes.Plant Physiol 135(2):773-782
Hong JK,Hwang BK(2009)The promoter of the pepper pathogeninduced membrane protein gene CaPIMP1 mediates environmental stress responses in plants.Planta 229(2):249-259
Hu YC,Schnyder H,Schmidhalter U(2000)Carbohydrate deposition and partitioning in elongating leaves of wheat under saline soil conditions.Aust J Plant Physiol 27(4):363-370
Hwang SG,Chen HC,Huang WY,Chu YC,Shii CT,Cheng WH(2010)Ectopic expression of rice OsNCED3 in Arabidopsis increases ABA level and alters leaf morphology.Plant Sci178(1):12-22
Iuchi S,Kobayashi M,Taji T,Naramoto M,Seki M,Kato T,Tabata S,Kakubari Y,Yamaguchi-Shinozaki K,Shinozaki K(2001)Regulation of drought tolerance by gene manipulation of 9-cisepoxycarotenoid dioxygenase,a key enzyme in abscisic acid biosynthesis in Arabidopsis.Plant J 27(4):325-333
Ji XM,Dong BD,Shiran B,Talbot MJ,Edlington JE,Hughes T,White RG,Gubler F,Dolferus R(2011)Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals.Plant Physiol156(2):647-662
Jia WS,Wang YQ,Zhang SQ,Zhang JH(2002)Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots.J Exp Bot 53(378):2201-2206
Jiang Y,Wu K,Lin F,Qu YN,Liu XX,Zhang Q(2014)Phosphatidic acid integrates calcium signaling and microtubule dynamics into regulating ABA-induced stomatal closure in Arabidopsis.Planta239(3):565-575
Kong XQ,Luo Z,Dong HZ,Eneji AE,Li WJ(2016)H2O2and ABAsignaling are responsible for the increased Na?efflux and water uptake in Gossypium hirsutum L.roots in the non-saline side under non-uniform root zone salinity.J Exp Bot67(8):2247-2261
Koomnneef M,Lon-Kloosterziel KM,Schwartz SH,Zeevaart JAD(1998)The genetic and molecular dissection of abscisic acid biosynthesis and signal transduction in Arabidopisis.Plant Physiol Biochem 36(1-2):83-89
Lee SY,Boon NJ,Webb AA,Tanaka RJ(2016)Synergistic activation of RD29A via integration of salinity stress and abscisic acid in Arabidopsis thaliana.Plant Cell Physiol57(10):2147-2160
Lefebvre V,North H,Frey A,Sotta B,Seo M,Okamoto M,Nambara E,Marion-Poll A(2006)Functional analysis of Arabidopsis NCED6 and NCED9 genes indicates that ABA synthesized in the endosperm is involved in the induction of seed dormancy.Plant J 45(3):309-319
Li QL,Yin H,Li D,Zhu HF,Zhang Y,Zhu WW(2007)Isolation and characterization of CMO gene promoter from Halophyte Suaeda liaotungensis K.J Genet Genomics 34(4):355-361
Liang JH,Yang LX,Chen X,Li L,Guo DL,Li HH,Zhang BY(2009)Cloning and characterization of the promoter of the 9-cisepoxycarotenoid dioxygenase gene in Arachishypogaea L.Biosci Biotechnol Biochem 73(9):2103-2106
Liu CL,Chen HP,Liu EE,Peng XX,Lu SY,Guo ZF(2003)Multiple tolerance of rice to abiotic stresses and its relationship with ABAaccumulation.Acta Agro Sin 29(5):725-729(in Chinese)
Mori IC,Murata Y(2011)ABA signaling in stomatal guard cells:lessons from Commelina and Vicia.J Plant Res 124(4):477-487
Mulholland BJ,Taylor IB,Jackson AC,Thompson AJ(2003)Can ABA mediate responses of salinity stressed tomato?Environ Exp Bot 50(1):17-28
Nakabayashi K,Okamoto M,Koshiba T,Kamiya Y,Nambara E(2005)Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination:epigenetic and genetic regulation of transcription in seed.Plant J 41(5):697-709
Park HC,Kim ML,Kang YH,Jeon JM,Yoo JH,Kim MC,Park CY,Jeong JC,Moon BC,Lee JH,Yoon HW,Lee SH,Chung WS,Lim CO,Lee SY,Hong JC,Cho MJ(2004)Pathogen-and NaClinduced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor.Plant Physiol 135(4):2150-2161
Parks GE,Dietrich M,Schumaker KS(2002)Increased vacuolar Na?/H?activity in Salicornia bigelovii Torr.in response to NaCl.J Exp Bot 53(371):1055-1065
Peng XC,Qiu DW,Zeng HM,Guo H,Yang XF,Liu Z(2015)Inducible and constitutive expression of an elicitor gene Hrip1from Alternaria tenuissima enhances stress tolerance in Arabidopsis.Transgenic Res 24(1):135-145
Qin XQ,Zeevaart JAD(2002)Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance.Plant Physiol 128(2):544-551
Schwartz SH,Tan BC,Gage DA,Zeevaart JA,McCarty DR(1997)Specific oxidative cleavage of carotenoids by VP14 of maize.Science 276(5320):1872-1874
Son S,Chitnis VR,Liu AH,Gao F,Nguyen TN,Ayele BT(2016)Abscisic acid metabolic genes of wheat(Triticum aestivum L.):identification and insights into their functionality in seed dormancy and dehydration tolerance.Planta 244(2):429-447
Soo YK(2007)Recent advances in ABA signaling.J Plant Bio50(2):117-121
Tan BC,Joseph LM,Deng WT,Liu LJ,Li QB,Cline K,McCarty DR(2003)Molecular characterization of the Arabidopsis 9-cisepoxyearotenoid dioxygenase gene family.Plant J 35(1):44-56
Teng KQ,Li JZ,Liu L,Han YC,Du YX,Zhang J,Sun HZ,Zhao QZ(2014)Exogenous ABA induces drought tolerance in upland rice:the role of chloroplast and ABA biosynthesis-related gene expression on photosystem II during PEG stress.Acta Physiol Plant 36(8):2219-2227
Vera-Estrella R,Barkla BJ,Garcia-Ramirez L,Pantoja O(2005)Salt stress in Thellungiella halophila activates Na?transport mechanisms required for salinity toletrance.Plant Physiol139(3):1507-1517
Wan XR,Li L(2006)Regulation of ABA level and water-stress tolerance of Arabidopsis by ectopic expression of a peanut 9-cisepoxycarotenoid dioxygenase gene.Biochem Biophys Res Commun 347(4):1030-1038
Wang YP,Wang YA,Ji K,Dai SJ,Hu Y,Sun L,Li Q,Chen P,Sun YF,Duan CR,Wu Y,Luo H,Zhang D,Guo YD,Leng P(2013)The role of abscisic acid in regulating cucumber fruit development and ripening and its transcriptional regulation.Plant Physiol Biochem 64:70-79
Wilkinson S,Davies WJ(2002)ABA-based chemical signalling:the co-ordination of responses to stress in plants.Plant Cell Environ25(2):195-210
Xian LH,Sun PP,Hu SS,Wu J,Liu JH(2014)Molecular cloning and characterization of CrNCED1,a gene encoding 9-cis-epoxycarotenoid dioxygenase in Citrus reshni,with functions in tolerance to multiple abiotic stresses.Planta 239(1):61-77
Yadav V,Kundu S,Chattopadhyay D,Negi P,Wei N,Deng XW,Chattopadhyay S(2002)Light regulated modulation of Z-box containing promoters by photoreceptors and downstream regulatory components,COP1 and HY5 Arabidopsis.Plant J31(6):741-753
Yang JF,Guo ZF(2007)Cloning of a 9-cis-epoxycarotenoid dioxygenase gene(SgNCED1)from Stylosanthes guianensis and its expression in response to abiotic stresses.Plant Cell Rep26(8):1383-1390
Zheng HQ,Lei Y,Lin SZ,Zhang Q,Zhang ZY(2011)Bidirectionalization of a methyl jasmonate-inducible plant promoter.Biotechnol Lett 33(2):387-393
Carol P,Lorenzo A,Champa SG(2004)Targeting transgene expression in research,agricultural,and environmental applications:promoters used in plant transformation.Vitro Cell Dev Biol Plant 40(1):1-22
Chaves MM,Flexas J,Pinheiro C(2009)Photosynthesis under drought and salt stress:regulation mechanisms from whole plant to cell.Ann Bot 103(4):551-560
Chernys JT,Zeevaart JAD(2000)Characterization of the 9-cisepoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado.Plant Physiol124(1):343-353
Cutler SR,Rodriguez PL,Finkelstein RR,Abrams SR(2010)Abscisic acid:emergence of a core signaling network.Annu Rev Plant Biol 61:651-679
Farifteh J,Farshad A,George RJ(2006)Assessing salt-affected soils using remote sensing,solute modeling,and geophysics.Geoderma 130(3-4):191-206
Gong W,Shen YP,Ma LG,Pan Y,Du YL,Wang DH,Yang JY,Hu LD,Liu XF,Dong CX(2004)Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes.Plant Physiol 135(2):773-782
Hong JK,Hwang BK(2009)The promoter of the pepper pathogeninduced membrane protein gene CaPIMP1 mediates environmental stress responses in plants.Planta 229(2):249-259
Hu YC,Schnyder H,Schmidhalter U(2000)Carbohydrate deposition and partitioning in elongating leaves of wheat under saline soil conditions.Aust J Plant Physiol 27(4):363-370
Hwang SG,Chen HC,Huang WY,Chu YC,Shii CT,Cheng WH(2010)Ectopic expression of rice OsNCED3 in Arabidopsis increases ABA level and alters leaf morphology.Plant Sci178(1):12-22
Iuchi S,Kobayashi M,Taji T,Naramoto M,Seki M,Kato T,Tabata S,Kakubari Y,Yamaguchi-Shinozaki K,Shinozaki K(2001)Regulation of drought tolerance by gene manipulation of 9-cisepoxycarotenoid dioxygenase,a key enzyme in abscisic acid biosynthesis in Arabidopsis.Plant J 27(4):325-333
Ji XM,Dong BD,Shiran B,Talbot MJ,Edlington JE,Hughes T,White RG,Gubler F,Dolferus R(2011)Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals.Plant Physiol156(2):647-662
Jia WS,Wang YQ,Zhang SQ,Zhang JH(2002)Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots.J Exp Bot 53(378):2201-2206
Jiang Y,Wu K,Lin F,Qu YN,Liu XX,Zhang Q(2014)Phosphatidic acid integrates calcium signaling and microtubule dynamics into regulating ABA-induced stomatal closure in Arabidopsis.Planta239(3):565-575
Kong XQ,Luo Z,Dong HZ,Eneji AE,Li WJ(2016)H2O2and ABAsignaling are responsible for the increased Na?efflux and water uptake in Gossypium hirsutum L.roots in the non-saline side under non-uniform root zone salinity.J Exp Bot67(8):2247-2261
Koomnneef M,Lon-Kloosterziel KM,Schwartz SH,Zeevaart JAD(1998)The genetic and molecular dissection of abscisic acid biosynthesis and signal transduction in Arabidopisis.Plant Physiol Biochem 36(1-2):83-89
Lee SY,Boon NJ,Webb AA,Tanaka RJ(2016)Synergistic activation of RD29A via integration of salinity stress and abscisic acid in Arabidopsis thaliana.Plant Cell Physiol57(10):2147-2160
Lefebvre V,North H,Frey A,Sotta B,Seo M,Okamoto M,Nambara E,Marion-Poll A(2006)Functional analysis of Arabidopsis NCED6 and NCED9 genes indicates that ABA synthesized in the endosperm is involved in the induction of seed dormancy.Plant J 45(3):309-319
Li QL,Yin H,Li D,Zhu HF,Zhang Y,Zhu WW(2007)Isolation and characterization of CMO gene promoter from Halophyte Suaeda liaotungensis K.J Genet Genomics 34(4):355-361
Liang JH,Yang LX,Chen X,Li L,Guo DL,Li HH,Zhang BY(2009)Cloning and characterization of the promoter of the 9-cisepoxycarotenoid dioxygenase gene in Arachishypogaea L.Biosci Biotechnol Biochem 73(9):2103-2106
Liu CL,Chen HP,Liu EE,Peng XX,Lu SY,Guo ZF(2003)Multiple tolerance of rice to abiotic stresses and its relationship with ABAaccumulation.Acta Agro Sin 29(5):725-729(in Chinese)
Mori IC,Murata Y(2011)ABA signaling in stomatal guard cells:lessons from Commelina and Vicia.J Plant Res 124(4):477-487
Mulholland BJ,Taylor IB,Jackson AC,Thompson AJ(2003)Can ABA mediate responses of salinity stressed tomato?Environ Exp Bot 50(1):17-28
Nakabayashi K,Okamoto M,Koshiba T,Kamiya Y,Nambara E(2005)Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination:epigenetic and genetic regulation of transcription in seed.Plant J 41(5):697-709
Park HC,Kim ML,Kang YH,Jeon JM,Yoo JH,Kim MC,Park CY,Jeong JC,Moon BC,Lee JH,Yoon HW,Lee SH,Chung WS,Lim CO,Lee SY,Hong JC,Cho MJ(2004)Pathogen-and NaClinduced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor.Plant Physiol 135(4):2150-2161
Parks GE,Dietrich M,Schumaker KS(2002)Increased vacuolar Na?/H?activity in Salicornia bigelovii Torr.in response to NaCl.J Exp Bot 53(371):1055-1065
Peng XC,Qiu DW,Zeng HM,Guo H,Yang XF,Liu Z(2015)Inducible and constitutive expression of an elicitor gene Hrip1from Alternaria tenuissima enhances stress tolerance in Arabidopsis.Transgenic Res 24(1):135-145
Qin XQ,Zeevaart JAD(2002)Overexpression of a 9-cis-epoxycarotenoid dioxygenase gene in Nicotiana plumbaginifolia increases abscisic acid and phaseic acid levels and enhances drought tolerance.Plant Physiol 128(2):544-551
Schwartz SH,Tan BC,Gage DA,Zeevaart JA,McCarty DR(1997)Specific oxidative cleavage of carotenoids by VP14 of maize.Science 276(5320):1872-1874
Son S,Chitnis VR,Liu AH,Gao F,Nguyen TN,Ayele BT(2016)Abscisic acid metabolic genes of wheat(Triticum aestivum L.):identification and insights into their functionality in seed dormancy and dehydration tolerance.Planta 244(2):429-447
Soo YK(2007)Recent advances in ABA signaling.J Plant Bio50(2):117-121
Tan BC,Joseph LM,Deng WT,Liu LJ,Li QB,Cline K,McCarty DR(2003)Molecular characterization of the Arabidopsis 9-cisepoxyearotenoid dioxygenase gene family.Plant J 35(1):44-56
Teng KQ,Li JZ,Liu L,Han YC,Du YX,Zhang J,Sun HZ,Zhao QZ(2014)Exogenous ABA induces drought tolerance in upland rice:the role of chloroplast and ABA biosynthesis-related gene expression on photosystem II during PEG stress.Acta Physiol Plant 36(8):2219-2227
Vera-Estrella R,Barkla BJ,Garcia-Ramirez L,Pantoja O(2005)Salt stress in Thellungiella halophila activates Na?transport mechanisms required for salinity toletrance.Plant Physiol139(3):1507-1517
Wan XR,Li L(2006)Regulation of ABA level and water-stress tolerance of Arabidopsis by ectopic expression of a peanut 9-cisepoxycarotenoid dioxygenase gene.Biochem Biophys Res Commun 347(4):1030-1038
Wang YP,Wang YA,Ji K,Dai SJ,Hu Y,Sun L,Li Q,Chen P,Sun YF,Duan CR,Wu Y,Luo H,Zhang D,Guo YD,Leng P(2013)The role of abscisic acid in regulating cucumber fruit development and ripening and its transcriptional regulation.Plant Physiol Biochem 64:70-79
Wilkinson S,Davies WJ(2002)ABA-based chemical signalling:the co-ordination of responses to stress in plants.Plant Cell Environ25(2):195-210
Xian LH,Sun PP,Hu SS,Wu J,Liu JH(2014)Molecular cloning and characterization of CrNCED1,a gene encoding 9-cis-epoxycarotenoid dioxygenase in Citrus reshni,with functions in tolerance to multiple abiotic stresses.Planta 239(1):61-77
Yadav V,Kundu S,Chattopadhyay D,Negi P,Wei N,Deng XW,Chattopadhyay S(2002)Light regulated modulation of Z-box containing promoters by photoreceptors and downstream regulatory components,COP1 and HY5 Arabidopsis.Plant J31(6):741-753
Yang JF,Guo ZF(2007)Cloning of a 9-cis-epoxycarotenoid dioxygenase gene(SgNCED1)from Stylosanthes guianensis and its expression in response to abiotic stresses.Plant Cell Rep26(8):1383-1390
Zheng HQ,Lei Y,Lin SZ,Zhang Q,Zhang ZY(2011)Bidirectionalization of a methyl jasmonate-inducible plant promoter.Biotechnol Lett 33(2):387-393