越橘VcNAC072克隆及其促进花青素积累的功能分析
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摘要
【目的】分离越橘VcNAC072(NAM,ATAF1/2,CUC2)转录因子,分析其表达模式并探讨其在调控花青素合成过程中的功能,为进一步研究越橘花青素积累的调控机理提供理论基础。【方法】以‘公爵’越橘(Vaccinium corymbosum ‘Duke’)为试材,克隆VcNAC072。通过农杆菌介导法获得转基因拟南芥,比较转基因和野生型拟南芥花青素积累的差异。利用酵母单杂交和瞬时表达试验,分析VcNAC072对MYB转录因子AtPAP1的转录调控。【结果】克隆获得越橘VcNAC072,该基因CDS为1 032 bp,编码含有343个氨基酸的蛋白质,含有1个保守的NAC结构域。表达分析显示,该基因在不同发育阶段的果实中均可表达,但表达差异明显,在粉色和蓝色果实中表达量较高,在绿色果实中表达量最低。随着VcNAC072表达的升高,果实中花青素含量呈递增的趋势。分析AtPAP1启动子序列,发现其序列中包含NAC转录因子的结合位点。酵母单杂交和烟草瞬时表达试验结果表明,VcNAC072可与AtPAP1的启动子相互作用,并激活其表达。在野生型拟南芥中异位表达VcNAC072,其种子中花青素积累量显著高于野生型。【结论】推测VcNAC072在越橘果实中正向调节花青素的积累。
【Objective】The objective of this study was to isolate a transcription factor VcNAC072(NAM, ATAF1/2, CUC2) from blueberry by PCR technology and studying its expression, and to identify its role in anthocyanin biosynthesis. This study laid the foundation for further study of the molecular mechanism of VcNAC072 affecting anthocyanin biosynthesis in blueberry. 【Method】The blueberry VcNAC072 gene was cloned by PCR technology from the fruits of blueberry(Vaccinium corymbosum ‘Duke'). The transgenic Arabidopsis thaliana were generated via Agrobacterium-mediated transformation. The differences in the anthocyanin accumulation were compared between transgenic and wild-type Arabidopsis. The yeast one-hybrid(Y1H) and transient expression assays were carried out to test the transcriptional regulation of MYB transcription factor AtPAP1 by Vc NAC072. 【Result】A blueberry Vc NAC072 was cloned from blueberry. Sequence analysis showed that the coding domain sequence(CDS) of VcNAC072 was 1 032 bp, which encoded 343 amino acids. Protein structure analysis showed that Vc NAC072 contained a NAC domain. Expression analysis showed that Vc NAC072 was expressed at different developmental stages of the blueberry fruits. However, the expression levels varied, with the highest expression level in pink fruits and blue fruits and the relatively low transcript levels in green fruits. The content of anthocyanin in fruits was increased with the elevation of relative expression of VcNAC072. The sequence of AtPAP1 promoter was analyzed and a NAC binding motif was found. The VcNAC072 protein could interact with the promoter of AtPAP1. Moreover, the VcNAC072 could induce the expression of AtPAP1 and favorably contributed to anthocyanin accumulation in seeds of transgenic Arabidopsis. 【Conclusion】It was speculated that Vc NAC072 up-regulated anthocyanin accumulation in fruits of blueberry.
【Objective】The objective of this study was to isolate a transcription factor VcNAC072(NAM, ATAF1/2, CUC2) from blueberry by PCR technology and studying its expression, and to identify its role in anthocyanin biosynthesis. This study laid the foundation for further study of the molecular mechanism of VcNAC072 affecting anthocyanin biosynthesis in blueberry. 【Method】The blueberry VcNAC072 gene was cloned by PCR technology from the fruits of blueberry(Vaccinium corymbosum ‘Duke'). The transgenic Arabidopsis thaliana were generated via Agrobacterium-mediated transformation. The differences in the anthocyanin accumulation were compared between transgenic and wild-type Arabidopsis. The yeast one-hybrid(Y1H) and transient expression assays were carried out to test the transcriptional regulation of MYB transcription factor AtPAP1 by Vc NAC072. 【Result】A blueberry Vc NAC072 was cloned from blueberry. Sequence analysis showed that the coding domain sequence(CDS) of VcNAC072 was 1 032 bp, which encoded 343 amino acids. Protein structure analysis showed that Vc NAC072 contained a NAC domain. Expression analysis showed that Vc NAC072 was expressed at different developmental stages of the blueberry fruits. However, the expression levels varied, with the highest expression level in pink fruits and blue fruits and the relatively low transcript levels in green fruits. The content of anthocyanin in fruits was increased with the elevation of relative expression of VcNAC072. The sequence of AtPAP1 promoter was analyzed and a NAC binding motif was found. The VcNAC072 protein could interact with the promoter of AtPAP1. Moreover, the VcNAC072 could induce the expression of AtPAP1 and favorably contributed to anthocyanin accumulation in seeds of transgenic Arabidopsis. 【Conclusion】It was speculated that Vc NAC072 up-regulated anthocyanin accumulation in fruits of blueberry.
引文
[1]李亚东,孙海悦,陈丽.我国蓝莓产业发展报告.中国果树,2016(5):1-10.LI Y D,SUN H Y,CHEN L.Report on the development of blueberry industry in China.China Fruits,2016(5):1-10.(in Chinese)
[2]吴林.中国蓝莓35年-科学研究与产业发展.吉林农业大学学报,2016,38(1):1-11.WU L.Thirty-five years of research and industry development of blueberry in China.Journal of Jilin Agricultural University,2016,38(1):1-11.(in Chinese)
[3]GORDILLO G,FANG H Q,KHANNA S,HARPER J,PHILIPS G,SEN C K.Oral administration of blueberry inhibits angiogenic tumor growth and enhances survival of mice with endothelial cell neoplasm.Antioxid Redox Signal,2009,11(1):47-58.
[4]BASU A,RHONE M,LYONS T J.Berries:Emerging impact on cardiovascular health.Nutrition Reviews,2010,68(3):168-177.
[5]SHAHNEJAT-BUSHEHRI S,TARKOWSKA D,SAKURABA Y,BALAZADEH S.Arabidopsis NAC transcription factor JUB1regulates GA/BR metabolism and signalling.Nature Plants,2016,2:16013.
[6]KIM H S,PARK B O,YOO J H,JUNG M S,LEE S M,HAN H J,KIM K E,KIM S H,LIM C O,YUN D J,LEE S Y,CHUNG W S.Identification of a calmodulin-binding NAC protein as a transcriptional repressor in Arabidopsis.Journal of Biological Chemistry,2007,282(50):36292-36302.
[7]HONG Y B,ZHANG H J,HUANG L,LI D Y,SONG F M.Overexpression of a stress-responsive NAC transcription factor gene ONAC022 improves drought and salt tolerance in rice.Frontiers in Plant Science,2016,7(e0116646):4.
[8]YU X W,LIU Y M,WANG S,TAO Y,WANG Z K,MIJITI A,WANG Z,ZHANG H,MA H.A chickpea stress-responsive NACtranscription factor,CarNAC5,confers enhanced tolerance to drought stress in transgenic.Plant Growth Regulation,2016,79(2):187-197.
[9]KO J H,YANG S H,PARK A H,LEROUXEL O,HAN K H.ANAC012,a member of the plant-specific NAC transcription factor family,negatively regulates xylary fiber development in Arabidopsis thaliana.The Plant Journal,2007,50(6):1035-1048.
[10]ZHONG R,DEMURA T,YE Z H.SND1,a NAC domain transcription factor,is a key regulator of secondary wall synthesis in fibers of Arabidopsis.The Plant Cell,2006,18(11):3158-3170.
[11]YAMAGUCHI M,KUBO M,FUKUDA H,DEMURA T.Vascular-related NAC-domain7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots.The Plant Journal,2008,55(4):652-664.
[12]ODA-YAMAMIZO C,MITSUDA N,SAKAMOTO S,OGAWA D,OHME-TAKAGI M,OHMIYA A.ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves.Scientific Reports,2016,6:23609.
[13]TAKASAKI H,MARUYAMA K,TAKAHASHI F,FUJITA M,YOSHIDA T,NAKASHIMA K,MYOUGA F,TOYOOKA K,YAMAGUCHI-SHINOZAKI K,SHINOZAKI K.SNAC-As,stress-responsive NAC transcription factors,mediate ABA-inducible leaf senescence.The Plant Journal,2015,84(6):1114-1123.
[14]ZHU Y,YAN J W,LIU W J,LIU L,SHENG Y,SUN Y,LI Y Y,SCHELLER H,JIANG M Y,HOU X L,NI L,ZHANG A Y.Phosphorylation of a NAC transcription factor by a calcium/calmodulin-dependent protein kinase regulates abscisic acid-induced antioxidant defense in maize.Plant Physiology,2016,171(3):1651-1664.
[15]ZHOU H,WANG K L,WANG H L,GU C,DARE A,ESPLEY R,HEH P,ALLAN A,HAN Y P.Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.The Plant Journal,2015,82(1):105-121.
[16]安建平,宋来庆,赵玲玲,由春香,王小非,郝玉金.苹果愈伤组织超表达MdNAC029促进花青苷积累.园艺学报,2018,45(5):845-854.AN J P,SONG L Q,ZHAO L L,YOU C X,WANG X F,HAO Y J.Overexpression of MdNAC029 promotes anthocyanin accumulation in apple calli.Acta Horticulturae Sinica,2018,45(5):845-854.(in Chinese)
[17]NURUZZAMAN M,MANIMEKALAI R,SHRRONI A M.Genome-wide analysis of NAC transcription factor family in rice.Gene,2010,465(1/2):30-44.
[18]KIM S G,LEE S,SEO P J,KIM S K,KIM J K,PARK C M.Genome-scale screening and molecular characterization of membranebound transcription factors in Arabidopsis and rice.Genomics,2010,95(1):56-65.
[19]SONG Y,LIU H D,ZHOU Q,ZHANG H J,ZHANG Z D,LI Y D,WANG H B,LIU F Z.High-throughput sequencing of highbush blueberry transcriptome and analysis of basic helix-loop-helix transcription factors.Journal of Integrative Agriculture,2017,16(3):591-604.
[20]YIN X R,ALLAN A C,CHEN K S,FERGUSON I B.Kiwifruit EILand ERF genes involved in regulating fruit ripening.Plant Physiology,2010,153(3):1280-1292.
[21]JEFFERSON R A,KAVANAGH T A,BEVAN M W.GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO Journal,1987,6(13),3901-3907.
[22]PERTUZATTI P B,BARCIA M T,REBELLO L P G,GóMEZ-ALONSO S,DUARTE R M T,DUARTE M C T,GODOY H T,HERMOSíN-GUTIéRREZ I.Antimicrobial activity and differentiation of anthocyanin profiles of rabbiteye and highbush blueberry using HPLC-DAD-ESI-MS and multivariate analysis.Journal of Functional Foods,2016,26:506-516.
[23]ZHU X Y,CHEN J Y,XIE Z K,GAO J,REN G D,GAO S,ZHOU X,KUAI B K.Jasmonic acid promotes degreening via MYB2/3/4-and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes.The Plant Journal,2015,84(3):597-610.
[24]LI S,GAO J,YAO L Y,REN G D,ZHU X Y,GAO S,QIU K,ZHOUX,KUAI B K.The role of ANAC072 in the regulation of chlorophyll degradation during age-and dark-induced leaf senescence.Plant Cell Reports,2016,35(8):1729-1741.
[25]HUANG J C,PIATER L A,DUBERY I A.The NAC transcription factor gene ANAC072 is differentially expressed in Arabidopsis thaliana in response to microbe-associated molecular pattern(MAMP)molecules.Physiological and Molecular Plant Pathology,2012,80(80):19-27.
[26]TRAN L S,NAKASHIMA K,SAKUMA Y,SIMPSON S D,FUJITAY,MARUYAMA K,FUJITA M,SEKI M,SHINOZAKI K,YAMAGUCHI-SHINOZAKI K.Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter.The Plant Cell,2004,16(9):2481-2498.
[27]LI X Y,SUN H Y,PEI J B,DONG Y Y,WANG F W,CHEN H,SUNY P,WANG N,LI H Y,LI Y D.De novo sequencing and comparative analysis of the blueberry transcriptome to discover putative genes related to antioxidant.Gene,2012,511(1):54-61.
[28]SUN H Y,LIU Y S,GAI Y Z,GENG J M,CHEN L,LIU H D,KANGL M,TIAN Y W,LI Y D.De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis,transport and regulation.BMC Genomics,2015,16(1):652.
[29]BOREVITZ J O,XIA Y J,BLOUNT J,DIXON R,LAMB C.Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.The Plant Cell,2000,12(12):2383-2393.
[30]TAKOS A,JAFFéF,JACOB S,BOGS J,ROBINSON S,WALKERA.Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples.Plant Physiology,2006,142(3):1216-1232.
[31]YAO G F,MING M L,ALLAN A,GU C,LI L T,WU X,WANG R Z,CHANG Y J,QI K J,ZHANG S L,WU J.Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis.The Plant Journal,2017,92:437-451.
[32]JAAKOLA L,POOLE M O,JONES M,K?M?R?INEN-KARPPINEN T,KOSKIM?KI J,HOHTOLA A,H?GGMAN H D,FRASER P,MANNING K J,KING G.A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits.Plant Physiology,2010,153(4):1619-1629.
[33]SAKURABA Y,KIM Y S,HAN S H,LEE B D,PAEK N C.The Arabidopsis transcription factor NAC016 promotes drought stress responses by repressing AREB1 transcription through a trifurcate feed-forward regulatory loop involving NAP.The Plant Cell,2015,27(6):1771-1787.
[34]QU Y T,DUAN M,ZHANG Z Q,DONG J L,WANG T.Overexpression of the Medicago falcata NAC transcription factor Mf NAC3 enhances cold tolerance in Medicago truncatula.Environmental and Experimental Botany,2016,129:67-76.
[2]吴林.中国蓝莓35年-科学研究与产业发展.吉林农业大学学报,2016,38(1):1-11.WU L.Thirty-five years of research and industry development of blueberry in China.Journal of Jilin Agricultural University,2016,38(1):1-11.(in Chinese)
[3]GORDILLO G,FANG H Q,KHANNA S,HARPER J,PHILIPS G,SEN C K.Oral administration of blueberry inhibits angiogenic tumor growth and enhances survival of mice with endothelial cell neoplasm.Antioxid Redox Signal,2009,11(1):47-58.
[4]BASU A,RHONE M,LYONS T J.Berries:Emerging impact on cardiovascular health.Nutrition Reviews,2010,68(3):168-177.
[5]SHAHNEJAT-BUSHEHRI S,TARKOWSKA D,SAKURABA Y,BALAZADEH S.Arabidopsis NAC transcription factor JUB1regulates GA/BR metabolism and signalling.Nature Plants,2016,2:16013.
[6]KIM H S,PARK B O,YOO J H,JUNG M S,LEE S M,HAN H J,KIM K E,KIM S H,LIM C O,YUN D J,LEE S Y,CHUNG W S.Identification of a calmodulin-binding NAC protein as a transcriptional repressor in Arabidopsis.Journal of Biological Chemistry,2007,282(50):36292-36302.
[7]HONG Y B,ZHANG H J,HUANG L,LI D Y,SONG F M.Overexpression of a stress-responsive NAC transcription factor gene ONAC022 improves drought and salt tolerance in rice.Frontiers in Plant Science,2016,7(e0116646):4.
[8]YU X W,LIU Y M,WANG S,TAO Y,WANG Z K,MIJITI A,WANG Z,ZHANG H,MA H.A chickpea stress-responsive NACtranscription factor,CarNAC5,confers enhanced tolerance to drought stress in transgenic.Plant Growth Regulation,2016,79(2):187-197.
[9]KO J H,YANG S H,PARK A H,LEROUXEL O,HAN K H.ANAC012,a member of the plant-specific NAC transcription factor family,negatively regulates xylary fiber development in Arabidopsis thaliana.The Plant Journal,2007,50(6):1035-1048.
[10]ZHONG R,DEMURA T,YE Z H.SND1,a NAC domain transcription factor,is a key regulator of secondary wall synthesis in fibers of Arabidopsis.The Plant Cell,2006,18(11):3158-3170.
[11]YAMAGUCHI M,KUBO M,FUKUDA H,DEMURA T.Vascular-related NAC-domain7 is involved in the differentiation of all types of xylem vessels in Arabidopsis roots and shoots.The Plant Journal,2008,55(4):652-664.
[12]ODA-YAMAMIZO C,MITSUDA N,SAKAMOTO S,OGAWA D,OHME-TAKAGI M,OHMIYA A.ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves.Scientific Reports,2016,6:23609.
[13]TAKASAKI H,MARUYAMA K,TAKAHASHI F,FUJITA M,YOSHIDA T,NAKASHIMA K,MYOUGA F,TOYOOKA K,YAMAGUCHI-SHINOZAKI K,SHINOZAKI K.SNAC-As,stress-responsive NAC transcription factors,mediate ABA-inducible leaf senescence.The Plant Journal,2015,84(6):1114-1123.
[14]ZHU Y,YAN J W,LIU W J,LIU L,SHENG Y,SUN Y,LI Y Y,SCHELLER H,JIANG M Y,HOU X L,NI L,ZHANG A Y.Phosphorylation of a NAC transcription factor by a calcium/calmodulin-dependent protein kinase regulates abscisic acid-induced antioxidant defense in maize.Plant Physiology,2016,171(3):1651-1664.
[15]ZHOU H,WANG K L,WANG H L,GU C,DARE A,ESPLEY R,HEH P,ALLAN A,HAN Y P.Molecular genetics of blood-fleshed peach reveals activation of anthocyanin biosynthesis by NAC transcription factors.The Plant Journal,2015,82(1):105-121.
[16]安建平,宋来庆,赵玲玲,由春香,王小非,郝玉金.苹果愈伤组织超表达MdNAC029促进花青苷积累.园艺学报,2018,45(5):845-854.AN J P,SONG L Q,ZHAO L L,YOU C X,WANG X F,HAO Y J.Overexpression of MdNAC029 promotes anthocyanin accumulation in apple calli.Acta Horticulturae Sinica,2018,45(5):845-854.(in Chinese)
[17]NURUZZAMAN M,MANIMEKALAI R,SHRRONI A M.Genome-wide analysis of NAC transcription factor family in rice.Gene,2010,465(1/2):30-44.
[18]KIM S G,LEE S,SEO P J,KIM S K,KIM J K,PARK C M.Genome-scale screening and molecular characterization of membranebound transcription factors in Arabidopsis and rice.Genomics,2010,95(1):56-65.
[19]SONG Y,LIU H D,ZHOU Q,ZHANG H J,ZHANG Z D,LI Y D,WANG H B,LIU F Z.High-throughput sequencing of highbush blueberry transcriptome and analysis of basic helix-loop-helix transcription factors.Journal of Integrative Agriculture,2017,16(3):591-604.
[20]YIN X R,ALLAN A C,CHEN K S,FERGUSON I B.Kiwifruit EILand ERF genes involved in regulating fruit ripening.Plant Physiology,2010,153(3):1280-1292.
[21]JEFFERSON R A,KAVANAGH T A,BEVAN M W.GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO Journal,1987,6(13),3901-3907.
[22]PERTUZATTI P B,BARCIA M T,REBELLO L P G,GóMEZ-ALONSO S,DUARTE R M T,DUARTE M C T,GODOY H T,HERMOSíN-GUTIéRREZ I.Antimicrobial activity and differentiation of anthocyanin profiles of rabbiteye and highbush blueberry using HPLC-DAD-ESI-MS and multivariate analysis.Journal of Functional Foods,2016,26:506-516.
[23]ZHU X Y,CHEN J Y,XIE Z K,GAO J,REN G D,GAO S,ZHOU X,KUAI B K.Jasmonic acid promotes degreening via MYB2/3/4-and ANAC019/055/072-mediated regulation of major chlorophyll catabolic genes.The Plant Journal,2015,84(3):597-610.
[24]LI S,GAO J,YAO L Y,REN G D,ZHU X Y,GAO S,QIU K,ZHOUX,KUAI B K.The role of ANAC072 in the regulation of chlorophyll degradation during age-and dark-induced leaf senescence.Plant Cell Reports,2016,35(8):1729-1741.
[25]HUANG J C,PIATER L A,DUBERY I A.The NAC transcription factor gene ANAC072 is differentially expressed in Arabidopsis thaliana in response to microbe-associated molecular pattern(MAMP)molecules.Physiological and Molecular Plant Pathology,2012,80(80):19-27.
[26]TRAN L S,NAKASHIMA K,SAKUMA Y,SIMPSON S D,FUJITAY,MARUYAMA K,FUJITA M,SEKI M,SHINOZAKI K,YAMAGUCHI-SHINOZAKI K.Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter.The Plant Cell,2004,16(9):2481-2498.
[27]LI X Y,SUN H Y,PEI J B,DONG Y Y,WANG F W,CHEN H,SUNY P,WANG N,LI H Y,LI Y D.De novo sequencing and comparative analysis of the blueberry transcriptome to discover putative genes related to antioxidant.Gene,2012,511(1):54-61.
[28]SUN H Y,LIU Y S,GAI Y Z,GENG J M,CHEN L,LIU H D,KANGL M,TIAN Y W,LI Y D.De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis,transport and regulation.BMC Genomics,2015,16(1):652.
[29]BOREVITZ J O,XIA Y J,BLOUNT J,DIXON R,LAMB C.Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis.The Plant Cell,2000,12(12):2383-2393.
[30]TAKOS A,JAFFéF,JACOB S,BOGS J,ROBINSON S,WALKERA.Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples.Plant Physiology,2006,142(3):1216-1232.
[31]YAO G F,MING M L,ALLAN A,GU C,LI L T,WU X,WANG R Z,CHANG Y J,QI K J,ZHANG S L,WU J.Map-based cloning of the pear gene MYB114 identifies an interaction with other transcription factors to coordinately regulate fruit anthocyanin biosynthesis.The Plant Journal,2017,92:437-451.
[32]JAAKOLA L,POOLE M O,JONES M,K?M?R?INEN-KARPPINEN T,KOSKIM?KI J,HOHTOLA A,H?GGMAN H D,FRASER P,MANNING K J,KING G.A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits.Plant Physiology,2010,153(4):1619-1629.
[33]SAKURABA Y,KIM Y S,HAN S H,LEE B D,PAEK N C.The Arabidopsis transcription factor NAC016 promotes drought stress responses by repressing AREB1 transcription through a trifurcate feed-forward regulatory loop involving NAP.The Plant Cell,2015,27(6):1771-1787.
[34]QU Y T,DUAN M,ZHANG Z Q,DONG J L,WANG T.Overexpression of the Medicago falcata NAC transcription factor Mf NAC3 enhances cold tolerance in Medicago truncatula.Environmental and Experimental Botany,2016,129:67-76.