青杄转录因子PwNAC30及其启动子序列的克隆与表达分析
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摘要
该研究以青杄(Picea wilsonii)为实验材料,通过PCR从青杄的cDNA文库中克隆得到一个NAC转录因子,命名为PwNAC30。生物信息学分析显示,PwNAC30开放阅读框1 179bp,共编码392个氨基酸,在其N端存在保守的NAM(no apical meristem)结构域,可分为A～E等5个亚结构域。多序列对比和系统进化树分析显示,PwNAC30蛋白与同为云杉属的北美云杉(Picea sitchensis)聚为一类。启动子克隆分析显示,PwNAC30基因启动子上存在脱落酸(ABA)、赤霉素(GA)、茉莉酸甲酯(MeJA)、TC-rich repeats等激素和逆境响应元件,在GA、ABA、MeJA、低温、干旱、盐的处理下,其启动子活性均明显增强。荧光定量PCR分析表明,PwNAC30在球果中的表达量最高,而在花粉和种子中的表达量最低。PwNAC30对于盐、干旱、低温、ABA、MeJA、GA处理均有响应,尤其对盐、干旱、MeJA的响应最为显著。亚细胞定位结果显示,PwNAC30蛋白定位于细胞核与细胞质,主要定位于细胞核中。酵母单杂及双杂结果表明,PwNAC30蛋白的全长和N端没有转录激活活性,而C端有转录激活活性,且PwNAC30自身能形成同源二聚体。研究表明,青杄PwNAC30基因可以作为一个转录因子发挥作用,其转录激活活性在C端,且自身能够形成同源二聚体结构;PwNAC30基因广泛参与了ABA、GA、MeJA等激素的信号通路,并对盐、干旱、低温处理有响应。
In this study,Picea wilsonii was used as experimental material to clone a NAC transcription factor from cDNA library by PCR,which was named PwNAC30.Bioinformatics analysis showed that PwNAC30 open reading frame was 1 179 bp,encoding a total of 392 amino acids,a conserved NAM(no apical meristem)domain at its N-terminus and can be divided into five sub-domains of A-E.Multi-sequence comparison and phylogenetic tree analysis showed that PwNAC30 protein was clustered with North American spruce(P.sitchensis)of the Picea.Promoter cloning analysis showed that there were hormones and stress response elements such as gibberellin(GA),abscisic acid(ABA),methyl jasmonate(MeJA),TC-rich repeats on the PwNAC30 promoter.Its promoter activity was significantly enhanced under ABA,GA,MeJA,low temperature,drought and salt treatments.Real-time PCR analysis showed that PwNAC30 had the highest expression in cones and the lowest in pollens and seeds.PwNAC30 responded to salt,drought,low temperature,ABA,MeJA,GA treatments,especially for salt,drought and MeJA.Subcellular localization experiments showed that PwNAC30 protein is localized in the nucleus and cytoplasm,mainly in the nucleus.Yeast one hybrid and two hybrid experiments showed that PwNAC30 protein had no transcriptional activation activity at its full length and N-terminus,whereas its C-terminus had transcriptional activation activity.PwNAC30 can form homodimers by itself.Studies have shown that PwNAC30 is a transcription factor with transcriptional activation activity at the C-terminus and is capable of forming a homodimeric structure by itself.It is widely involved in the signaling pathways of ABA,GA,MeJA and other hormones,and responds to salt,drought and low temperature.
In this study,Picea wilsonii was used as experimental material to clone a NAC transcription factor from cDNA library by PCR,which was named PwNAC30.Bioinformatics analysis showed that PwNAC30 open reading frame was 1 179 bp,encoding a total of 392 amino acids,a conserved NAM(no apical meristem)domain at its N-terminus and can be divided into five sub-domains of A-E.Multi-sequence comparison and phylogenetic tree analysis showed that PwNAC30 protein was clustered with North American spruce(P.sitchensis)of the Picea.Promoter cloning analysis showed that there were hormones and stress response elements such as gibberellin(GA),abscisic acid(ABA),methyl jasmonate(MeJA),TC-rich repeats on the PwNAC30 promoter.Its promoter activity was significantly enhanced under ABA,GA,MeJA,low temperature,drought and salt treatments.Real-time PCR analysis showed that PwNAC30 had the highest expression in cones and the lowest in pollens and seeds.PwNAC30 responded to salt,drought,low temperature,ABA,MeJA,GA treatments,especially for salt,drought and MeJA.Subcellular localization experiments showed that PwNAC30 protein is localized in the nucleus and cytoplasm,mainly in the nucleus.Yeast one hybrid and two hybrid experiments showed that PwNAC30 protein had no transcriptional activation activity at its full length and N-terminus,whereas its C-terminus had transcriptional activation activity.PwNAC30 can form homodimers by itself.Studies have shown that PwNAC30 is a transcription factor with transcriptional activation activity at the C-terminus and is capable of forming a homodimeric structure by itself.It is widely involved in the signaling pathways of ABA,GA,MeJA and other hormones,and responds to salt,drought and low temperature.
引文
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[2]SOUER E,HOUWELINGEN A V,KLOOS D,et al.The no apical meristem,gene of petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries[J].Cell,1996,85(2):159-170.
[3]SHEN H,YIN Y,CHEN F,et al.A bioinformatic analysis of NAC genes for plant cell wall development in relation to lignocellulosic bioenergy production[J].Bioenergy Research,2009,2(4):217-232.
[4]DUVAL M,HSIEH T F,KIM S Y,et al.Molecular characterization of AtNAM:a member of the Arabidopsis NAC domain superfamily[J].Plant Molecular Biology,2002,50(2):237-248.
[5]OOKA H,SATOH K,DOI K,et al.Comprehensive analysis of NAC family genes in Oryza sativaand Arabidopsis thaliana[J].DNA Research,2003,10(6):239-247.
[6]JIA D,GONG X,LI M,et al.Overexpression of a novel apple NAC transcription factor gene,MdNAC1,confers the dwarf phenotype in transgenic apple(Malus domestica)[J].Genes,2018,9(5):229.
[7]KOU X,ZHAO Y,WU C,et al.SNAC4,and SNAC9,transcription factors show contrasting effects on tomato carotenoids biosynthesis and softening[J].Postharvest Biology&Technology,2018,144:9-19.
[8]DALMAN K,WIND J J,NEMESIOGORRIZ M,et al.Overexpression of PaNAC03,a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development[J].Bmc Plant Biology,2017,17(1):6.
[9]ZHANG J,HUANG G,et al.The cotton(Gossypium hirsutum)NAC transcription factor(FSN1)as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers[J].New Phytologist,2017,217(2):625-640.
[10]YU S,HUANG A,LI J,et al.OsNAC45,plays complex roles by mediating POD activity and the expression of development-related genes under various abiotic stresses in rice root[J].Plant Growth Regulation,2018,84(3):519-531.
[11]MAO C,DING J,ZHANG B,et al.OsNAC2positively affects salt-induced cell death and binds to the OsAP37and OsCOX11 promoters[J].Plant Journal,2018,94(3):454-468.
[12]YANG X W,WANG X Y,LU J,et al.Overexpression of a Miscanthus lutarioriparius NAC gene MlNAC5confers enhanced drought and cold tolerance in Arabidopsis[J].Plant Cell Reports,2015,34(6):943.
[13]YU X,LIU Y,WANG S,et al.CarNAC4,a NAC-type chickpea transcription factor conferring enhanced drought and salt stress tolerances in Arabidopsis[J].Plant Cell Reports,2015,35(3):1-15.
[14]刘钊金.山西关帝山区青育苗与移植技术[J].山西林业,2017,(3):32-33.LIU Z J.Green seedling and tansplanting technology in Guandi Mountain area of Shanxi Province[J].Shanxi Forestry,2017,(3):32-33.
[15]张大勇,赵松岭,张鹏云,等.青杄林恢复演替过程中的邻体竞争效应及邻体干扰指数的改进模型[J].生态学报,1989,9(1):52-58.ZHANG D Y,ZHAO S L,ZHANG P Y,et al.Improved model of adjacent competition and adjacent interference index in restoration succession of Qinglan forest[J].Chinese Journal of Ecology,1989,9(1):52-58.
[16]ZHANG T,ZHANG D,LIU Y,et al.Overexpression of a NF-YB3transcription factor fromPicea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana[J].Plant Physiology&Biochemistry,2015,94:153-164.
[17]周燕妮,李艳芳,张通,等.青杄PwUSP2基因的克隆和表达分析[J].植物生理学报,2015,51(8):1 307-1 314.ZHOU Y N,LI Y F,ZHANG T,et al.Cloning and expression analysis of PwUSP2gene in Picea wilsonii[J].Chinese Journal of Plant Physiology,2015,51(8):1 307-1 314.
[18]HELLENS R P,ALLAN A C,FRIEL E N,et al.Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants[J].Plant Methods,2005,1(1):13-13.
[19]QI T,WANG J,HUANG H,et al.Regulation of jasmonate-induced leaf senescence by antagonism between bHLH subgroup IIIe and IIId factors in Arabidopsis[J].Plant Cell,2015,27(6):1 634-1 649.
[20]YU Y,LI Y,HUANG G,et al.PwHAP5,a CCAAT-binding transcription factor,interacts with PwFKBP12and plays a role in pollen tube growth orientation in Picea wilsonii.[J].Journal of Experimental Botany,2011,62(14):4 805.
[21]HAO Y J,SONG Q X,CHEN H W,et al.Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation[J].Planta,2010,232(5):1 033-1 043.
[22]ERNST H A,OLSEN A N,SKRIVER K,et al.Structure of the conserved domain of ANAC,a member of the NACfamily of transcription factors[J].Embo Reports,2004,5(3):297-303.
[23]HE X J,MU R L,CAO W H,et al.AtNAC2,a transcription factor downstream of ethylene and auxin signaling pathways,is involved in salt stress response and lateral root development[J].Plant Journal,2005,44(6):903-916.
[24]JEONG J S,PARK Y T,JUNG H,et al.Rice NAC proteins act as homodimers and heterodimers[J].Plant Biotechnology Reports,2009,3(2):127-134.
[25]CHEN D,CHAI S,MCINTYRE C L,et al.Overexpression of a predominantly root-expressed NAC transcription factor in wheat roots enhances root length,biomass and drought tolerance[J].Plant Cell Reports,2018,37(2):225-237.
[26]LIU C,WANG B,LI Z,et al.TsNAC1is a key transcription factor in abiotic stress resistance and growth[J].Plant Physiology,2017,176(1):742-756.
[27]QU Y,DUAN M,ZHANG Z,et al.Overexpression of the Medicago falcata,NAC transcription factor MfNAC3,enhances cold tolerance in Medicago truncatula[J].Environmental&Experimental Botany,2016,129:67-76.
[28]HUANG L,HONG Y,ZHANG H,et al.Rice NAC transcription factor ONAC095plays opposite roles in drought and cold stress tolerance[J].Bmc Plant Biology,2016,16(1):203.
[29]DELESSERT C,KAZAN K,et al.The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis[J].Plant Journal,2010,43(5):745-757.
[30]WANG G,ZHANG S,MA X,et al.A stress‐associated NAC transcription factor(SlNAC35)from tomato plays a positive role in biotic and abiotic stresses[J].Physiologia Plantarum,2016,158(1):45-64.
[31]WU R,DUAN L,PRUNEDAPAZ J,et al.The 6xABREsynthetic promoter enables the spatiotemporal analysis of ABA-mediated transcriptional regulation[J].Plant Physiology,2018,177(4):1 650-1 665.
[2]SOUER E,HOUWELINGEN A V,KLOOS D,et al.The no apical meristem,gene of petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries[J].Cell,1996,85(2):159-170.
[3]SHEN H,YIN Y,CHEN F,et al.A bioinformatic analysis of NAC genes for plant cell wall development in relation to lignocellulosic bioenergy production[J].Bioenergy Research,2009,2(4):217-232.
[4]DUVAL M,HSIEH T F,KIM S Y,et al.Molecular characterization of AtNAM:a member of the Arabidopsis NAC domain superfamily[J].Plant Molecular Biology,2002,50(2):237-248.
[5]OOKA H,SATOH K,DOI K,et al.Comprehensive analysis of NAC family genes in Oryza sativaand Arabidopsis thaliana[J].DNA Research,2003,10(6):239-247.
[6]JIA D,GONG X,LI M,et al.Overexpression of a novel apple NAC transcription factor gene,MdNAC1,confers the dwarf phenotype in transgenic apple(Malus domestica)[J].Genes,2018,9(5):229.
[7]KOU X,ZHAO Y,WU C,et al.SNAC4,and SNAC9,transcription factors show contrasting effects on tomato carotenoids biosynthesis and softening[J].Postharvest Biology&Technology,2018,144:9-19.
[8]DALMAN K,WIND J J,NEMESIOGORRIZ M,et al.Overexpression of PaNAC03,a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development[J].Bmc Plant Biology,2017,17(1):6.
[9]ZHANG J,HUANG G,et al.The cotton(Gossypium hirsutum)NAC transcription factor(FSN1)as a positive regulator participates in controlling secondary cell wall biosynthesis and modification of fibers[J].New Phytologist,2017,217(2):625-640.
[10]YU S,HUANG A,LI J,et al.OsNAC45,plays complex roles by mediating POD activity and the expression of development-related genes under various abiotic stresses in rice root[J].Plant Growth Regulation,2018,84(3):519-531.
[11]MAO C,DING J,ZHANG B,et al.OsNAC2positively affects salt-induced cell death and binds to the OsAP37and OsCOX11 promoters[J].Plant Journal,2018,94(3):454-468.
[12]YANG X W,WANG X Y,LU J,et al.Overexpression of a Miscanthus lutarioriparius NAC gene MlNAC5confers enhanced drought and cold tolerance in Arabidopsis[J].Plant Cell Reports,2015,34(6):943.
[13]YU X,LIU Y,WANG S,et al.CarNAC4,a NAC-type chickpea transcription factor conferring enhanced drought and salt stress tolerances in Arabidopsis[J].Plant Cell Reports,2015,35(3):1-15.
[14]刘钊金.山西关帝山区青育苗与移植技术[J].山西林业,2017,(3):32-33.LIU Z J.Green seedling and tansplanting technology in Guandi Mountain area of Shanxi Province[J].Shanxi Forestry,2017,(3):32-33.
[15]张大勇,赵松岭,张鹏云,等.青杄林恢复演替过程中的邻体竞争效应及邻体干扰指数的改进模型[J].生态学报,1989,9(1):52-58.ZHANG D Y,ZHAO S L,ZHANG P Y,et al.Improved model of adjacent competition and adjacent interference index in restoration succession of Qinglan forest[J].Chinese Journal of Ecology,1989,9(1):52-58.
[16]ZHANG T,ZHANG D,LIU Y,et al.Overexpression of a NF-YB3transcription factor fromPicea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana[J].Plant Physiology&Biochemistry,2015,94:153-164.
[17]周燕妮,李艳芳,张通,等.青杄PwUSP2基因的克隆和表达分析[J].植物生理学报,2015,51(8):1 307-1 314.ZHOU Y N,LI Y F,ZHANG T,et al.Cloning and expression analysis of PwUSP2gene in Picea wilsonii[J].Chinese Journal of Plant Physiology,2015,51(8):1 307-1 314.
[18]HELLENS R P,ALLAN A C,FRIEL E N,et al.Transient expression vectors for functional genomics,quantification of promoter activity and RNA silencing in plants[J].Plant Methods,2005,1(1):13-13.
[19]QI T,WANG J,HUANG H,et al.Regulation of jasmonate-induced leaf senescence by antagonism between bHLH subgroup IIIe and IIId factors in Arabidopsis[J].Plant Cell,2015,27(6):1 634-1 649.
[20]YU Y,LI Y,HUANG G,et al.PwHAP5,a CCAAT-binding transcription factor,interacts with PwFKBP12and plays a role in pollen tube growth orientation in Picea wilsonii.[J].Journal of Experimental Botany,2011,62(14):4 805.
[21]HAO Y J,SONG Q X,CHEN H W,et al.Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation[J].Planta,2010,232(5):1 033-1 043.
[22]ERNST H A,OLSEN A N,SKRIVER K,et al.Structure of the conserved domain of ANAC,a member of the NACfamily of transcription factors[J].Embo Reports,2004,5(3):297-303.
[23]HE X J,MU R L,CAO W H,et al.AtNAC2,a transcription factor downstream of ethylene and auxin signaling pathways,is involved in salt stress response and lateral root development[J].Plant Journal,2005,44(6):903-916.
[24]JEONG J S,PARK Y T,JUNG H,et al.Rice NAC proteins act as homodimers and heterodimers[J].Plant Biotechnology Reports,2009,3(2):127-134.
[25]CHEN D,CHAI S,MCINTYRE C L,et al.Overexpression of a predominantly root-expressed NAC transcription factor in wheat roots enhances root length,biomass and drought tolerance[J].Plant Cell Reports,2018,37(2):225-237.
[26]LIU C,WANG B,LI Z,et al.TsNAC1is a key transcription factor in abiotic stress resistance and growth[J].Plant Physiology,2017,176(1):742-756.
[27]QU Y,DUAN M,ZHANG Z,et al.Overexpression of the Medicago falcata,NAC transcription factor MfNAC3,enhances cold tolerance in Medicago truncatula[J].Environmental&Experimental Botany,2016,129:67-76.
[28]HUANG L,HONG Y,ZHANG H,et al.Rice NAC transcription factor ONAC095plays opposite roles in drought and cold stress tolerance[J].Bmc Plant Biology,2016,16(1):203.
[29]DELESSERT C,KAZAN K,et al.The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis[J].Plant Journal,2010,43(5):745-757.
[30]WANG G,ZHANG S,MA X,et al.A stress‐associated NAC transcription factor(SlNAC35)from tomato plays a positive role in biotic and abiotic stresses[J].Physiologia Plantarum,2016,158(1):45-64.
[31]WU R,DUAN L,PRUNEDAPAZ J,et al.The 6xABREsynthetic promoter enables the spatiotemporal analysis of ABA-mediated transcriptional regulation[J].Plant Physiology,2018,177(4):1 650-1 665.