中间锦鸡儿CiATAF1基因的亚细胞定位及表达分析
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摘要
为研究NAC转录因子在非生物胁迫条件下的表达,以中间锦鸡儿为材料,克隆并分析了NAC转录因子家族CiATAF1基因。该基因ORF全长为873 bp,编码291个氨基酸,由2个内含子和3个外显子构成,具有典型NAC结构域。染色体步移法获得CiATAF1基因启动子826 bp,该启动子含有光、激素、厌氧诱导等多种响应元件。经拟南芥叶片原生质体瞬时表达分析亚细胞定位,CiATAF1基因主要定位于细胞核中。荧光定量分析表明:CiATAF1基因表达具有组织特异性,相比于中间锦鸡儿根和茎,在叶部表达量最高;在干旱、盐、冷、ABA处理下,CiATAF1基因表达量增加,推测该基因可能与非生物胁迫应答相关。CiATAF1同模式植物拟南芥及豆科其他植物的同源基因做多重序列分析,序列一致性为81.14%,表明ATAF1基因序列结构相对保守。生物信息学分析CiATAF1蛋白质属于不稳定的亲水蛋白,二级结构主要由α-螺旋、β折叠、无规则卷曲构成。研究结果对挖掘中间锦鸡儿抗非生物胁迫的基因和深入分析逆境胁迫机理具有重要意义。
To study the expression of NAC under abiotic stress,CiATAF1 gene from Caragana intermedia was cloned and analyzed. It had an ORF of 873 bp and encoded 291 amino acids,consisting of two introns and three exons,and presenting a typical NAC domain. The genome walking method was used to obtain the CiATAF1 promoter with a length of 826 bp,which contained various response elements to light,hormone and anaerobic induction. The subcellular localization was conducted by analysis of the transient expression of Arabidopsis leaf protoplasts,showing that the CiATAF1 gene was mainly located in the nuclei. Quantitative fluorescence RT-PCR analysis showed that the expression of CiATAF1 was tissue-specific,having the highest expression level in the leaves of Caragana intermedia and relatively low level in roots and stems. Under the treatments of drought,salt,cold and ABA,the expression level of CiATAF1 increased differentially. It was speculated that the CiATAF1 gene is related to the abiotic stress regulations. Multiple sequence analysis was carried out between CiATAF1 with homologues of Arabidopsis and other leguminous plants,and their consistency of the sequences was 81.14%,indicating that the ATAF1 was relatively conservative. Bioinformatics analysis showed that CiATAF1 protein was an unstable hydrophilic protein,and its secondary structure was composed of alpha helixes,beta sheets and random coils. This study had the significance for excavating genes resistant to abiotic stress in Caragana intermedia and for analyzing the mechanism of environmental stresses in depth.
To study the expression of NAC under abiotic stress,CiATAF1 gene from Caragana intermedia was cloned and analyzed. It had an ORF of 873 bp and encoded 291 amino acids,consisting of two introns and three exons,and presenting a typical NAC domain. The genome walking method was used to obtain the CiATAF1 promoter with a length of 826 bp,which contained various response elements to light,hormone and anaerobic induction. The subcellular localization was conducted by analysis of the transient expression of Arabidopsis leaf protoplasts,showing that the CiATAF1 gene was mainly located in the nuclei. Quantitative fluorescence RT-PCR analysis showed that the expression of CiATAF1 was tissue-specific,having the highest expression level in the leaves of Caragana intermedia and relatively low level in roots and stems. Under the treatments of drought,salt,cold and ABA,the expression level of CiATAF1 increased differentially. It was speculated that the CiATAF1 gene is related to the abiotic stress regulations. Multiple sequence analysis was carried out between CiATAF1 with homologues of Arabidopsis and other leguminous plants,and their consistency of the sequences was 81.14%,indicating that the ATAF1 was relatively conservative. Bioinformatics analysis showed that CiATAF1 protein was an unstable hydrophilic protein,and its secondary structure was composed of alpha helixes,beta sheets and random coils. This study had the significance for excavating genes resistant to abiotic stress in Caragana intermedia and for analyzing the mechanism of environmental stresses in depth.
引文
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[10] Mahmood K,Xu Z H,El-Kereamy A,Casaretto J A,Rothstein S J.The Arabidopsis transcription factor ANAC032 represses anthocyanin biosynthesis in response to high sucrose and oxidative and abiotic stresses[J].Frontiers in Plant Science,2016(7):1-15.doi:10.3389/Fpls.2016.01548.
[11] Oda-Yamamizo C,Mitsuda N,Sakamoto S,Ogawa D,Ohme-Takagi M,Ohmiya A.The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves[J].Sci Rep,2016(6):1-12.doi:10.1038/srep23609.
[12] Chen D,Chai S,McIntyre C L,Xue G P.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.doi:10.1007/s00299-017-2224-y.
[13] 岳俊燕,岳文冉,杨杞,于秀敏,杨飞芸,王光霞,李国婧,王瑞刚,丛靖宇.中间锦鸡儿转录因子基因CiNAC1的克隆及功能分析[J].西北植物学报,2016,36(7):1285-1293.doi:10.7606/j.issn.1000-4025.2016.07.1285.Yue J Y,Yue W R,Yang Q,Yu X M,Yang F Y,Wang G X,Li G J,Wang R G,Cong J Y.Cloning and functional analysis of CiNAC1 from Caragana intermedia[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(7):1285-1293.
[14] Han X,Feng Z,Xing D,Yang Q,Wang R,Qi L,Li G.Two NAC transcription factors from Caragana intermedia altered salt tolerance of the transgenic Arabidopsis[J].Bmc Plant Biology,2015,15(1):208-211.doi:10.1186/s12870-015-0591-5.
[15] Lescot M,Dehais P,Thijs G,Marchal K,Moreau Y,Van de Peer Y,Rouze P,Rombauts S.PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327.doi:10.1093/nar/30.1.325.
[16] Artimo P,Jonnalagedda M,Arnold K,Baratin D,Csardi G,de Castro E,Duvaud S,Flegel V,Fortier A,Gasteiger E,Grosdidier A,Hernandez C,Ioannidis V,Kuznetsov D,Liechti R,Moretti S,Mostaguir K,Redaschi N,Rossier G,Xenarios I,Stockinger H.ExPASy:SIB bioinformatics resource portal[J].Nucleic Acids Research,2012,40(W1):W597-W603.doi:10.1093/nar/gks400.
[17] Lu P L,Chen N Z,An R,Su Z,Qi B S,Ren F,Chen J,Wang X C.A novel drought-inducible gene,ATAF1,encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis[J].Plant Molecular Biology,2007,63(2):289-305.doi:10.1007/s11103-006-9089-8.
[18] Wu Y R,Deng Z Y,Lai J B,Zhang Y Y,Yang C P,Yin B J,Zhao Q Z,Zhang L,Li Y,Yang C W,Xie Q.Dual function of Arabidopsis ATAF1 in abiotic and biotic stress responses[J].Cell Research,2009,19(11):1279-1290.doi:10.1038/cr.2009.108.
[19] Liu Y C,Sun J,and Wu Y R.Arabidopsis ATAF1 enhances the tolerance to salt stress and ABA in transgenic rice[J].Journal of Plant Research,2016,129(5):955-962.doi:10.1007/s10265-016-0833-0.
[20] Garapati P,Xue G P,Munne-Bosch S,Balazadeh S.Transcription Factor ATAF1 in Arabidopsis promotes senescence by direct regulation of key chloroplast maintenance and senescence transcriptional cascades[J].Plant Physiology,2015,168(3):1122-1139.doi:10.1104/pp.15.00567.
[21] Jensen M K,Lindemose S,de Masi F,Reimer J J,Nielsen M,Perera V,Workman C T,Turck F,Grant M R,Mundy J,Petersen M,Skriver K.ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana[J].Febs Open Bio,2013(3):321-327.doi:10.1016/j.fob.2013.07.006.
[22] He X,Zhu L,Xu L,Guo W F,Zhang X L.GhATAF1,a NAC transcription factor,confers abiotic and biotic stress responses by regulating phytohormonal signaling networks[J].Plant Cell Reports,2016,35(10):2167-2179.doi:10.1007/s00299-016-2027-6.
[2] 张红梅,刘晓庆,陈华涛,袁星星,崔晓艳,张智民,黄中文,陈新.大豆转录因子GmWRKY58亚细胞定位及在非生物胁迫下的表达分析[J].华北农学报,2018,33(2):49-57.doi:10.7668/hbnxb.2018.02.008.Zhang H M,Liu X Q,Chen H T,Yuan X X,Cui X Y,Zhang Z M,Huang Z W,Chen X.Subcellular localization and expression analysis of a soybean transcription factor GmWRKY58 in response to abiotic stresses[J].Acta Agriculturae Boreali-Sinica,2018,33(2):49-57.
[3] 朱长保,刘志,徐辰峰,刘仁梅,林中正,成立瑞.全基因组分析盐胁迫下水稻转录因子的表达[J].北方农业学报,2018,46(6):38-43.doi:10.3969/j.issn.2096-1197.2018.06.08.Zhu C B,Liu Z,Xu C F,Liu R M,Lin Z Z,Cheng L R.Global genome expression analysis of transcription factors in rice under salt stress[J].Journal of Northern Agriculture,2018,46(6):38-43.
[4] 田李,孙景宽,刘京涛.植物转录因子家族在耐盐抗旱调控网络中的作用[J].生命科学,2015,27(2):217-227.doi:10.13376/j.cbls/2015031.Tian L,Sun J K,Liu J T.Role of different transcription factor families in the regulatory networks ofdrought and salinity tolerance in plants[J].Chinese Bulletin of Life Sciences,2015,27(2):217-227.
[5] Soutourina J.Transcription regulation by the Mediator complex[J].Nat Rev Mol Cell Biol,2018,19(4):262-274.doi:10.1038/nrm.2017.115.
[6] Wu D,Sun Y,Wang H,Shi H,Su M,Shan H,Li T,Li Q.The SlNAC8 gene of the halophyte Suaeda liaotungensis enhances drought and salt stress tolerance in transgenic Arabidopsis thaliana[J].Gene,2018,662:10-20.doi:10.1016/j.gene.2018.04.012.
[7] Kamiuchi Y,Yamamoto K,Furutani M,Tasaka M,Aida M.The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development[J].Frontiers in Plant Science,2014(5):1-9.doi:10.3389/fpls.2014.00165.
[8] Olsen A N,Ernst H A,Leggio L L,Skriver,K.NAC transcription factors:structurally distinct,functionally diverse[J].Trends in Plant Science,2005,10(2):79-87.doi:10.1016/j.tplants.2004.12.010.
[9] Mahmood K,El-Kereamy A,Kim S H,Nambara E,Rothstein S J.ANAC032 positively regulates age-dependent and stress-induced senescence in Arabidopsis thaliana[J].Plant and Cell Physiology,2016,57(10):2029-2046.doi:10.1093/pcp/pcw120.
[10] Mahmood K,Xu Z H,El-Kereamy A,Casaretto J A,Rothstein S J.The Arabidopsis transcription factor ANAC032 represses anthocyanin biosynthesis in response to high sucrose and oxidative and abiotic stresses[J].Frontiers in Plant Science,2016(7):1-15.doi:10.3389/Fpls.2016.01548.
[11] Oda-Yamamizo C,Mitsuda N,Sakamoto S,Ogawa D,Ohme-Takagi M,Ohmiya A.The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves[J].Sci Rep,2016(6):1-12.doi:10.1038/srep23609.
[12] Chen D,Chai S,McIntyre C L,Xue G P.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.doi:10.1007/s00299-017-2224-y.
[13] 岳俊燕,岳文冉,杨杞,于秀敏,杨飞芸,王光霞,李国婧,王瑞刚,丛靖宇.中间锦鸡儿转录因子基因CiNAC1的克隆及功能分析[J].西北植物学报,2016,36(7):1285-1293.doi:10.7606/j.issn.1000-4025.2016.07.1285.Yue J Y,Yue W R,Yang Q,Yu X M,Yang F Y,Wang G X,Li G J,Wang R G,Cong J Y.Cloning and functional analysis of CiNAC1 from Caragana intermedia[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(7):1285-1293.
[14] Han X,Feng Z,Xing D,Yang Q,Wang R,Qi L,Li G.Two NAC transcription factors from Caragana intermedia altered salt tolerance of the transgenic Arabidopsis[J].Bmc Plant Biology,2015,15(1):208-211.doi:10.1186/s12870-015-0591-5.
[15] Lescot M,Dehais P,Thijs G,Marchal K,Moreau Y,Van de Peer Y,Rouze P,Rombauts S.PlantCARE,a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J].Nucleic Acids Research,2002,30(1):325-327.doi:10.1093/nar/30.1.325.
[16] Artimo P,Jonnalagedda M,Arnold K,Baratin D,Csardi G,de Castro E,Duvaud S,Flegel V,Fortier A,Gasteiger E,Grosdidier A,Hernandez C,Ioannidis V,Kuznetsov D,Liechti R,Moretti S,Mostaguir K,Redaschi N,Rossier G,Xenarios I,Stockinger H.ExPASy:SIB bioinformatics resource portal[J].Nucleic Acids Research,2012,40(W1):W597-W603.doi:10.1093/nar/gks400.
[17] Lu P L,Chen N Z,An R,Su Z,Qi B S,Ren F,Chen J,Wang X C.A novel drought-inducible gene,ATAF1,encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in Arabidopsis[J].Plant Molecular Biology,2007,63(2):289-305.doi:10.1007/s11103-006-9089-8.
[18] Wu Y R,Deng Z Y,Lai J B,Zhang Y Y,Yang C P,Yin B J,Zhao Q Z,Zhang L,Li Y,Yang C W,Xie Q.Dual function of Arabidopsis ATAF1 in abiotic and biotic stress responses[J].Cell Research,2009,19(11):1279-1290.doi:10.1038/cr.2009.108.
[19] Liu Y C,Sun J,and Wu Y R.Arabidopsis ATAF1 enhances the tolerance to salt stress and ABA in transgenic rice[J].Journal of Plant Research,2016,129(5):955-962.doi:10.1007/s10265-016-0833-0.
[20] Garapati P,Xue G P,Munne-Bosch S,Balazadeh S.Transcription Factor ATAF1 in Arabidopsis promotes senescence by direct regulation of key chloroplast maintenance and senescence transcriptional cascades[J].Plant Physiology,2015,168(3):1122-1139.doi:10.1104/pp.15.00567.
[21] Jensen M K,Lindemose S,de Masi F,Reimer J J,Nielsen M,Perera V,Workman C T,Turck F,Grant M R,Mundy J,Petersen M,Skriver K.ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana[J].Febs Open Bio,2013(3):321-327.doi:10.1016/j.fob.2013.07.006.
[22] He X,Zhu L,Xu L,Guo W F,Zhang X L.GhATAF1,a NAC transcription factor,confers abiotic and biotic stress responses by regulating phytohormonal signaling networks[J].Plant Cell Reports,2016,35(10):2167-2179.doi:10.1007/s00299-016-2027-6.