草莓FvARF5基因的克隆、生物信息学及表达分析
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摘要
生长素响应因子ARF是一类新的转录因子,通过激活或抑制生长素响应基因的表达调控植物的生长发育过程。为解析草莓生长素响应因子FvARF5在草莓形成过程中的功能,以草莓为试验材料,克隆了FvARF5基因并进行了生物信息学分析,利用qPCR分析了FvARF5在草莓不同组织及植物激素处理下的表达模式。结果表明,FvARF5基因开放阅读框为2 745 bp,编码914个氨基酸,蛋白质分子质量为100.65 ku,等电点为5.25。多序列比对和系统进化树分析表明,FvARF5基因具有保守的B3 DNA结合结构域和生长素响应因子保守结构域,与蔷薇科月季ARF5基因的进化关系最近,相似度达95%。亚细胞定位预测分析表明,FvARF5蛋白定位于细胞核中。启动子分析显示,FvARF5基因具有多样化的激素应答元件。qPCR结果表明,FvARF5存在组织表达差异,在茎和绿果中表达量较高。另外,FvARF5基因表达受到生长素和赤霉素的诱导,初步推测该基因可能参与了生长素和赤霉素调控草莓果实的形成过程。为进一步研究FvARF5基因的功能奠定基础。
Auxin response factor ARF is a new class of transcription factors. They play an important role during plant development as an activation or inhibition in auxin-related gene expression. In order to identify the function of FvARF5 gene during strawberry development,FvARF5 gene was cloned by RT-PCR from Fragaria vesca and analyzed by bioinformatics. Furthermore,Real-time quantitative PCR(qPCR)assay was performed to investigate the tissue-specific expression of FvARF5 gene,and its expression patterns under various phytohormone treatments. The results showed that the open reading frame of FvARF5 gene was 2 745 bp,encoding a protein of 914 amino acids. The molecular weight of FvARF5 protein was 100.65 ku,and the theoretical pI was 5.25. Multiple sequence alignment and phylogenetic tree analysis showed that FvARF5 was closely related to ARF5 from Rosa chinensis with 95% identity. FvARF5 was predicated to be the auxin response gene that was located in the nucleus. Promoter analysis revealed that the sequence of FvARF5 contained multiple hormone response elements. Real-time PCR analysis showed that FvARF5 had specific expression difference in different organs,and it had a higher expression in stem and green fruit. Also,FvARF5 had significant response to exogenous IAA and GA treatment. The results showed that the FvARF5 might play an important role in auxin and GA signal transduction during strawberry development. This research provided a theoretical basis for further study of the function and molecular regulation mechanism of FvARF5.
Auxin response factor ARF is a new class of transcription factors. They play an important role during plant development as an activation or inhibition in auxin-related gene expression. In order to identify the function of FvARF5 gene during strawberry development,FvARF5 gene was cloned by RT-PCR from Fragaria vesca and analyzed by bioinformatics. Furthermore,Real-time quantitative PCR(qPCR)assay was performed to investigate the tissue-specific expression of FvARF5 gene,and its expression patterns under various phytohormone treatments. The results showed that the open reading frame of FvARF5 gene was 2 745 bp,encoding a protein of 914 amino acids. The molecular weight of FvARF5 protein was 100.65 ku,and the theoretical pI was 5.25. Multiple sequence alignment and phylogenetic tree analysis showed that FvARF5 was closely related to ARF5 from Rosa chinensis with 95% identity. FvARF5 was predicated to be the auxin response gene that was located in the nucleus. Promoter analysis revealed that the sequence of FvARF5 contained multiple hormone response elements. Real-time PCR analysis showed that FvARF5 had specific expression difference in different organs,and it had a higher expression in stem and green fruit. Also,FvARF5 had significant response to exogenous IAA and GA treatment. The results showed that the FvARF5 might play an important role in auxin and GA signal transduction during strawberry development. This research provided a theoretical basis for further study of the function and molecular regulation mechanism of FvARF5.
引文
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[7] Li S B,Ouyang W Z,Hou X J,Xie L L,Hu C G,Zhang J Z.Genome-wide identification,isolation and expression analysis of auxin response factor(ARF)gene family in sweet orange(Citrus sinensis)[J].Frontiers in Plant Science,2015,6:119.doi:10.3389/fpls.2015.00119.
[8] Wan S,Li W,Zhu Y,Yang S H,Song Y J,Wang J H.Genome-wide identification,characterization and expression analysis of the auxin response factor gene family in Vitis vinifera[J].Plant Cell Reports,2014,33(8):1365-1375.doi:10.1007/s00299-014-1622-7.
[9] Wei H,Jiao Z,Hou X W,Yan Y,Wei Y X,Liu J H,Li M Y,Xu B Y,Jin Z Q.The auxin response factor gene family in banana:genome-wide identification and expression analyses during development,ripening,and abiotic stress[J].Frontiers in Plant Science,2015,6(742):742.doi:10.3389/fpls.2015.00742.
[10] Zouine M,Fu Y,Chateigner-Boutin A L,Mila I,Frasse P,Wang H,Audran C,Roustan J P,Bouzayen M.Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing[J].PLoS One,2014,9(1):e84203.doi:10.1371/journal.pone.0084203.
[11] Ellis C M,Nagpal P,Young J C,Hagen G,Guilfoyle T J,Reed J W.AUXIN RESPONSE FACTOR 1 and AUXIN RESPONSE FACTOR 2 regulate senescence and floral organ abscission in Arabidopsis thaliana[J].Development,2005,132(20):4563-4574.doi:10.1242/dev.02012.
[12] Goetz M,Vivian-Smith A,Johnson S D,Koltunow A M.AUXIN RESPONSE FACTOR 8 is a negative regulator of fruit initiation in Arabidopsis[J].Plant Cell,2006,18(8):1873-1886.doi:10.1105/tpc.105.037192.
[13] Wilmoth J C,Wang S,Tiwari S B,Joshi A D,Hagen G,Guilfoyle T J,Alonso J M,Ecker J R,Reed J W.NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root for mation[J].Plant Journal,2005,43(1):118-130.doi:10.1111/j.1365-313X.2005.02432.x.
[14] Okushima Y,Fukaki H,Onoda M,Theologis A,Tasaka M.ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis[J].Plant Cell,2007,19(1):118-130.doi:10.1105/tpc.106.047761.
[15] Sagar M,Chervin C,Mila I,Hao Y W,Roustan J P,Benichou M,Gibon Y,Biais B,Maury P,Latche A,Pech J C,Bouzayen M,Zouine M.Sl-ARF4,an auxin response factor involved in the control of sugar metabolism during tomato fruit development[J].Plant Physiology,2013,161(3):1362-1374.doi:10.1104/pp.113.213843.
[16] Aida M,Vernoux T,Furutani M,Traas J,Tasaka M.Roles of PIN-FORMED1 and MONOPTEROS in pattern formation of the apical region of the Arabidopsis embryo[J].Development,2002,129(17):3965.doi:10.1101/gad.242202.
[17] Wenzel C L,Schuetz M,Yu Q,Mattsson J.Dynamics of MONOPTEROS and PIN‐FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana[J].Plant Journal,2010,49(3):387-398.doi:10.1111/j.1365-313X.2006.02977.x.
[18] 李青青,南文斌,张汉马.ARF5/MONOPTEROS(MP)调控作用研究进展[J].西北植物学报,2016,36(1):197-203.doi:10.7606/j.issn.1000-4025.2016.01.0197.Li Q Q,Nan W B,Zhang H M.Progress on the functions of ARF5/MONOPTEROS(MP)[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(1):197-203.
[19] Ckurshumova W,Smirnova T,Marcos D,Zayed Y,Berleth T.Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation[J].New Phytologist,2015,204(3):556-566.doi:10.1111/nph.13014.
[20] Krogan N T,Berleth T.A dominant mutation reveals asymmetry in MP/ARF5 function along the adaxial-abaxial axis of shoot lateral organs[J].Plant Signaling & Behavior,2012,7(8):940-943.doi:10.1111/nph.13014.
[21] Liu S,Zhang Y,Feng Q,Qin L,Pan C,Lamin-Samu A T,Lu G.ONSE FACTOR 5 regulates fruit set and development via the Tomato AUXIN RESP mediation of auxin and gibberellin signaling[J].Scientific Reports,2018,8(1):2971.doi:10.1038/s41598-018-21315-y.
[22] 苏丽艳,田爱梅,陶贵荣,李蒙.苹果MdCaM的克隆及其对果实釆后非生物胁迫的响应[J].华北农学报,2017,32(1):47-52.doi:10.7668/hbnxb.2017.01.008.Su L Y,Tian A M,Tao G R,Li M.Cloning of MdCaM and its expression responses under abiotic stresses during Malus domestica postharvest storage[J].Acta Agriculturae Boreali-Sinica,2017,32(1):47-52.
[23] Kumar R,Tyagi A K,Sharma A K.Genome-wide analysis of auxin response factor(ARF)gene family from tomato and analysis of their role in flower and fruit development[J].Molecular Genetics & Genomics,2011,285(3):245-260.doi:10.1007/s00438-011-0602-7.
[24] Shen C,Yue R,Sun T,Zhang L,Xu L Q,Tie S G,Wang H Z,Yang Y J.Genome-wide identification and expression analysis of auxin response factor gene family in Medicago truncatula[J].Frontiers in Plant Science,2015,6(11):73.doi:10.3389/fpls.2015.00073.
[25] De J M,Woltersarts M,Garcíamartínez J L,Mariani C,Vriezen W H.The Solanum lycopersicum AUXIN RESPONSE FACTOR 7(SlARF7)mediates cross-talk between auxin and gibberellin signalling during tomato fruit set and development[J].Journal of Experimental Botany,2011,62(2):617.doi:10.1093/jxb/erq293.
[26] Liu N,Wu S,Van J H,Wang Y,Ding B,Fei Z,Clarke T H,Reed J W,van der Knaap E.Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[J].Journal of Experimental Botany,2014,65(9):2507-2520.doi:10.1093/jxb/eru141.
[2] 张运涛,王桂霞,董静,王萍.草莓畸形果形成的原因分析[J].落叶果树,2004,36(6):10-13.doi:10.3969/j.issn.1002-2910.2004.06.004.Zhang Y T,Wang G X,Dong J,Wang P.Analysis of the reason of the formation of malformed fruits in strawberry[J].Deciduous Fruits,2004,36(6):10-13.
[3] Estrada-johnson E,Csukasi F,Pizarro C M,Vallarino J G,Kiryakova Y,Vioque A,Brumos J,Medina-Escobar N,Botella1 M A,Alonso J M,Fernie A R,Sánchez-Sevilla J F,Osorio S,Valpuesta V.Transcriptomic analysis in strawberry fruits reveals active auxin biosynthesis and signaling in the ripe receptacle[J].Frontiers in Plant Science,2017,8:889.doi:10.3389/fpls.2017.00889.
[4] Guilfoyle T J,Hagen G.Auxin response factors[J].Plant Growth Regulation,2007,10(5):453.doi:10.1016/j.pbi.2007.08.014.
[5] Li S B,Xie Z Z,Hu C G,Zhang J Z.A review of auxin response factors(ARFs)in plants[J].Frontiers in Plant Science,2016,7(742):47.doi:10.3389/fpls.2016.00047.
[6] Okushima Y,Overvoorde P J,Arima K,Alonso J M,Chan A,Chang C,Ecker J R,Hughes B,Lui A,Nguyen D,Onodera C,Quach H,Smith A,Yu G,Theologis A.Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in arabidopsis thaliana:unique and overlapping functions of ARF 7 and ARF 19[J].Plant Cell,2005,17(2):444-463.doi:10.1105/tpc.104.028316.
[7] Li S B,Ouyang W Z,Hou X J,Xie L L,Hu C G,Zhang J Z.Genome-wide identification,isolation and expression analysis of auxin response factor(ARF)gene family in sweet orange(Citrus sinensis)[J].Frontiers in Plant Science,2015,6:119.doi:10.3389/fpls.2015.00119.
[8] Wan S,Li W,Zhu Y,Yang S H,Song Y J,Wang J H.Genome-wide identification,characterization and expression analysis of the auxin response factor gene family in Vitis vinifera[J].Plant Cell Reports,2014,33(8):1365-1375.doi:10.1007/s00299-014-1622-7.
[9] Wei H,Jiao Z,Hou X W,Yan Y,Wei Y X,Liu J H,Li M Y,Xu B Y,Jin Z Q.The auxin response factor gene family in banana:genome-wide identification and expression analyses during development,ripening,and abiotic stress[J].Frontiers in Plant Science,2015,6(742):742.doi:10.3389/fpls.2015.00742.
[10] Zouine M,Fu Y,Chateigner-Boutin A L,Mila I,Frasse P,Wang H,Audran C,Roustan J P,Bouzayen M.Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing[J].PLoS One,2014,9(1):e84203.doi:10.1371/journal.pone.0084203.
[11] Ellis C M,Nagpal P,Young J C,Hagen G,Guilfoyle T J,Reed J W.AUXIN RESPONSE FACTOR 1 and AUXIN RESPONSE FACTOR 2 regulate senescence and floral organ abscission in Arabidopsis thaliana[J].Development,2005,132(20):4563-4574.doi:10.1242/dev.02012.
[12] Goetz M,Vivian-Smith A,Johnson S D,Koltunow A M.AUXIN RESPONSE FACTOR 8 is a negative regulator of fruit initiation in Arabidopsis[J].Plant Cell,2006,18(8):1873-1886.doi:10.1105/tpc.105.037192.
[13] Wilmoth J C,Wang S,Tiwari S B,Joshi A D,Hagen G,Guilfoyle T J,Alonso J M,Ecker J R,Reed J W.NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root for mation[J].Plant Journal,2005,43(1):118-130.doi:10.1111/j.1365-313X.2005.02432.x.
[14] Okushima Y,Fukaki H,Onoda M,Theologis A,Tasaka M.ARF7 and ARF19 regulate lateral root formation via direct activation of LBD/ASL genes in Arabidopsis[J].Plant Cell,2007,19(1):118-130.doi:10.1105/tpc.106.047761.
[15] Sagar M,Chervin C,Mila I,Hao Y W,Roustan J P,Benichou M,Gibon Y,Biais B,Maury P,Latche A,Pech J C,Bouzayen M,Zouine M.Sl-ARF4,an auxin response factor involved in the control of sugar metabolism during tomato fruit development[J].Plant Physiology,2013,161(3):1362-1374.doi:10.1104/pp.113.213843.
[16] Aida M,Vernoux T,Furutani M,Traas J,Tasaka M.Roles of PIN-FORMED1 and MONOPTEROS in pattern formation of the apical region of the Arabidopsis embryo[J].Development,2002,129(17):3965.doi:10.1101/gad.242202.
[17] Wenzel C L,Schuetz M,Yu Q,Mattsson J.Dynamics of MONOPTEROS and PIN‐FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana[J].Plant Journal,2010,49(3):387-398.doi:10.1111/j.1365-313X.2006.02977.x.
[18] 李青青,南文斌,张汉马.ARF5/MONOPTEROS(MP)调控作用研究进展[J].西北植物学报,2016,36(1):197-203.doi:10.7606/j.issn.1000-4025.2016.01.0197.Li Q Q,Nan W B,Zhang H M.Progress on the functions of ARF5/MONOPTEROS(MP)[J].Acta Botanica Boreali-Occidentalia Sinica,2016,36(1):197-203.
[19] Ckurshumova W,Smirnova T,Marcos D,Zayed Y,Berleth T.Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation[J].New Phytologist,2015,204(3):556-566.doi:10.1111/nph.13014.
[20] Krogan N T,Berleth T.A dominant mutation reveals asymmetry in MP/ARF5 function along the adaxial-abaxial axis of shoot lateral organs[J].Plant Signaling & Behavior,2012,7(8):940-943.doi:10.1111/nph.13014.
[21] Liu S,Zhang Y,Feng Q,Qin L,Pan C,Lamin-Samu A T,Lu G.ONSE FACTOR 5 regulates fruit set and development via the Tomato AUXIN RESP mediation of auxin and gibberellin signaling[J].Scientific Reports,2018,8(1):2971.doi:10.1038/s41598-018-21315-y.
[22] 苏丽艳,田爱梅,陶贵荣,李蒙.苹果MdCaM的克隆及其对果实釆后非生物胁迫的响应[J].华北农学报,2017,32(1):47-52.doi:10.7668/hbnxb.2017.01.008.Su L Y,Tian A M,Tao G R,Li M.Cloning of MdCaM and its expression responses under abiotic stresses during Malus domestica postharvest storage[J].Acta Agriculturae Boreali-Sinica,2017,32(1):47-52.
[23] Kumar R,Tyagi A K,Sharma A K.Genome-wide analysis of auxin response factor(ARF)gene family from tomato and analysis of their role in flower and fruit development[J].Molecular Genetics & Genomics,2011,285(3):245-260.doi:10.1007/s00438-011-0602-7.
[24] Shen C,Yue R,Sun T,Zhang L,Xu L Q,Tie S G,Wang H Z,Yang Y J.Genome-wide identification and expression analysis of auxin response factor gene family in Medicago truncatula[J].Frontiers in Plant Science,2015,6(11):73.doi:10.3389/fpls.2015.00073.
[25] De J M,Woltersarts M,Garcíamartínez J L,Mariani C,Vriezen W H.The Solanum lycopersicum AUXIN RESPONSE FACTOR 7(SlARF7)mediates cross-talk between auxin and gibberellin signalling during tomato fruit set and development[J].Journal of Experimental Botany,2011,62(2):617.doi:10.1093/jxb/erq293.
[26] Liu N,Wu S,Van J H,Wang Y,Ding B,Fei Z,Clarke T H,Reed J W,van der Knaap E.Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato[J].Journal of Experimental Botany,2014,65(9):2507-2520.doi:10.1093/jxb/eru141.
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