拟南芥转录因子基因WRKY72的特性分析及其抗逆功能鉴定
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摘要
拟南芥(Arabidopsis thaliana)WRKY72转录因子属于Ⅱb亚组成员。为探明WRKY72基因的特性及其在非生物胁迫中的作用,本研究采用多种细胞分子生物学方法进行研究。亚细胞定位发现WRKY72转录因子定位在细胞核中。qRT-PCR检测WRKY72对多种逆境与激素处理的响应,结果表明在热害、盐害、脱落酸、低氮、葡萄糖等非生物胁迫处理3 h后,WRKY72的转录水平显著受到抑制。酵母体内转录活性分析以及双荧光素酶报告系统分析显示WRKY72是一个转录抑制子。酵母双杂交(yeast two-hybrid, Y2H)和双分子荧光互补(bimolecular fluorescence complementation, BiFC)发现,WRKY72不仅能与近源的WRKY9、WRKY36、WRKY47、WRKY61、WRKY72转录因子发生互作,还能与自身发生互作。利用该基因T-DNA插入突变体进行表型分析,结果显示,wrky72突变体在百草枯(methyl viologen,MV)和低氮(low nitrogen, LN)处理下主根延伸长度显著变短,说明WRKY72对MV和LN敏感,提示WRKY72参与调控活性氧耐受和氮营养吸收或利用过程。综上所述,WRKY72可能与自身及一些近源WRKY转录因子互作,以多聚体的形式发挥其转录抑制子的功能,并参与特定的非生物胁迫相关的信号转导过程。本研究为深入解析WRKY72转录因子的抗逆功能与分子调控机制提供了资料基础。
WRKY transcription factors constitute a large family in plants and play important roles in many different physiological processes. Many members of WRKY genes have been studied in various processes including abiotic and biotic stress tolerance in Arabidopsis thaliana. However, the functions and molecular mechanisms of many WRKY genes are still unknown. In this report, the WRKY72 gene in Arabidopsis, which is a member of group Ⅱb is studied. In order to explore the characteristics of WRKY72 gene and its role in abiotic stress tolerance, various cellular and molecular biology approaches were employed. Subcellular localization using green fluorescence protein reporter gene(GFP) in leaves of Nicotiana benthamiana showed that the WRKY72 transcription factor was localized in the nucleus of leaf epidermal cells. qRT-PCR was used toexamine its response to multiple stress and phytohormone treatments at 3 time-points. The results demonstrated that WRKY72 transcription was inhibited by cold, heat, salt, abscisic acid(ABA), methyl viologen(MV), low nitrogen(LN) and glucose(Glc) treatments, which showed that WRKY72 may play a role in these stress signaling pathways. The transcriptional activity of WRKY72 was assessed through in vivo yeast assay,revealing that WRKY72 was a transcriptional repressor. Through a dual luciferase reporter system, WRKY72 was identified to significantly repress the transcription of firefly luciferase gene under the control of tandem repeats of W-box cis-elements, compared to the control. Yeast two-hybrid(Y2H) and bimolecular fluorescence complementation(BiFC) assays were used to screen and confirm the interacting proteins of WRKY72 and the results showed that WRKY72 interacted with homologous WRKY9, WRKY36, WRKY47, WRKY61 and itself. These indicated that WRKY72 was able to form both heterodimers and homodimers in vivo. Finally, 2 TDNA insertion mutants of WRKY72 gene were identified through PCR, and semi qRT-PCR assay indicated lossof-function mutation. Phenotypic assay was done to compare the response of the knock-out mutant to wild-type Col-0 under different abiotic stress conditions at the post-germination level. Root elongation assay showed that the elongation of wrky72 mutant roots were significantly shorter under MV and LN treatments whereas no significant difference was observed under any other stress treatment, suggesting that WRKY72 might participate in the signaling pathways of oxidative stress tolerance and nitrogen absorption and/or utilization. In summary, the experimental evidences provided here support that WRKY72 was a transcriptional repressor and played a role in abiotic stress signaling by forming a complex with homologous WRKY9, WRKY36,WRKY47 and WRKY61. These data have laid a foundation for in-depth dissection of function and molecular mechanism of WRKY72 transcription factor gene in the context of abiotic stress signaling.
WRKY transcription factors constitute a large family in plants and play important roles in many different physiological processes. Many members of WRKY genes have been studied in various processes including abiotic and biotic stress tolerance in Arabidopsis thaliana. However, the functions and molecular mechanisms of many WRKY genes are still unknown. In this report, the WRKY72 gene in Arabidopsis, which is a member of group Ⅱb is studied. In order to explore the characteristics of WRKY72 gene and its role in abiotic stress tolerance, various cellular and molecular biology approaches were employed. Subcellular localization using green fluorescence protein reporter gene(GFP) in leaves of Nicotiana benthamiana showed that the WRKY72 transcription factor was localized in the nucleus of leaf epidermal cells. qRT-PCR was used toexamine its response to multiple stress and phytohormone treatments at 3 time-points. The results demonstrated that WRKY72 transcription was inhibited by cold, heat, salt, abscisic acid(ABA), methyl viologen(MV), low nitrogen(LN) and glucose(Glc) treatments, which showed that WRKY72 may play a role in these stress signaling pathways. The transcriptional activity of WRKY72 was assessed through in vivo yeast assay,revealing that WRKY72 was a transcriptional repressor. Through a dual luciferase reporter system, WRKY72 was identified to significantly repress the transcription of firefly luciferase gene under the control of tandem repeats of W-box cis-elements, compared to the control. Yeast two-hybrid(Y2H) and bimolecular fluorescence complementation(BiFC) assays were used to screen and confirm the interacting proteins of WRKY72 and the results showed that WRKY72 interacted with homologous WRKY9, WRKY36, WRKY47, WRKY61 and itself. These indicated that WRKY72 was able to form both heterodimers and homodimers in vivo. Finally, 2 TDNA insertion mutants of WRKY72 gene were identified through PCR, and semi qRT-PCR assay indicated lossof-function mutation. Phenotypic assay was done to compare the response of the knock-out mutant to wild-type Col-0 under different abiotic stress conditions at the post-germination level. Root elongation assay showed that the elongation of wrky72 mutant roots were significantly shorter under MV and LN treatments whereas no significant difference was observed under any other stress treatment, suggesting that WRKY72 might participate in the signaling pathways of oxidative stress tolerance and nitrogen absorption and/or utilization. In summary, the experimental evidences provided here support that WRKY72 was a transcriptional repressor and played a role in abiotic stress signaling by forming a complex with homologous WRKY9, WRKY36,WRKY47 and WRKY61. These data have laid a foundation for in-depth dissection of function and molecular mechanism of WRKY72 transcription factor gene in the context of abiotic stress signaling.
引文
陈思雀,翁群清,曹红瑞,等.2017.WRKY转录因子在生物和非生物胁迫中的功能和调控机理的研究进展[J].农业生物技术学报,25(4):668-682.(Chen S Q,Weng QQ,Cao H R,et al.2017.The functions and regulation mechanisms of WRKY transcription factors in response to biotic and abiotic stresses[J].Journal of Agricultural Biotechnology,25(4):668-682.)
范晓江,郭小华,牛芳芳,等.2018.拟南芥WRKY61转录因子的转录活性与互作蛋白分析[J].西北植物学报,2018(1).(Fan X J,Guo X H,Niu F F,et al.2018.Exploring the transcriptional activity and interacting proteins of WRKY61 transcriptional factor in Arabidopsis thaliana[J].Acta Botanica Boreali-Occidentalia Sinica,2018(1).)
司爱君,余渝,陈红,等.2017.棉花逆境应答GhWRKY2基因的结构与功能预测[J].农业生物技术学报,25(2):222-230.(Si A J,Yu Y,Chen H,et al.2017.Functional prediction of stress response GhWRKY2 gene in cotton(Gossypium hirsutum)[J].Journal of Agricultural Biotechnology,25(2):222-230.)
王瑞,吴华玲,张铭,等.2013.小麦TaWRKY51基因的克隆、表达分析和转基因功能鉴定[J].农业生物技术学报,21(9):1019-1027.(Wang R,Wu H L,Zhang M,et al.2013.Cloning,characterization and transgenic function analysis of wheat(Triticum aestivum L.)TaWRKY51gene[J].Journal of Agricultural Biotechnology,21(9):1019-1027.)
Babitha K C,Ramu S V,Pruthvi V,et al.2013.Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis[J].Transgenic Research,22(2):327-41.
Banerjee A,Roychoudhury A.2015.WRKY proteins:Signaling and regulation of expression during abiotic stress responses[J].Scientific World Journal,2015(807560):807560.
Besseau S,Li J,Palva E T.2012.WRKY54 and WRKY70 cooperate as negative regulators of leaf senescence in Arabidopsis thaliana[J].Journal of Experimental Botany,63(7):2667-79.
Bhattarai K K,Atamian H S,Kaloshian I,et al.2010.WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-forgene resistance mediated by the tomato R gene Mi-1[J].The Plant Journal,63(2):229-240.
Chen B,Niu F,Liu W Z,et al.2016.Identification,cloning and characterization of R2R3-MYB gene family in canola(Brassica napus L.)identify a novel member modulating ROS accumulation and hypersensitive-like cell death[J].DNA Research,23(2):101-114.
Chen J,Nolan T M,Ye H,et al.2017.Arabidopsis WRKY46,WRKY54,and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses[J].Plant Cell,29(6):1425-1439.
Chen L,Song Y,Li S,et al.2012.The role of WRKY transcription factors in plant abiotic stresses[J].Biochimica et Biophysica Acta,1819(2):120-128.
Chen X,Liu J,Lin G,et al.2013.Overexpression of AtWRKY28 and AtWRKY75 in Arabidopsis enhances resistance to oxalic acid and Sclerotinia sclerotiorum[J]Plant Cell Reports,32(10):1589-1599.
Chen Y F,Li L Q,Xu Q,et al.2009.The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis[J].Plant Cell,21(11):3554-3566.
Devaiah B N,Karthikeyan A S,Raghothama K G.2007WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis[J].Plant Physiology,143(4):1789-801.
Eulgem T,Rushton P J,Robatzek S,et al.2000.The WRKYsuperfamily of plant transcription factors[J].Trends Plant Science,5(5):199-206.
Eulgem T,Somssich I E.2007.Networks of WRKY transcription factors in defense signaling[J].Current Opinion Plant Biology,10(4):366-371.
Gao R,Liu P,Yong Y,et al.2016.Genome-wide transcriptomic analysis reveals correlation between higher WRKY61expression and reduced symptom severity in Turnip crinkle virus infected Arabidopsis thaliana[J].Science Reports,6(24604):24604.
Jiang Y,Deyholos M K.2009.Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33transcription factors in abiotic stresses[J].Plant Molecular Biology,69(1-2):91-105.
Jiang Y,Liang G,Yu D.2012.Activated expression of WRKY57 confers drought tolerance in Arabidopsis[J]Molecular Plant,5(6):1375-1388.
Li S,Fu Q,Chen L,et al.2011.Arabidopsis thaliana WRKY25,WRKY26,and WRKY33 coordinate induction of plant thermotolerance[J].Planta,233(6):1237-1252.
Liang W,Yang B,Yu B J,et al.2013.Identification and analysis of MKK and MPK gene families in canola(Brassica napus L.)[J].BMC Genomics,14(1):392.
Mao G,Meng X,Liu Y,et al.2011.Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis[J].Plant Cell,23(4):1639-1653.
Rushton P J,Macdonald H,Huttly A K,et al.1995.Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of alpha-Amy2genes[J].Plant Molecular Biology,29(4):691-702.
Rushton P J,Somssich I E,Ringler P,et al.2010.WRKY transcription factors[J].Trends in Plant Science,15(5):247-258.
Scarpeci T E,Zanor M I,Mueller-Roeber B,et al.2013.Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana[J].Plant Molecular Biology,83(3):265-277.
Su T,Xu Q,Zhang F C,et al.2015.WRKY42 modulates phosphate homeostasis through regulating phosphate translocation and acquisition in Arabidopsis[J].Plant Physiology,167(4):1579-1591.
Sun Y,Yu D.2015.Activated expression of AtWRKY53 negatively regulates drought tolerance by mediating stomatal movement[J].Plant Cell Reports,34(8):1295-1306.
Ulker B,Somssich I E.2004.WRKY transcription factors:From DNA binding towards biological function[J].Current Opinion in Plant Biology,7(5):491-498.
Xie Z,Zhang Z L,Zou X,et al.2006.Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells[J].The Plant Journal,46(2):231-242.
Xu X,Chen C,Fan B,et al.2006.Physical and functional interactions between pathogen-induced Arabidopsis WRKY18,WRKY40,and WRKY60 transcription factors[J].Plant Cell,18(5):1310-1326.
Yamasaki K,Kigawa T,Watanabe S,et al.2012.Structural basis for sequence-specific DNA recognition by an Arabidopsis WRKY transcription factor[J].Journal of Biological Chemistry,287(10):7683-7691.
Yan J,Niu F,Liu W Z,et al.2014.Arabidopsis CIPK14 positively regulates glucose response[J].Biochemical&Biophysical Research Communications,450(4):1679-1683.
Yoo S D,Cho Y H,Sheen J.2007.Arabidopsis mesophyll protoplasts:A versatile cell system for transient gene expression analysis[J].Nature Protocols,2(7):1565-1572.
Zhang H,Liu W Z,Zhang Y,et al.2014.Identification,expression and interaction analyses of calcium-dependent protein kinase(CPK)genes in canola(Brassica napus L.)[J].BMC Genomics,15(1):211.
范晓江,郭小华,牛芳芳,等.2018.拟南芥WRKY61转录因子的转录活性与互作蛋白分析[J].西北植物学报,2018(1).(Fan X J,Guo X H,Niu F F,et al.2018.Exploring the transcriptional activity and interacting proteins of WRKY61 transcriptional factor in Arabidopsis thaliana[J].Acta Botanica Boreali-Occidentalia Sinica,2018(1).)
司爱君,余渝,陈红,等.2017.棉花逆境应答GhWRKY2基因的结构与功能预测[J].农业生物技术学报,25(2):222-230.(Si A J,Yu Y,Chen H,et al.2017.Functional prediction of stress response GhWRKY2 gene in cotton(Gossypium hirsutum)[J].Journal of Agricultural Biotechnology,25(2):222-230.)
王瑞,吴华玲,张铭,等.2013.小麦TaWRKY51基因的克隆、表达分析和转基因功能鉴定[J].农业生物技术学报,21(9):1019-1027.(Wang R,Wu H L,Zhang M,et al.2013.Cloning,characterization and transgenic function analysis of wheat(Triticum aestivum L.)TaWRKY51gene[J].Journal of Agricultural Biotechnology,21(9):1019-1027.)
Babitha K C,Ramu S V,Pruthvi V,et al.2013.Co-expression of AtbHLH17 and AtWRKY28 confers resistance to abiotic stress in Arabidopsis[J].Transgenic Research,22(2):327-41.
Banerjee A,Roychoudhury A.2015.WRKY proteins:Signaling and regulation of expression during abiotic stress responses[J].Scientific World Journal,2015(807560):807560.
Besseau S,Li J,Palva E T.2012.WRKY54 and WRKY70 cooperate as negative regulators of leaf senescence in Arabidopsis thaliana[J].Journal of Experimental Botany,63(7):2667-79.
Bhattarai K K,Atamian H S,Kaloshian I,et al.2010.WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-forgene resistance mediated by the tomato R gene Mi-1[J].The Plant Journal,63(2):229-240.
Chen B,Niu F,Liu W Z,et al.2016.Identification,cloning and characterization of R2R3-MYB gene family in canola(Brassica napus L.)identify a novel member modulating ROS accumulation and hypersensitive-like cell death[J].DNA Research,23(2):101-114.
Chen J,Nolan T M,Ye H,et al.2017.Arabidopsis WRKY46,WRKY54,and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses[J].Plant Cell,29(6):1425-1439.
Chen L,Song Y,Li S,et al.2012.The role of WRKY transcription factors in plant abiotic stresses[J].Biochimica et Biophysica Acta,1819(2):120-128.
Chen X,Liu J,Lin G,et al.2013.Overexpression of AtWRKY28 and AtWRKY75 in Arabidopsis enhances resistance to oxalic acid and Sclerotinia sclerotiorum[J]Plant Cell Reports,32(10):1589-1599.
Chen Y F,Li L Q,Xu Q,et al.2009.The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis[J].Plant Cell,21(11):3554-3566.
Devaiah B N,Karthikeyan A S,Raghothama K G.2007WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis[J].Plant Physiology,143(4):1789-801.
Eulgem T,Rushton P J,Robatzek S,et al.2000.The WRKYsuperfamily of plant transcription factors[J].Trends Plant Science,5(5):199-206.
Eulgem T,Somssich I E.2007.Networks of WRKY transcription factors in defense signaling[J].Current Opinion Plant Biology,10(4):366-371.
Gao R,Liu P,Yong Y,et al.2016.Genome-wide transcriptomic analysis reveals correlation between higher WRKY61expression and reduced symptom severity in Turnip crinkle virus infected Arabidopsis thaliana[J].Science Reports,6(24604):24604.
Jiang Y,Deyholos M K.2009.Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33transcription factors in abiotic stresses[J].Plant Molecular Biology,69(1-2):91-105.
Jiang Y,Liang G,Yu D.2012.Activated expression of WRKY57 confers drought tolerance in Arabidopsis[J]Molecular Plant,5(6):1375-1388.
Li S,Fu Q,Chen L,et al.2011.Arabidopsis thaliana WRKY25,WRKY26,and WRKY33 coordinate induction of plant thermotolerance[J].Planta,233(6):1237-1252.
Liang W,Yang B,Yu B J,et al.2013.Identification and analysis of MKK and MPK gene families in canola(Brassica napus L.)[J].BMC Genomics,14(1):392.
Mao G,Meng X,Liu Y,et al.2011.Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis[J].Plant Cell,23(4):1639-1653.
Rushton P J,Macdonald H,Huttly A K,et al.1995.Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of alpha-Amy2genes[J].Plant Molecular Biology,29(4):691-702.
Rushton P J,Somssich I E,Ringler P,et al.2010.WRKY transcription factors[J].Trends in Plant Science,15(5):247-258.
Scarpeci T E,Zanor M I,Mueller-Roeber B,et al.2013.Overexpression of AtWRKY30 enhances abiotic stress tolerance during early growth stages in Arabidopsis thaliana[J].Plant Molecular Biology,83(3):265-277.
Su T,Xu Q,Zhang F C,et al.2015.WRKY42 modulates phosphate homeostasis through regulating phosphate translocation and acquisition in Arabidopsis[J].Plant Physiology,167(4):1579-1591.
Sun Y,Yu D.2015.Activated expression of AtWRKY53 negatively regulates drought tolerance by mediating stomatal movement[J].Plant Cell Reports,34(8):1295-1306.
Ulker B,Somssich I E.2004.WRKY transcription factors:From DNA binding towards biological function[J].Current Opinion in Plant Biology,7(5):491-498.
Xie Z,Zhang Z L,Zou X,et al.2006.Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells[J].The Plant Journal,46(2):231-242.
Xu X,Chen C,Fan B,et al.2006.Physical and functional interactions between pathogen-induced Arabidopsis WRKY18,WRKY40,and WRKY60 transcription factors[J].Plant Cell,18(5):1310-1326.
Yamasaki K,Kigawa T,Watanabe S,et al.2012.Structural basis for sequence-specific DNA recognition by an Arabidopsis WRKY transcription factor[J].Journal of Biological Chemistry,287(10):7683-7691.
Yan J,Niu F,Liu W Z,et al.2014.Arabidopsis CIPK14 positively regulates glucose response[J].Biochemical&Biophysical Research Communications,450(4):1679-1683.
Yoo S D,Cho Y H,Sheen J.2007.Arabidopsis mesophyll protoplasts:A versatile cell system for transient gene expression analysis[J].Nature Protocols,2(7):1565-1572.
Zhang H,Liu W Z,Zhang Y,et al.2014.Identification,expression and interaction analyses of calcium-dependent protein kinase(CPK)genes in canola(Brassica napus L.)[J].BMC Genomics,15(1):211.