药用野生稻ADF基因的克隆及其抗逆性分析
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摘要
为了研究ADF基因对药用野生稻非生物胁迫的影响,利用药用野生稻干旱胁迫转录组数据分析的基础,通过PCR技术克隆OoADF1基因,序列分析结果表明,OoADF1的开放阅读框长度为453 bp,无内含子,编码150个氨基酸残基,具有典型的ADF结构域。qRT-PCR技术分析表明,OoADF1在四叶期的叶片、叶鞘和根,以及抽穗期的叶片、叶鞘、茎和幼穗等7个组织中均表达,四叶期根部表达量最高,抽穗期叶鞘表达量最高。干旱和高盐胁迫后,OoADF1表达量分别上调了17.9,40.0倍。将OoADF1完整的编码序列融合到原核表达载体p ET-28a(+)中,通过IPTG诱导及SDS-PASGE检测表明,OoADF1编码的蛋白分子量约为16 kDa,与预测的相符。OoADF1在大肠杆菌中的表达增加了大肠杆菌对高盐的抗性,表明OoADF1对高盐胁迫具有一定程度的响应能力,从而使得细胞免受高盐的毒害。
In order to investigate the effect of ADF gene on abiotic stress in Oryza officinalis.Based on the analysis on the drought-stressed transcriptome of Oryza officinalis,OoADF1 was cloned by RT-PCR technique and sequence analysis showed that OoADF1 contains 453 bp-length ORF and was intronless,and OoADF1 was predicted to contain 150 amino acids and have conserved ADF structure.Quantitative RT-PCR analysis showed that OoADF1 was expressed in all seven tissues,including leaf blade,leaf sheath and root at four-leaves stage,leaf blade,leaf sheath,stem and young panicle at heading stage in Oryza officinalis.The highest expression level of OoADF1 was detected in the root at four-leaves stage,while leaf sheath at heading stage.The fold of expression of OoADF1 amounted to 17.9 and 40.0 in the drought-or high Na Cl-stressed young seedling,respectively.The coding sequence of OoADF1 was fused to the prokaroytic expression vector p ET-28 a(+),and the combinant vectors were induced by IPTG and detected by SDS-PAGE.The molecular weight of protein OoADF1 was detected to be 16 kDa,which was consistent with theoretical one.The results of stress tolerance assay showed that the recombinant strain producing OoADF1 fusion protein improved the resistance to the high concentration,indicating that OoADF1 may exert a role in respond to salt stress and protect cells from high-salt harm.These results provides that OoADF1 has a certain degree of response to high salt stress,so that cells from high salt poisoning.
In order to investigate the effect of ADF gene on abiotic stress in Oryza officinalis.Based on the analysis on the drought-stressed transcriptome of Oryza officinalis,OoADF1 was cloned by RT-PCR technique and sequence analysis showed that OoADF1 contains 453 bp-length ORF and was intronless,and OoADF1 was predicted to contain 150 amino acids and have conserved ADF structure.Quantitative RT-PCR analysis showed that OoADF1 was expressed in all seven tissues,including leaf blade,leaf sheath and root at four-leaves stage,leaf blade,leaf sheath,stem and young panicle at heading stage in Oryza officinalis.The highest expression level of OoADF1 was detected in the root at four-leaves stage,while leaf sheath at heading stage.The fold of expression of OoADF1 amounted to 17.9 and 40.0 in the drought-or high Na Cl-stressed young seedling,respectively.The coding sequence of OoADF1 was fused to the prokaroytic expression vector p ET-28 a(+),and the combinant vectors were induced by IPTG and detected by SDS-PAGE.The molecular weight of protein OoADF1 was detected to be 16 kDa,which was consistent with theoretical one.The results of stress tolerance assay showed that the recombinant strain producing OoADF1 fusion protein improved the resistance to the high concentration,indicating that OoADF1 may exert a role in respond to salt stress and protect cells from high-salt harm.These results provides that OoADF1 has a certain degree of response to high salt stress,so that cells from high salt poisoning.
引文
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[14]王楠,吕香玲,李亮,等.植物肌动蛋白解聚因子ADF的研究进展[J].西北植物学报,2015,35(11):2349-2354.
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[17]Singh B K,Sattler J M,Chatterjee M,et al.Crystal structures explain functional differences in the two actin deploymerization factors of the malaria parasite[J].The Journal of Biological Chemistry,2011,286(32):28256-28264.
[18]Wong W,AI Webb,Maya A Olshina,et al.A mechanism for actinfilament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface[J].Biol Chem,2014,289(7):4043-4054.
[19]Huang Y C,Huang W L,Hong C Y,et al.Comprehensive analysis of differentially expressed rice actin depolymerizing factor gene family and heterologous overexpression of Os ADF3 confers Arabidopsis thaliana drought tolerance[J].Rice,2012,5(1):33.
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[22]Fu Y,Duan X,Tang C,et al.Ta ADF7,an actin-depolymerizing factor,contributes to wheat resistance against Puccinia striiformis f.sp.tritici[J].The Plant Journal:for Cell and Molecular Biology,2014,78(1):16-30.
[23]Henty-Ridilla J L,Li J,Day B,et al.ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis[J].The Plant Cell,2014,26(1):340-352.
[24]陈琼,黄善金,于荣.植物微丝骨架动态变化的调节[J].植物生理学报,2011,47(1):18-26.
[25]Zheng Y,Xie Y,Jiang Y et al.Arabidopsis actin-depolymerizing factor7 severs actin filaments and regulates catin cable turnover to promote normal pollen tube growth[J].Plant Cell,2013,5:3405-3423.
[26]Dong C H,Hong Y.Arabidopsis CDPK6 phosphorylates ADF1 at N-terminal serine 6 predominantly[J].Plant Cell Reports,2013,32(11):1715-1728.
[27]Fan T T,Ni J J,Dong W C,et al.Effect of low temperature on profilins and ADFs transcription and actin cytoskeleton reorganization in Arabidopsis[J].Biol Plant,2015,59(4):793-796.
[28]Fan T W,Cao S.Heat stress induces actin cytoskeletal reorganization and transcript profiles of vegetative profilins and actin deploy merizing factors(ADFs)in Arabidopsis[J].Acta Physiol plant,2016,38:37.
[29]Zhao S,Jiang Y,Zhao Y,et al.Casein KINASE1-LIKE PROTEIN2 regulates actin filament stability and stomatal closure via phosphorylation of actin depolymerizing factor[J].Plant Cell,2016,28(6):1422-1439.
[30]Samayoa L F,Malvar R A,Olukolu B A,et al.Genomewide association study reveals a set of genes associated with resistance to the Mediterranean corn borer(Sesamia nonagrioides L.)in a maize diversity panel[J].BMC Plant Biology,2015,15(1):35.
[31]Kijima S T,Hirose K,Kong S G,et al.Distinct biochemical properties of Arabidopsis thaliana actin isoforms[J].Plant Cell Physiol,2016,57(1):46-56.
[32]Gunning P W,Ghoshdastider U,Whitaker S,et al.The evolution of compositionally and functionally distinct actin filaments[J].J Cell Sci,2015,128(11):2009-2019.
[33]Fu Y,Duan X,Tang C,et al.Ta ADF7,an actin-depolymerizing factor,contributes to wheat resistance against Puccinia striiformis f.sp.tritici[J].The Plant Journal:for Cell and Molecular Biology,2014,78(1):16-30.
[34]Li L,Li Y,Song S,et al.An anther development Fbox(ADF)protein regulated by tapetum degeneration retardation(TDR)controls rice anther development[J].Planta,2015,241(1):157-166.
[35]Hong L,Xu L,Ma J X,et al.Cloning and expression analysis of a novel tetratricopeptide repeat gene(Ta TPR1)from Triticum L.[J].Journal of Triticeae Crops,2014,34(9):1161-1169.
[36]Feng Y,Liu Q,Xue Q.Comparative study of rice and Arabidopsis actin-depolymerizing factors gene families[J].Journal of Plant Physiology,2006,163(1):69-79.
[37]Solanke A U,Sharma A K.Signal transduction during cold stress in plants[J].Physiology and Molecular Biology of Plants:an International Journal of Functional Plant Biology,2008,14(1/2):69-79.
[38]Drobak B K,Franklin-Tong V E,Staiger C T.The role of the actin cytoskeleton in plant cell signaling[J].New Phytol,2004,163(1):13-30.
[39]Ali G M,Komatsu S.Proteomic analysis of rice leaf sheath during drought stress[J].Journal of Proteome Research,2006,5(2):396-403.
[40]Yan S,Tang Z,Su W,et al.Proteomic analysis of salt stress-responsive proteins in rice root[J].Proteomics,2005,5(1):235-244.
[41]Chen C W,Yang Y W,Lur H S,et al.A novel function of abscisic acid in the regulation of rice(Oryza sativa L.)root growth and development[J].Plant&Cell Physiology,2006,47(1):1-13.
[2]孙传清,王象坤,吉村淳,等.普通野生稻和亚洲栽培稻遗传多样性的研究[J].遗传学报,2000,27(3):227-234.
[3]董玉琛.作物野生种质资源及其利用[C]//中国野生稻研究与利用.第一届全国野生稻大会论文集,2003:3-7.
[4]李容柏,秦学毅.广西野生稻抗病虫性鉴定研究的主要进展[J].广西科学,1994,1(1):83-85.
[5]陈成斌,李道远.广西野生稻优异种质评价与利用研究进展[J].广西农业科学,1995(5):193-195.
[6]蓝伟侦,何光存,吴士筠,等.利用水稻C0t-1 DNA和基因组DNA对栽培稻,药用野生稻和疣粒野生稻基因组的比较分析[J].中国农业科学,2006,39(6):1083-1090.
[7]Liu Y G,Nagaki K,Fujita M,et al.Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome(TAC)vector[J].The Plant Journal:for Cell and Molecular Biology,2000,23(5):687-695.
[8]Song J,Dong F,Lilly J W,et al.Instability of bacterial artificial chromosome(BAC)clones containing tandemly repeated DNA sequences[J].Genome,2001,44(3):463-469.
[9]Jena K K,Kochert G,Khush G S.RFLP analysis of rice(Oryza sativa L.)introgression lines[J].Theoretical and Applied Genetics,1992,84(5/6):608-616.
[10]何瑞锋.药用野生稻基因组文库构建与大片段DNA转化[D].武汉:武汉大学,2003.
[11]潘小芬.基于新型TAC载体的药用野生稻文库构建与性能分析[D].广州:华南农业大学,2006:3-14.
[12]刘蕊,张欢欢,陈志雄,等.筛选和转化药用野生稻TAC克隆获得耐旱水稻[J].中国农业科学,2014,47(8):1445-1457.
[13]谭碧兰.药用野生稻抗旱转录谱分析[D].广州:华南农业大学,2014.
[14]王楠,吕香玲,李亮,等.植物肌动蛋白解聚因子ADF的研究进展[J].西北植物学报,2015,35(11):2349-2354.
[15]Bamburg J R.Proteins of the ADF/cofilin family:essential regulators of actin dynamics[J].Annual Review of Cell and Developmental Biology,1999,15(1):185-230.
[16]Maciver S K,Hussey P J.The ADF/cofilin family:actinremodeling proteins[J].Genome Biology,2002,3(5):reviews,3007.1-3007.12.
[17]Singh B K,Sattler J M,Chatterjee M,et al.Crystal structures explain functional differences in the two actin deploymerization factors of the malaria parasite[J].The Journal of Biological Chemistry,2011,286(32):28256-28264.
[18]Wong W,AI Webb,Maya A Olshina,et al.A mechanism for actinfilament severing by malaria parasite actin depolymerizing factor 1 via a low affinity binding interface[J].Biol Chem,2014,289(7):4043-4054.
[19]Huang Y C,Huang W L,Hong C Y,et al.Comprehensive analysis of differentially expressed rice actin depolymerizing factor gene family and heterologous overexpression of Os ADF3 confers Arabidopsis thaliana drought tolerance[J].Rice,2012,5(1):33.
[20]叶佳.低温胁迫下拟南芥肌动蛋白解聚因子ADF5的功能研究[D].甘肃:兰州大学,2010.
[21]Clément M,Ketelaar T,Rodiuc N,et al.Actin-depolymerizing factor2-mediated actin dynamics are essential for root-knot nematode infection of Arabidopsis[J].The Plant Cell,2009,21(9):2963-2979.
[22]Fu Y,Duan X,Tang C,et al.Ta ADF7,an actin-depolymerizing factor,contributes to wheat resistance against Puccinia striiformis f.sp.tritici[J].The Plant Journal:for Cell and Molecular Biology,2014,78(1):16-30.
[23]Henty-Ridilla J L,Li J,Day B,et al.ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis[J].The Plant Cell,2014,26(1):340-352.
[24]陈琼,黄善金,于荣.植物微丝骨架动态变化的调节[J].植物生理学报,2011,47(1):18-26.
[25]Zheng Y,Xie Y,Jiang Y et al.Arabidopsis actin-depolymerizing factor7 severs actin filaments and regulates catin cable turnover to promote normal pollen tube growth[J].Plant Cell,2013,5:3405-3423.
[26]Dong C H,Hong Y.Arabidopsis CDPK6 phosphorylates ADF1 at N-terminal serine 6 predominantly[J].Plant Cell Reports,2013,32(11):1715-1728.
[27]Fan T T,Ni J J,Dong W C,et al.Effect of low temperature on profilins and ADFs transcription and actin cytoskeleton reorganization in Arabidopsis[J].Biol Plant,2015,59(4):793-796.
[28]Fan T W,Cao S.Heat stress induces actin cytoskeletal reorganization and transcript profiles of vegetative profilins and actin deploy merizing factors(ADFs)in Arabidopsis[J].Acta Physiol plant,2016,38:37.
[29]Zhao S,Jiang Y,Zhao Y,et al.Casein KINASE1-LIKE PROTEIN2 regulates actin filament stability and stomatal closure via phosphorylation of actin depolymerizing factor[J].Plant Cell,2016,28(6):1422-1439.
[30]Samayoa L F,Malvar R A,Olukolu B A,et al.Genomewide association study reveals a set of genes associated with resistance to the Mediterranean corn borer(Sesamia nonagrioides L.)in a maize diversity panel[J].BMC Plant Biology,2015,15(1):35.
[31]Kijima S T,Hirose K,Kong S G,et al.Distinct biochemical properties of Arabidopsis thaliana actin isoforms[J].Plant Cell Physiol,2016,57(1):46-56.
[32]Gunning P W,Ghoshdastider U,Whitaker S,et al.The evolution of compositionally and functionally distinct actin filaments[J].J Cell Sci,2015,128(11):2009-2019.
[33]Fu Y,Duan X,Tang C,et al.Ta ADF7,an actin-depolymerizing factor,contributes to wheat resistance against Puccinia striiformis f.sp.tritici[J].The Plant Journal:for Cell and Molecular Biology,2014,78(1):16-30.
[34]Li L,Li Y,Song S,et al.An anther development Fbox(ADF)protein regulated by tapetum degeneration retardation(TDR)controls rice anther development[J].Planta,2015,241(1):157-166.
[35]Hong L,Xu L,Ma J X,et al.Cloning and expression analysis of a novel tetratricopeptide repeat gene(Ta TPR1)from Triticum L.[J].Journal of Triticeae Crops,2014,34(9):1161-1169.
[36]Feng Y,Liu Q,Xue Q.Comparative study of rice and Arabidopsis actin-depolymerizing factors gene families[J].Journal of Plant Physiology,2006,163(1):69-79.
[37]Solanke A U,Sharma A K.Signal transduction during cold stress in plants[J].Physiology and Molecular Biology of Plants:an International Journal of Functional Plant Biology,2008,14(1/2):69-79.
[38]Drobak B K,Franklin-Tong V E,Staiger C T.The role of the actin cytoskeleton in plant cell signaling[J].New Phytol,2004,163(1):13-30.
[39]Ali G M,Komatsu S.Proteomic analysis of rice leaf sheath during drought stress[J].Journal of Proteome Research,2006,5(2):396-403.
[40]Yan S,Tang Z,Su W,et al.Proteomic analysis of salt stress-responsive proteins in rice root[J].Proteomics,2005,5(1):235-244.
[41]Chen C W,Yang Y W,Lur H S,et al.A novel function of abscisic acid in the regulation of rice(Oryza sativa L.)root growth and development[J].Plant&Cell Physiology,2006,47(1):1-13.