拟南芥信号肽AtRALF1的表达、纯化及活性分析
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摘要
利用RT-PCR扩增获得拟南芥At RALF1基因的全长cDNA序列,将其构建入携带His标签的原核表达载体p ET28b中,获得重组表达载体p ET28b-At RALF1,并将其转入到大肠杆菌BL21(DE3)中。随后在不同的IPTG浓度、温度和诱导时间条件下,进行At RALF1蛋白的表达研究,建立了At RALF1融合蛋白的高效表达体系。结果表明,在30℃、1 mmol/L IPTG的条件下诱导4 h,At RALF1融合蛋白的表达量最大。进一步用获得的At RALF1融合蛋白处理苗龄5 d的野生型拟南芥(Col-0)植株,发现其根的生长受到了抑制,表明我们获得了具有活性的At RALF1小肽,为进一步研究该小肽奠定了基础。
The full-length c DNA sequence of At RALF1 was amplified from Arabidopsis thaliana by RT-PCR.The verified sequence of At RALF1 was inserted into the corresponding region of p ET28 b vector to construct the recombinant plasmid p ET28b-At RALF1.The plasmid p ET28b-At RALF1 was then introduced into the bacterial host E.coli BL21(DE3) to analyze the expression of At RALF1.Through the analysis of different concentration of IPTG,temperature and time course,the expression conditions were optimized,and30 ℃ for 4 h with 1 mmol/L IPTG induction was found to be the optimal condition for prokaryotic expression.The At RALF1 protein was then purified for further research.When the purified At RALF1 protein was added to 5-day-old seedlings(Col-0),the growth of the seedling root was obviously inhibited in Arabidopsis thaliana.It is suggested that the recombinant At RALF1 protein has the inhibition activity of signaling peptides,which can be used for further study.
The full-length c DNA sequence of At RALF1 was amplified from Arabidopsis thaliana by RT-PCR.The verified sequence of At RALF1 was inserted into the corresponding region of p ET28 b vector to construct the recombinant plasmid p ET28b-At RALF1.The plasmid p ET28b-At RALF1 was then introduced into the bacterial host E.coli BL21(DE3) to analyze the expression of At RALF1.Through the analysis of different concentration of IPTG,temperature and time course,the expression conditions were optimized,and30 ℃ for 4 h with 1 mmol/L IPTG induction was found to be the optimal condition for prokaryotic expression.The At RALF1 protein was then purified for further research.When the purified At RALF1 protein was added to 5-day-old seedlings(Col-0),the growth of the seedling root was obviously inhibited in Arabidopsis thaliana.It is suggested that the recombinant At RALF1 protein has the inhibition activity of signaling peptides,which can be used for further study.
引文
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[12]PEARCE G,YAMAGUCHI Y,MUNSKE G,et al.Structure activity studies of RALF,rapid alkalinization factor,reveal an essential YISY motif[J].Peptides,2010,31(11):1973-1977.
[13]MORATO DO CANTO A,CECILIATO P H O,RIBEIRO B,et al.Biological activity of nine recombinant At RALF peptides:implications for their perception and function in Arabidopsis[J].Plant Physiology&Biochemistry,2014,75:45-54.
[14]HARUTA M,SABAT G,STECKER K,et al.A peptide hormone and its receptor protein kinase regulate plant cell expansion[J].Science,2014,343:408-411.
[15]HARUTA M,MONSHAUSEN G,GILROY S,et al.A cytoplasmic Ca2+functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings:identification of At RALF1 peptide[J].Biochemistry,2008,47(24):6311-6321.
[16]XIAO B Y,FAN S Q,ZENG Z Y,et al.Purification of novel UBAP1 protein and its decreased expression on nasopharyngeal carcinoma tissue microarray[J].Protein Expression&Purification,2006,47(1):60-67.
[17]聂新民,张必成,向娟娟,等.鼻咽癌侯选抑瘤基因BRD7原核表达载体的构建及其表达[J].生物化学与生物物理进展(NIE Xin-min,ZHANG Bi-cheng,XIANG Juan-juan,et al.Construction of prokaryotic expression vector of BRD7 and its expression in E.coli.[J].Progress in Biochemistry and Biophysics),2002,29(4):631-634.
[18]张艳霞,贾海英,司志飞,等.水稻甲基结合蛋白基因MBD701的克隆及原核表达[J].生命科学研究(ZHANG Yan-xia,JIA Hai-ying,SI Zhi-fei,et al.Cloning of methyl-binding domain protein gene MBD701 and its prokaryotic expression[J].Life Science Research),2012,16(2):120-124.
[2]MATSUBAYASHI Y,SAKAGAMI Y.Peptide hormones in plants[J].Annual Review of Plant Biology,2006,57:649-674.
[3]SUGANO S S,SHIMADA T,IMAI Y,et al.Stomagen positively regulates stomatal density in Arabidopsis[J].Nature,2010,463(7278):241-244.
[4]KONDO Y,HIRAKAWA Y,KIEBER J J,et al.CLE peptides can negatively regulate protoxylem vessel formation via cytokinin signaling[J].Plant and Cell Physiology,2011,52(1):37-48.
[5]WHITFORD R,FERNANDEZ A,TEJOS R,et al.GOLVEN secretory peptides regulate auxin carrier turnover during plant gravitropic responses[J].Developmental Cell,2012,22(3):678-685.
[6]PEARCE G,MOURA D S,STRATMANN J,et al.Production of multiple plant hormones from a single polyprotein precursor[J].Nature,2001,411(6839):817-820.
[7]FRANSSEN H J,BISSELING T.Peptide signaling in plants[J].Proceedings of the National Academy of Sciences USA,2001,98(23):12855-12856.
[8]RYAN C A,PEARCE G.Systemins:a functionally defined family of peptide signals that regulate defensive genes in Solanaceae species[J].Proceedings of the National Academy of Sciences USA,2003,25(100):14577-14580.
[9]YANG H,MATSUBAYASHI Y,HANAI H,et al.Molecular cloning and characterization of Os PSK,a gene encoding a precursor for phytosulfokine-α,required for rice cell proliferation[J].Plant Molecular Biology,2000,44(5):635-647.
[10]RYAN C A,PEARCE G.Polypeptide hormones[J].Plant Physiology,2001,125(1):65-68.
[11]MATSUBAYASHI Y,YANG H,SAKAGAMI Y.Peptide signals and their receptors in higher plants[J].Trends in Plant Science,2001,6(12):573-577.
[12]PEARCE G,YAMAGUCHI Y,MUNSKE G,et al.Structure activity studies of RALF,rapid alkalinization factor,reveal an essential YISY motif[J].Peptides,2010,31(11):1973-1977.
[13]MORATO DO CANTO A,CECILIATO P H O,RIBEIRO B,et al.Biological activity of nine recombinant At RALF peptides:implications for their perception and function in Arabidopsis[J].Plant Physiology&Biochemistry,2014,75:45-54.
[14]HARUTA M,SABAT G,STECKER K,et al.A peptide hormone and its receptor protein kinase regulate plant cell expansion[J].Science,2014,343:408-411.
[15]HARUTA M,MONSHAUSEN G,GILROY S,et al.A cytoplasmic Ca2+functional assay for identifying and purifying endogenous cell signaling peptides in Arabidopsis seedlings:identification of At RALF1 peptide[J].Biochemistry,2008,47(24):6311-6321.
[16]XIAO B Y,FAN S Q,ZENG Z Y,et al.Purification of novel UBAP1 protein and its decreased expression on nasopharyngeal carcinoma tissue microarray[J].Protein Expression&Purification,2006,47(1):60-67.
[17]聂新民,张必成,向娟娟,等.鼻咽癌侯选抑瘤基因BRD7原核表达载体的构建及其表达[J].生物化学与生物物理进展(NIE Xin-min,ZHANG Bi-cheng,XIANG Juan-juan,et al.Construction of prokaryotic expression vector of BRD7 and its expression in E.coli.[J].Progress in Biochemistry and Biophysics),2002,29(4):631-634.
[18]张艳霞,贾海英,司志飞,等.水稻甲基结合蛋白基因MBD701的克隆及原核表达[J].生命科学研究(ZHANG Yan-xia,JIA Hai-ying,SI Zhi-fei,et al.Cloning of methyl-binding domain protein gene MBD701 and its prokaryotic expression[J].Life Science Research),2012,16(2):120-124.