杜仲Dirigent1基因的原核表达及蛋白纯化
|
摘要
植物Dirigent蛋白认为指导木脂素和木质素的生物合成,同时在生物防御应答,次生代谢调控和病原体抗性的过程中起到了重要的作用。本研究以实验室前期克隆得到杜仲Dirigent1基因(EuDIR1)为基础,利用基因重组技术构建pET-30a-EuDIR1原核表达载体。重组载体转化大肠杆菌BL21 (DE3)中,在15℃下0.1 mmol/L异丙基硫代β-D-半乳糖苷诱导6 h后,蛋白的沉积量达到最高值,表达模式主要为包涵体。对包涵体进行溶解,使用镍-亚氨基二乙酸亲和层析柱进行纯化。收集纯度较高的洗脱组分进行透析复性,并用15%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western Blot鉴定重组蛋白。结果表明,成功构建了pET-30a-EuDIR1原核表达载体,表达菌株诱导后在相对分子质量约18 kD处有1条特异性蛋白条带,包涵体纯化、复性后,经Western Blot鉴定,获得可溶性且纯度较高的重组蛋白。实验中得到的EuDIR1蛋白为体外生化实验提供了科学依据。
The plant dirigent(DIR) protein are proposed to as an improve role in guiding the biosynthesis of lignan and lignin, as well as in the process of biological defense responses, regulation of secondary metabolism, and pathogen resistance. This study was based on the previous experiments about cloning EuDIR1 gene in laboratory and then the prokaryotic expressing vector of pET-30 a-EuDIR1 was constructed by the gene recombination technique. During the transformation of Escherichia coli BL21(DE3) by recombinant vector, the protein deposition reached the highest level after 6 h induction with 0.1 mmol/L isopropyl β-D-thiogalactopyranoside(IPTG) at 15℃,and the expression pattern was mainly inclusion body. After dissolution, the inclusion bodies were purified by Ni-IDA(Nickel-iminodiacetic acid) affinity chromatography columu. The eluted fractions of high purity were collected for dialysis renaturation, and the recombinant protein was identified by 15% SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western Blot. The results showed that we successfully constructed the pET-30 a-EuDIR1 prokaryotic expressing vector and there was a specific protein band in the relative molecular weight of about 18 kD after the expression strain was inducted. The inclusion bodies were putificated and renaturated, then verified by Western Blot. We obtained the highly pure and soluble recombinant proteins. The EuDIR1 protein obtained in the experiment would provide a scientific basis for biochemical experiments in vitro.
The plant dirigent(DIR) protein are proposed to as an improve role in guiding the biosynthesis of lignan and lignin, as well as in the process of biological defense responses, regulation of secondary metabolism, and pathogen resistance. This study was based on the previous experiments about cloning EuDIR1 gene in laboratory and then the prokaryotic expressing vector of pET-30 a-EuDIR1 was constructed by the gene recombination technique. During the transformation of Escherichia coli BL21(DE3) by recombinant vector, the protein deposition reached the highest level after 6 h induction with 0.1 mmol/L isopropyl β-D-thiogalactopyranoside(IPTG) at 15℃,and the expression pattern was mainly inclusion body. After dissolution, the inclusion bodies were purified by Ni-IDA(Nickel-iminodiacetic acid) affinity chromatography columu. The eluted fractions of high purity were collected for dialysis renaturation, and the recombinant protein was identified by 15% SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western Blot. The results showed that we successfully constructed the pET-30 a-EuDIR1 prokaryotic expressing vector and there was a specific protein band in the relative molecular weight of about 18 kD after the expression strain was inducted. The inclusion bodies were putificated and renaturated, then verified by Western Blot. We obtained the highly pure and soluble recombinant proteins. The EuDIR1 protein obtained in the experiment would provide a scientific basis for biochemical experiments in vitro.
引文
Alvarez A.,Montesa no M.,Schmelz E.,and León I.P.D.,2016,Activation of shikimate,phenylpropanoid,oxylipins,and auxin pathways in pectobacterium carotovorum elicitors-treated moss,Front.Plant Sci.,7:483
Burlat V.,Kwon M.,Davin L.B.,and Lewis N.G.,2001,Dirigent proteins and dirigent sites in lignifying tissues,Phytochemistry,57(6):883-897
Davin L.B.,Wang H.B.,Crowell A.L.,Bedgar D.L.,Martin D.M.,Sarkanen S.,and Lewis N.G.,1997,Stereoselective bimolecular phenoxy radical coupling by an auxiliary(dirigent)protein without an active center,Science,275(5298):362-366
Effenberger I.,Zhang B.,Li L.,Wang Q.,Liu Y.,Klaiber I.,Pfannstiel J.,and Schaller A.,2015,Dirigent proteins from cotton(Gossypium sp.)for the atropselective synthesis of gossypol,Angew.Chem.Int.Ed.Engl.,54(49):14660-14663
Espieira J.M.,Novo Uzal E.,Gómez Ros L.V.,Carrión J.S.,Merino F.,Ros Barcelo A.,and Pomar F.,2011,Distribution of lignin monomers and the evolution of lignification among lower plants,Plant Biol.,13(1):59-68
Fang M.,and Huang H.L.,2001,Advances in in vitro refolding of inclusion body proteins,Shengwu Gongcheng Xuebao(Chinese Journal of Biotechnology),17(6):608-612(方敏,黄华樑,2001,包涵体蛋白体外复性的研究进展,生物工程学报,17(6):608-612)
Gasper R.,Effenberger I.,Kolesinski P.,Terlecka B.,Hofmann E.,and Schaller A.,2016,Dirigent protein mode of action revealed by the crystal structure of At DIR6,Plant Physiol.,172(4):2165-2175
Guan S.Y.,and Su W.W.,2003,Chemical composition and pharmacological research progress of Eucommia ulmoides Zhongyaocai(Traditional Chinese Medicinal Materials),26(2):124-129(管淑玉,苏薇薇,2003,杜仲化学成分与药理研究进展,中药材,26(2):124-129)
Hosmani P.S.,Kamiya T.,Danku J.,Naseer S.,Geldner N.Guerinot M.L.,and Salt D.E.,2013,Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based casparian strip in the root,Proc Natl.Acad.Sci.USA,110(35):14498-14503
Kazenwadel C.,Klebensberger J.,Richter S.,Pfannstiel J.Gerken U.,Pickel B.,Schaller A.,and Hauer B.,2013,Optimized expression of the dirigent protein AtDIR6 in Pichia pastoris and impact of glycosylation on protein structure and function,Appl.Microbiol.Biotechnol.,97(16):7215-7227
Kiefhaber T.,Rudolph R.,Kohler H.H.,and Buchner J.,1991Protein aggregation in vitro and in vivo:a quantitative mode of the kinetic competition between folding and aggregation Nat.Biotechnol.,9(9):825-829
Kim M.K.,Jeon J.H.,Fujita M.,Davin L.B.,and Lewis N.G.2002,The western red cedar(Thuja plicata)8-8'dirigent family displays diverse expression patterns and conserved monolignol coupling specificity,Plant Mol.Biol.,49(2)199-214
Li N.,Zhao M.,Liu T.,Dong L.,Cheng Q.,Wu J.,Wang L.Chen X.,Zhang C.,Lu W.,Xu P.,and Zhang S.,2017,Anovel soybean dirigent gene GmDIR22 contributes to promotion of lignan biosynthesis and enhances resistance to Phytophthora sojae,Front.Plant Sci.,8:1185
Luo L.F.,Wu W.H.,Zhou Y.J.,Yan J.,Yang G.P.,and Ouyang D.S.,2010,Antihypertensive effect of Eucommia ulmoides oliv.extracts in spontaneously hypertensive rats,J.Ethnopharmacol.,129(2):238-243
Ma J.Y.,Yang J.D.,Li Q.,and Zhang L.,2017,Discovery and bioinformatics analysis of Dirigent multigene family in Salvia miltiorrhiza,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1594-1610(马金洋,杨瑾冬,李卿,张磊,2017,丹参Dirigent基因家族的发现与生物信息学分析,基因组学与应用生物学,36(4):1594-1610)
Ma Q.H.,and Liu Y.C.,2015,TaDIR13,a dirigent protein from wheat,promotes lignan biosynthesis and enhances pathogen resistance,Plant Mol.Biol.Rep.,33(1):143-152
Ralph S.,Park J.Y.,Bohlmann J.,and Mansfield S.D.,2006,Dirigent proteins in conifer defense:gene discovery,phylogeny and differential wound-and insect-induced expression of a family of DIR and DIR-like genes in spruce(Picea spp.),Plant Mol.Biol.,60(1):21-40
Ralph S.G.,Jancsik S.,and Bohlmann J.,2007,Dirigent proteins in conifer defenseⅡ:extended gene discovery,phylogeny,and constitutive and stress-induced gene expression in spruce(Picea spp.),Phytochemistry,68(14):1975-1991
Rang W.Z.,and Zhou Q.M.,2013,Bioinformatic analysis of the Dirigent gene family in rice,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University),39(2):111-120(穰中文,周清明,2013,水稻Dirigent基因家族生物信息学分析,湖南农业大学学报,39(2):111-120)
Shi H.,Liu Z.,Zhu L.,Zhang C.,Chen Y.,Zhou Y.,Li F.,and Li X.,2012,Overexpression of cotton(Gossypium hirsutum)dirigent1 gene enhances lignification that blocks the spread of verticillium dahliae,Acta Biochim.Biophys.Sin.,44(7):555-564
Wang W.D.,2017,Cloning and expression analysis of Dirigent protein in Eucommia ulmoides,Thesis for M.S.,College of Life Sciences,Guizhou University,Supervisor:Zhao D.G.,pp.25-35(王维东,2017,杜仲Dirigent编码基因克隆及表达分析,硕士学位论文,贵州大学生命科学学院,导师:赵德刚,pp.25-35)
Xia Z.Q.,Costa M.A.,Proctor J.,Davin L.B.,and Lewis N.G.,2000,Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum and Podophyllum peltatum,Phytochemistry,55(6):537-549
Burlat V.,Kwon M.,Davin L.B.,and Lewis N.G.,2001,Dirigent proteins and dirigent sites in lignifying tissues,Phytochemistry,57(6):883-897
Davin L.B.,Wang H.B.,Crowell A.L.,Bedgar D.L.,Martin D.M.,Sarkanen S.,and Lewis N.G.,1997,Stereoselective bimolecular phenoxy radical coupling by an auxiliary(dirigent)protein without an active center,Science,275(5298):362-366
Effenberger I.,Zhang B.,Li L.,Wang Q.,Liu Y.,Klaiber I.,Pfannstiel J.,and Schaller A.,2015,Dirigent proteins from cotton(Gossypium sp.)for the atropselective synthesis of gossypol,Angew.Chem.Int.Ed.Engl.,54(49):14660-14663
Espieira J.M.,Novo Uzal E.,Gómez Ros L.V.,Carrión J.S.,Merino F.,Ros Barcelo A.,and Pomar F.,2011,Distribution of lignin monomers and the evolution of lignification among lower plants,Plant Biol.,13(1):59-68
Fang M.,and Huang H.L.,2001,Advances in in vitro refolding of inclusion body proteins,Shengwu Gongcheng Xuebao(Chinese Journal of Biotechnology),17(6):608-612(方敏,黄华樑,2001,包涵体蛋白体外复性的研究进展,生物工程学报,17(6):608-612)
Gasper R.,Effenberger I.,Kolesinski P.,Terlecka B.,Hofmann E.,and Schaller A.,2016,Dirigent protein mode of action revealed by the crystal structure of At DIR6,Plant Physiol.,172(4):2165-2175
Guan S.Y.,and Su W.W.,2003,Chemical composition and pharmacological research progress of Eucommia ulmoides Zhongyaocai(Traditional Chinese Medicinal Materials),26(2):124-129(管淑玉,苏薇薇,2003,杜仲化学成分与药理研究进展,中药材,26(2):124-129)
Hosmani P.S.,Kamiya T.,Danku J.,Naseer S.,Geldner N.Guerinot M.L.,and Salt D.E.,2013,Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based casparian strip in the root,Proc Natl.Acad.Sci.USA,110(35):14498-14503
Kazenwadel C.,Klebensberger J.,Richter S.,Pfannstiel J.Gerken U.,Pickel B.,Schaller A.,and Hauer B.,2013,Optimized expression of the dirigent protein AtDIR6 in Pichia pastoris and impact of glycosylation on protein structure and function,Appl.Microbiol.Biotechnol.,97(16):7215-7227
Kiefhaber T.,Rudolph R.,Kohler H.H.,and Buchner J.,1991Protein aggregation in vitro and in vivo:a quantitative mode of the kinetic competition between folding and aggregation Nat.Biotechnol.,9(9):825-829
Kim M.K.,Jeon J.H.,Fujita M.,Davin L.B.,and Lewis N.G.2002,The western red cedar(Thuja plicata)8-8'dirigent family displays diverse expression patterns and conserved monolignol coupling specificity,Plant Mol.Biol.,49(2)199-214
Li N.,Zhao M.,Liu T.,Dong L.,Cheng Q.,Wu J.,Wang L.Chen X.,Zhang C.,Lu W.,Xu P.,and Zhang S.,2017,Anovel soybean dirigent gene GmDIR22 contributes to promotion of lignan biosynthesis and enhances resistance to Phytophthora sojae,Front.Plant Sci.,8:1185
Luo L.F.,Wu W.H.,Zhou Y.J.,Yan J.,Yang G.P.,and Ouyang D.S.,2010,Antihypertensive effect of Eucommia ulmoides oliv.extracts in spontaneously hypertensive rats,J.Ethnopharmacol.,129(2):238-243
Ma J.Y.,Yang J.D.,Li Q.,and Zhang L.,2017,Discovery and bioinformatics analysis of Dirigent multigene family in Salvia miltiorrhiza,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(4):1594-1610(马金洋,杨瑾冬,李卿,张磊,2017,丹参Dirigent基因家族的发现与生物信息学分析,基因组学与应用生物学,36(4):1594-1610)
Ma Q.H.,and Liu Y.C.,2015,TaDIR13,a dirigent protein from wheat,promotes lignan biosynthesis and enhances pathogen resistance,Plant Mol.Biol.Rep.,33(1):143-152
Ralph S.,Park J.Y.,Bohlmann J.,and Mansfield S.D.,2006,Dirigent proteins in conifer defense:gene discovery,phylogeny and differential wound-and insect-induced expression of a family of DIR and DIR-like genes in spruce(Picea spp.),Plant Mol.Biol.,60(1):21-40
Ralph S.G.,Jancsik S.,and Bohlmann J.,2007,Dirigent proteins in conifer defenseⅡ:extended gene discovery,phylogeny,and constitutive and stress-induced gene expression in spruce(Picea spp.),Phytochemistry,68(14):1975-1991
Rang W.Z.,and Zhou Q.M.,2013,Bioinformatic analysis of the Dirigent gene family in rice,Hunan Nongye Daxue Xuebao(Journal of Hunan Agricultural University),39(2):111-120(穰中文,周清明,2013,水稻Dirigent基因家族生物信息学分析,湖南农业大学学报,39(2):111-120)
Shi H.,Liu Z.,Zhu L.,Zhang C.,Chen Y.,Zhou Y.,Li F.,and Li X.,2012,Overexpression of cotton(Gossypium hirsutum)dirigent1 gene enhances lignification that blocks the spread of verticillium dahliae,Acta Biochim.Biophys.Sin.,44(7):555-564
Wang W.D.,2017,Cloning and expression analysis of Dirigent protein in Eucommia ulmoides,Thesis for M.S.,College of Life Sciences,Guizhou University,Supervisor:Zhao D.G.,pp.25-35(王维东,2017,杜仲Dirigent编码基因克隆及表达分析,硕士学位论文,贵州大学生命科学学院,导师:赵德刚,pp.25-35)
Xia Z.Q.,Costa M.A.,Proctor J.,Davin L.B.,and Lewis N.G.,2000,Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum and Podophyllum peltatum,Phytochemistry,55(6):537-549