番茄果实转录因子CNR的原核表达与纯化及其抗体的制备
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摘要
转录因子CNR(Colourless non-ripening)在果实的发育和成熟过程中起重要作用。目前关于番茄转录因子CNR的研究主要集中在转录水平,然而蛋白水平才是CNR发挥作用的水平。通过对CNR进行原核表达和纯化,并制备CNR的多克隆抗体,在蛋白水平上确定番茄转录因子CNR的表达模式。结果表明:pET-CNR重组载体具有正确的读码框,且与已发表的番茄CNR编码序列完全一致,并且在27 kD处有CNR蛋白表达,证明pET-CNR原核表达载体构建成功。诱导剂IPTG的最适宜浓度为1.0mmol/L,当用咪唑浓度为500 mmol/L的洗脱缓冲液洗脱层析柱时得到单一的CNR蛋白,并以此作为抗原免疫小鼠,制得效价高且特异性好的CNR蛋白多克隆抗体。最后研究不同成熟时期番茄果实CNR蛋白的表达模式,结果发现转录因子CNR在番茄果实的破色期起重要作用。
Transcription factor CNR(Colourless non-ripening)plays a key role in the process of fruit development and maturity.At present,the study on tomato transcription factor CNR is mainly at the transcriptional level;however,the protein level is where CNR functions. In this study,the expression pattern of transcription factor CNR in tomato fruit was studied at protein level by prokaryotic expression,purification of CNR,and preparation of polyclonal antibody. The results confirmed that the pET-CNR recombinant vector had the correct reading frame and the sequence was in full agreement with the published CNR coding sequence in tomato. There was CNR protein expression at27 kD,indicating that pET-CNR prokaryotic expression vector was successfully constructed. The optimal concentration of IPTG inducer was1.0 mmol/L. The purified CNR protein was collected while eluting chromatographic column by the elution buffer of 500 mmol/L imidazole,then used as antigen to immunize mice,and the CNR protein polyclonal antibody with high potency and favorable specificity was obtained. Finally,the expression patterns of CNR protein in tomato fruit at different maturity stages were studied,and it was discovered that transcription factor CNR played a crucial role in the color-breaking stage of tomato fruit.
Transcription factor CNR(Colourless non-ripening)plays a key role in the process of fruit development and maturity.At present,the study on tomato transcription factor CNR is mainly at the transcriptional level;however,the protein level is where CNR functions. In this study,the expression pattern of transcription factor CNR in tomato fruit was studied at protein level by prokaryotic expression,purification of CNR,and preparation of polyclonal antibody. The results confirmed that the pET-CNR recombinant vector had the correct reading frame and the sequence was in full agreement with the published CNR coding sequence in tomato. There was CNR protein expression at27 kD,indicating that pET-CNR prokaryotic expression vector was successfully constructed. The optimal concentration of IPTG inducer was1.0 mmol/L. The purified CNR protein was collected while eluting chromatographic column by the elution buffer of 500 mmol/L imidazole,then used as antigen to immunize mice,and the CNR protein polyclonal antibody with high potency and favorable specificity was obtained. Finally,the expression patterns of CNR protein in tomato fruit at different maturity stages were studied,and it was discovered that transcription factor CNR played a crucial role in the color-breaking stage of tomato fruit.
引文
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[16]李玲.转录因子LeMADS-RIN调控番茄果实乙烯生物合成机制的研究[D].北京:中国农业大学, 2011:22-37.
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[20]秦伟,黄昆仑,贺晓云,等.水稻密码子优化的cry2A*基因在大肠杆菌中的表达及其表达产物的纯化[J].食品科学,2008, 29(7):267-271.
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[22]李军. Nanog基因的原核表达、蛋白纯化及抗体制备[D].杨凌:西北农林科技大学, 2007:27-50.
[23]宋振.人SUMO-1蛋白多克隆抗体的制备与鉴定及亚细胞结构的定位[D].杨凌:西北农林科技大学, 2008:21-28.
[24]袁蓓,李京生,吴燕民.利用植物生产药用蛋白的进展、局限及应对策略[J].草业学报, 2013, 22(4):283-299.
[25]刘嘉霖.重组冷休克蛋白的表达纯化及其神经保护机制的研究[D].北京:中国人民解放军医学院. 2015:3-22.
[26]Zhang W, Li B, Yu B. Genome-wide identification, phylogeny and expression analysis of the SBP-box gene family in maize(Zea mays)[J]. Journal of Integrative Agriculture, 2016, 15(1):29-41.
[27]Chen WW, Kong JH, Lai TF, et al. Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening[J]. Scientific Reports, 2015, 5:7852.
[28]Martel C, Vrebalov J, Tafelmeyer P, et al. The tomato MADSboxtranscriptionfactorRIPENINGINHIBITORinteracts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENING-dependent manner[J]. Plant Physiology, 2011, 157:1568-1579.
[29]Chen WW, Yu ZM, Kong JH, et al. Comparative WGBS identifies genes that influence non-ripe phenotype in tomato epimutant Colourless non-ripening[J]. Science China-Life Sciences, 2018,61:244-252.
[2]Huijser P, Schmid M. The control of developmental phase transitions in plants[J]. Development, 2011, 138:4117-4129.
[3]Jung JH, Seo PJ, Kang SK, et al. miR172 signals are incorporated into the miR156 signaling pathway at the SPL3/4/5 genes in Arabidopsis developmental transitions[J]. Plant Molecular Biology, 2011, 76:35-45.
[4]Jung JH, Ju Y, Seo PJ, et al. The SOC1-SPL module integrates photoperiod and gibberellic acid signals to control flowering time in Arabidopsis[J]. Plant Journal, 2012, 69:577-588.
[5]Yu S, Galvao VC, Zhang YC, et al. Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE transcription factors[J]. Plant Cell,2012, 24:3320-3332.
[6]Zhang SD, Ling LZ. Genome-wide identification and evolutionary analysis of the SBP-box gene family in castor bean[J]. PLoS One,2014, 9(1):e86688.
[7]Tan HW, Song XM, Duan WK, et al. Genome-wide analysis of the SBP-box gene family in Chinese cabbage(Brassica rapa subsp pekinensis)[J]. Genome, 2015, 58(11):463-477.
[8]Xu Z, Sun L, Zhou Y, et al. Identification and expression analysis of the SQUAMOSA promoter-binding protein(SBP)-box gene family in Prunus mume[J]. Mol Genet Genomics, 2015, 290(5):1701-1715.
[9]Zhang SD, ling LZ, Yi TS. Evolution and divergence of SBP-box genes in land plants[J]. BMC Genomics, 2015, 16:787-796.
[10]Zhang W, Li B, Yu B. Genome-wide identification, phylogeny and expression analysis of the SBP-box gene family in maize(Zea mays)[J]. Journal of Integrative Agriculture, 2016, 15(1):29-41.
[11]Fraser P, Bramley P, Seymour GB. Effect of the Cnr mutation on carotenoid formation during tomato fruit ripening[J].Phytochemistry, 2001, 58:75-79.
[12]Orfila C, Huisman MM, Willats WG, et al. Altered cell wall disassembly during ripening of Cnr tomato fruit:implications for cell adhesion and fruit softening[J]. Planta, 2002, 215:440-447.
[13]Eriksson EM, Bovy A, Manning K, et al. Effect of the colorless nonripening mutation on cell wall biochemistry and gene expression during tomato fruit development and ripening[J]. Plant Physiology, 2004, 136:4184-4197.
[14]Manning K, T?r M, Poole M, et al. A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[J]. Nature Genetics, 2006, 38(8):948-952.
[15]Chen WW, Jin JF, Lou HQ, et al. LeSPL-CNR negatively regulates Cd acquisition through repressing nitrate reductase-mediated nitric oxide production in tomato[J]. Planta, 2018, 248(4):893-907.
[16]李玲.转录因子LeMADS-RIN调控番茄果实乙烯生物合成机制的研究[D].北京:中国农业大学, 2011:22-37.
[17]王贺,杨瑞金,华霄,等.密苏里游动放线菌葡萄糖异构酶基因xylA的克隆及其表达条件的优化[J].食品科学, 2010, 31(15):171-176.
[18]郭尧君.蛋白质电泳实验技术[M].第2版.北京:科学出版社,2005:92-118, 255-268.
[19]朱鸿亮.番茄果实成熟相关转录因子LeMADS-RIN的表达和功能分析[D].北京:中国农业大学, 2008:30.
[20]秦伟,黄昆仑,贺晓云,等.水稻密码子优化的cry2A*基因在大肠杆菌中的表达及其表达产物的纯化[J].食品科学,2008, 29(7):267-271.
[21]杨川,胡敏.斑马鱼SFPQ蛋白的原核表达及纯化[J].生物技术通报, 2016, 32(1):163-168.
[22]李军. Nanog基因的原核表达、蛋白纯化及抗体制备[D].杨凌:西北农林科技大学, 2007:27-50.
[23]宋振.人SUMO-1蛋白多克隆抗体的制备与鉴定及亚细胞结构的定位[D].杨凌:西北农林科技大学, 2008:21-28.
[24]袁蓓,李京生,吴燕民.利用植物生产药用蛋白的进展、局限及应对策略[J].草业学报, 2013, 22(4):283-299.
[25]刘嘉霖.重组冷休克蛋白的表达纯化及其神经保护机制的研究[D].北京:中国人民解放军医学院. 2015:3-22.
[26]Zhang W, Li B, Yu B. Genome-wide identification, phylogeny and expression analysis of the SBP-box gene family in maize(Zea mays)[J]. Journal of Integrative Agriculture, 2016, 15(1):29-41.
[27]Chen WW, Kong JH, Lai TF, et al. Tuning LeSPL-CNR expression by SlymiR157 affects tomato fruit ripening[J]. Scientific Reports, 2015, 5:7852.
[28]Martel C, Vrebalov J, Tafelmeyer P, et al. The tomato MADSboxtranscriptionfactorRIPENINGINHIBITORinteracts with promoters involved in numerous ripening processes in a COLORLESS NONRIPENING-dependent manner[J]. Plant Physiology, 2011, 157:1568-1579.
[29]Chen WW, Yu ZM, Kong JH, et al. Comparative WGBS identifies genes that influence non-ripe phenotype in tomato epimutant Colourless non-ripening[J]. Science China-Life Sciences, 2018,61:244-252.