番茄果实转录因子CNR的原核表达与纯化及其抗体的制备

doi:10.13560/j.cnki.biotech.bull.1985.2018-0664

摘要/Abstract

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1.0 mmol/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。

关键词: 番茄, 转录因子CNR, 原核表达, 蛋白纯化, 多克隆抗体

Abstract: 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 at 27 kD,indicating that pET-CNRprokaryotic expression vector was successfully constructed. The optimal concentration of IPTG inducer was 1.0 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.

Key words: tomato, transcription factor CNR, prokaryotic expression, protein purification, polyclonal antibody

李玲, 白娟娟, 郭梅, 王思禹, 王晓玥. 番茄果实转录因子CNR的原核表达与纯化及其抗体的制备[J]. 生物技术通报, 2019, 35(2): 1-8.

LI Ling, BAI Juan-juan, GUO Mei, WANG Si-yu, WANG Xiao-yue. Prokaryotic Expression,Purification and Antibody Preparation of Transcription Factor CNR in Tomato Fruit[J]. Biotechnology Bulletin, 2019, 35(2): 1-8.

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