Biotechnology Bulletin ›› 2019, Vol. 35 ›› Issue (2): 1-8.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0664

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Prokaryotic Expression,Purification and Antibody Preparation of Transcription Factor CNR in Tomato Fruit

LI Ling, BAI Juan-juan, GUO Mei, WANG Si-yu, WANG Xiao-yue   

  1. College of Food Science and Biotechnology,Tianjin Agricultural University,Tianjin 300384
  • Received:2018-07-22 Online:2019-02-26 Published:2019-03-07

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