Prokaryotic Expression, Purification And Preparation Of Antibody Of Transcription Factor CNR In Tomato Fruit

Colourless non-ripening(cnr)tomato is a typica l fruit ripening mutant.And this mutation is mainly due to the high degree of cytosine methylation of 286 bp sequence in the upstream of LeSPL-CNR promoter in tomato mutant cnr which leads to the change of DNA methylation,but there is no difference in nucleic acid sequence.At present,the study of tomato transcriptional factor CNR is mainly at the transcriptional level.However,after the protein synthesis,there is a post translation level modification,and the protein level is the level of the transcription factor CNR.In this study,tomato transcription factor CNR was studied on protein level by expression,purification of tomato transcription factor CNR and preparation of polyclonal antibody.The main results are as follows:(1)Construction of pET-CNR prokaryotic expression vector: primers were designed according to the sequence of tomato CNR gene published in GenBank,and used for CNR gene amplification.The total RNA of tomato was used as a template and reverse transcribed into cDNA.Then we amplified the CNR gene with cDNA as template to obtained the fragment which was in agreement with the expected CNR coding gene size.The fragment and the expression vector of pET-30 a were cut into two sticky ends with the restriction endonuclease BamHI and XhoI.After purification,the T4 ligase was used for directional connection and transferred into the Escherichia coli receptor state Rosetta for expression.It was confirmed by sequencing that the pET-CNR recombinant vector had the correct coding frame,the sequencing results were in full agreement with the published CNR coding sequence of the tomato,and it is preliminarily proved that the prokaryotic expression vector pET-CNR has been successfully constructed.A small amount of expression was induced by IPTG and the results were analyzed at 27 kD with the expression of CNR protein,which proved that pETCNR could express the protein and the pET-CNR prokaryotic expression vector was constructed.(2)The prokaryotic expression and purification of CNR protein in Escherichia coli: single positive colonies in solid medium containing recombinant plasmid pET-CNR were picked up in culture medium in 37? and induced by IPTG as inducers.The inducement concentration of IPTG inducers were 0.1,0.4,0.8 and 1.0mmol/L respectively.After 3h,the bacteria werecollected respectively and broke up with the ultrasonic technique,and the supernatant and precipitation were retained respectively.The the expression of protein were analyzed by12%SDS-PAGE,and determined that the best concentration of IPTG inducer was 1.0mmol/L.The induced expression of CNR protein was expressed in the supernatant of the lysate,but almost no expression in the precipitate of the lysate.Therefore,the supernatant was filtered directly into the Ni affinity chromatography column after 0.22 mu m filter membrane and purified to collect the proteins eluted by the imidazole concentration of 500 mmol/L elution buffer.Each tube was collected 200 ?L.The protein collected by each tube was analyzed by BCA kit method,and the protein concentration was mixed in the collection tube of 1mg/ml,and the concentration of total protein was used for antibody preparation.(3)The preparation of CNR protein polyclonal antibody: 100?g the purified CNR protein was emulsified by 1:1 mixed with freund's complete adjuvant,and the mice were immunized under the abdominal subcutaneous 4-6 points,and then the immunization was done 3 times,and the whole blood of the mice was collected after the last 1 immunizations.The immune serum was placed at 4? for the night,and was centrifuged with 5000 rpm for 10 min.The supernatant was extracted,separated for the serum titer and Western blot test respectively to obtain the high titer polyclonal antibody was obtained.The results showed that transcription factor CNR played an important role in the breaking stage of tomato fruits by extracting total protein from tomato fruits in different periods with Western blot.