| Nitrate is nitrosamines' precursors which is the strong carcinogen.The carcinogenesis of digestive organs will be induced by excessive intake of nitrate.And seriously endanger human health.The human body takes in nitrate most from the vegetables.Leafy vegetables belong to nitrate-easily-enrich crops,particularly vegetables such as spinach.They are the main source for people to take in nitrate. Thus to control the content of nitrate human take in is the first necessary to control the content of nitrate in vegetables.Nitrate can be reduced to NO2- by nitrate reductase(NR).Enhancing nitrate reductase activity(NRA)in vegetables can not only reduce the nitrate content of vegetables but also increase the output of vegetable under the same conditions of vegetable fertilizer and consequently improve the utilization of fertilizers.We studied the influence of the spinach nitrate's accumulation reply to different nitrogen levels through potted experiment.And then low nitrate spinach varieties was screened by salicylic acid method.In 400 ppm high nitrogen level we had screened low nitrate spinach varieties——GZ round leaf spinach varietie.And their leaves were used as material for extracting the total RNA,then we reverse transcribed the RNA into cDNA,designed PCR primers according to the sequence of NR had been reported,and amplified its open reading frame.Then we constructed pET-NR recombinant and expressed in the BL21.The fusion protein was examined by SDS-PAGE gel electrophoresis.Specific results were as follows:1.In this study we had screened the following two spinach varieties from 40 of them:①In 182 ppm low nitrogen level low nitrate spinach varieties——HN white and big leaf spinach varieties(115.073 mg/kg)was screened,were the percent of commercial variety CZ large leaf spinach(251.716 mg/kg)45.7;②In 400 ppm high nitrogen level low nitrate spinach varieties——GZ round leaf spinach varieties (453.721 mg/kg)was screened,were the percent of commercial variety CZ large leaf spinach(999.265 mg/kg)45.4.2.We had obtained the ORF sequence of nitrate reductase gene:In 400 ppm high nitrogen level we had screened low nitrate spinach varieties——GZ round leaf spinach varieties.And their leaves were used as material for extracting the total RNA, then we reverse transcribed the RNA into cDNA,designed PCR primers according to the sequence of NR had been reported,and amplified its open reading frame.Then we constructed pET-NR recombinant and expressed in the BL21.The fusion protein was examined by SDS-PAGE gel electrophoresis.The sequence of amino acid was deduced by DNAstar,and it gets as high as 99%match to the reportorial NR sequence.3.Prokaryotic expression:the ORF of NR gene which had been amplified was recombined with pET-28a expression vector out of the body.Then the recombined prokaryotic expression vector pET-NR was constructed,and the recombinant was expressed in E.coli BL21,and SDS-PAGE was used to detect the molecular weight of fusion protein which was induced by IPTG.The results indicated that the size of fusion protein was 104 kD.It was consistent with the expectant molecular weight. |
