Physiology And Molecular Biology Research Of NO₃^- Metabolism In Non-heading Chinese Cabbage

Nitrate is the most important source of mineral N for plants growing in aerobic soils, especially for leaf vegetables. Research on NO₃^- metabolism pathway of vegetable plays an important role in nitrogenous fertilizing, increasing yield and quality and developing no public damage vegetable. In this study, using nitrate sensitive non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar 'Suzhouqing' as a test material, studied on the effect of different NO₃^--N concentration on yield, nutrient quality and nitrate content; molecular cloning and characterization the genes of enzymes in NO₃^- metabolism pathway; function identifying of the nitrate reductase gene which is a rate-limiting enzyme gene in the pathway and analysis the expressions of each gene under different nitrogen treatments. The results were as follows:1. Effect of different NO₃^--N concentration on yield, nutrient quality and nitrate content of non-heading Chinese cabbageNitrate sensitive non-heading Chinese cabbage 'Suzhouqing' (Brassica campestris ssp. chinensis Makino) was cultivated in soilless subtrate irrigating with nutrient solution with five nitrate concentrations (0, 4, 8, 12, 16 and 20 mmol·L^-1). After fourteen days, the yield, nutrient quality, nitrate content and nitrate reductase activity (NRA) of non-heading Chinese cabbage were determined. The results showed that the shoot fresh weight increased most rapidly with NO₃^--N supply except the treatment of 20mmol·L^-1. Vitamin C and soluble sugar content changed similarly, and they presented negative correlation with nitrogen concentration except the treatment of 0mmol·L^-1. However, soluble protein content reached the highest at 16mmol·L^-1 and had a little decrease at 20mmol·L^-1. Nitrate content in root, petiole and leave increased with NO₃^--N supply. Generally, the maximum nitrate concentration was observed in the petiole while the lowest in the root. The highest nitrate reductase activity (NRA) was found at 4 mmol·L^-1. In addition, there is no obvious relationship between NRA and nitrate concentration in the plants. In conclusion, the NO₃^--N of 12 mmol·L^-1 is the best concentration for non-heading Chinese cabbage growing.2. Molecular Cloning, characterization and expression of nitrate reductase gene BcNR from non-heading Chinese cabbage in Escherichia coliBy RT-PCR and 5'/3' RACE strategy, a full length cDNA encoding nitrate reductase was cloned from non-hesding Chinese cabbage (Brassica campestris ssp. chinensis), It was 3049bp and contained a opening-reading frame (ORF) 2733 bp encoding 910 amino acids. The deduced amino sequence was highly homology to Arabidopsis thaliana, Nicotiana benthamiana,Brassica napus and Solarium tuberosum and so on. The protein structure domain analysis indicated that, deduced amino acid sequence has the integrity NR structure. we also proved that this clone is belong to NADH-NR. The Southern blot indicated this gene has three copies at least in Brassica campestris ssp. chinensis. This sequence has been submitted to the GenBank database and the accession number is EF492053. The coding sequence of NR (2733 bp) for protein expression was amplified by PCR technique. Then the recombinant plasmid pET-BcNR was constructed. The recombinant protein was expressed in E. coli BL21 (DE3) after induced by IPTG. SDS-PA GE was used to analyze expression of the target protein. The result showed that there was a specific band at about 103 KDa in size, which was identical with the expected molecular weight of the recombinant protein.3.Fuction analysis of BcNR from non-heading Chinese cabbageThis section was about a fuctional research of BcNR from non-heading Chinese cabbage (Brassica campestris ssp.chinensis). An efficiently expression vector of pCAM-BcNR was constructed and transformed into Arabidopsis thaliana by Agrobacterium mediated depressor permeating method. Transgenic Arabidopsis thaliana plants in TO were obtained by resistance screening and PCR identification. The expression of BcNR. in positive plants which treated with 30mmol·L^-1 KNO₃ was detected by real-time PCR method, and the nitrate reductase activities of transgenic plants and wild type plants were determined. The results showed that compared with wild Arabidopsis thaliana. The transgenic plants exhibited an enhanced level of NR and nitrate reductase activity (NRA) of leaves under NO₃^- inducement. 4.Molecular Cloning and characterization of nitrite reductase gene BcNiR from non-heading Chinese cabbageThe BcNiR gene was cloned using RT-PCR with cDNA isolated from non-heading Chinese cabbage (Brassica campestris ssp.chinensis), which was nitrate-induced for 4 hours. The cDNA of BcNiR was 1852 bp and contained a 1749 bp opening-reading frame (ORF) encoding 583 amino acids. The further comparison showed that BcNiR identity to Arabidopsis thaliana NiR1 gene and Nicotiana tabacum nii2 gene were 83% and 76%, respectively. The predicted BcNiR protein was found to have a hemoprotein beta-compnent (ferrodoxin-like), a siroheme-binding site and 4Fe-4S region. A similar 3D structural were obtained from the SWiSS-MODEL database. The Genbank accession number of BcNiR is EU499384.5.Molecular cloning, characterization and subcell location of cytoplasmic glutamine synthetase gene BcGS1 from non-heading Chinese cabbageThe cytoplasmic glutamine synthetase gene BcGS1 was cloned using RT-PCR with cDNA isolated from the root of non-heading Chinese cabbage (Brassica campestris ssp.chinensis), It was 1413 bp and contained a opening-reading frame (ORF) 1071 bp encoding 356 amino acids. The deduced amino sequence was highly homology to Arabidopsis thaliana, Brassica napus, Brassica rapa, Spinacia oleracea and so on. This sequence has been submitted to the GenBank database and the accession number is EU499383. BcGS1 gene linked with gfp gene was transformed into the cell of onion cuticular. Onion cuticular cell containing gfp was used as control, the distribution of BcGS1 in onion cuticular cells was observed with fluorescent microscope. It is suggested that BcGS1 localized in cytoplasm.6.Molecular Cloning, characterization and expression of chloroplast glutamine synthetase gene BcGS2 from non-heading Chinese cabbage in Escherichia coliThe cytoplasmic glutamine synthetase gene BcGS1 was cloned using RT-PCR with cDNA isolated from the leaves of non-heading Chinese cabbage (Brassica campestris ssp.chinensis), It was 1497bp and contained a opening-reading frame (ORF) 1287bp encoding 428 amino acids. The deduced amino sequence was highly homology to Arabidopsis thaliana, Nicotiana benthamiana, Spinacia olerace, Cucumis melo and so on. This sequence has been submitted to the GenBank database and the accession number is EU239243. The coding sequence of BcGS2 (1287 bp) for protein expression was amplified by PCR technique. Then the recombinant plasmid pET- BcGS2 was constructed. The recombinant protein was expressed in E. coli BL21 (DE3) after induced by IPTG. SDS-PA GE was used to analyze expression of the target protein. The result showed that there was a specific band at about 43 kDa in size, which was identical with the expected molecular weight of the recombinant protein.7. Expression analysis of the genes encoding enzymes related to nitrogen metabolizition in non-heading Chinese cabbagemRNA levels of the genes BcNR, BcNiR, BcGS1 and BcGS2 (encoding enzymes nitrate reductase, nitrite reductase, cytoplasmic glutamine synthetase and chloroplast glutamine synthetase, respectively) related to nitrogen metabolizition in the root and leaves of non-heading Chinese cabbage (Brassica campestris ssp.chinensis) were detected by real time PCR methord to observe the effects of different nitrate resource, concentration and inducing time on the expression of these genes. The results showed that, the maxmum expression of BcNR and BcNiR in roots and leaves were induced by 30 mmol·L^-1 NO₃^--N, while the maxmum expression of BcGS1 and BcGS2 were induced by 20 mmol·L^-1 NO₃^--N. Under the treatment of NO₃^--N, Expression of the four genes were significantly increased at earlier stage, but decreased later. NH₄⁺-N can enhanced the expression of BcGS1 and BcGS2, while high level of NH₄⁺-N treatment restrained the expression of BcNR and BcNiR.