关键词: 不结球白菜, 硝酸还原酶, 克隆, 功能鉴定

Abstract:

【Objective】 A full length cDNA of nitrate reductase gene (BcNR) was isolated from the non-heading Chinese cabbage (Brassica campestris ssp. chinensis) cultivar Suzhouqing, and transformed into Arobidopsis thaliana for functional identification. 【Method】 The full-length cDNA was isolated by RT-PCR, subsection PCR and (5′/3′)-RACE techniques. Bioinformatics methods were used to sequence analysis and protein structure analysis. An efficiently expression vector pCAM-BcNR was constructed and transformed into Arabidopsis thaliana by Agrobacterium mediated depressor permeating method. Transgenic Arabidopsis thaliana plants in T0 were obtained by resistance screening and PCR identification. The expression of BcNR in positive plants treated with 30 mmol?L-1 KNO3 was detected by real-time PCR method, and the nitrate reductase activities of transgenic plants and wild type plants were determined. 【Result】 The full-length cDNA sequence of 3 049 bp in length contained an open reading frame of 2 733 bp encoding 910 amino acids. The deduced amino sequence was highly homology to Arabidopsis thaliana, Nicotiana benthamiana, Brassica napus and Solanum tuberosum and other high plants. This protein shares common structural features with NRs from other higher plants and other eukaryotes. It was also proved that this clone was the style of NADH-NR. The sequence was accepted by GenBank (Accession number EU662272). GUS PCR analysis revealed that the recombinant plasmid was integrated into the transgenic Arabidopsis thaliana plants in T0. Compared with wild Arabidopsis thaliana, the transgenic plants exhibited an enhanced level of NR and nitrate reductase activity (NRA) of leaves under NO3- inducement. 【Conclusion】 Full length BcNR gene was firstly isolated and characterized from non-heading Chinese cabbage and introduced into Arabidopsis thaliana for preliminary functional identification. This study lays a foundation for further utilization of this gene in genetic modification of nitrate content in vegetable production.

Key words: Brassica campestris ssp. chinensis, nitrate reductase, cloning, functional identification