不结球白菜硝酸还原酶基因BcNR的克隆及其在拟南芥中的转化

Molecular Cloning of A Non-Heading Chinese Cabbage Nitrate Reductase Gene (BcNR) and Transformation into Arabidopsis thaliana

【目的】克隆不结球白菜硝酸还原酶基因(BcNR),并转化拟南芥做初步的功能鉴定。【方法】采用分段RT-PCR及3′RACE和5′RACE技术克隆了BcNR基因的全长cDNA,利用生物信息学方法进行序列分析和蛋白结构分析。构建pCAM-BcNR植物过量表达载体,通过农杆菌介导的真空渗透法将其转入野生拟南芥中,经抗性筛选及PCR验证获得T0代转基因植株,用Real-timePCR方法对30mmol·L-1KNO3溶液处理的转基因拟南芥的硝酸还原酶基因表达进行检测,并测定转基因植株的硝酸还原酶活性。【结果】获得了编码不结球白菜硝酸还原酶基因(BcNR)的cDNA全序列3049bp,包含有2733bp的开放阅读框,编码910个氨基酸。其氨基酸序列与拟南芥、烟草、甘蓝型油菜、马铃薯等编码的氨基酸序列具有较高同源性。蛋白质结构域分析表明,所推导的氨基酸序列具有完整的硝酸还原酶结构,同时证明从不结球白菜中克隆的这条基因属于NADH-NR型。GenBank登录号为EU662272。转基因拟南芥T0代植株进行GUS基因PCR检测,证明重组质粒已整合到转基因植株基因组中。硝酸盐处理后的转基因植株硝酸还原酶基因的表达比野生型显著提高,且硝酸还原酶活性有不同程度的提高。【结论】本研究首次从不结球白菜中克隆获得BcNR基因的全长cDNA序列,揭示了其序列特征并对其进行了初步的功能鉴定,为进一步利用该基因开展蔬菜硝酸盐含量的基因调控研究奠定了基础。

[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 TO 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.