Shang Yimin, Guo Wenfang, Liu Xiaomeng, Ma Lei, Liu Dehu, Chen Sanfeng
College of Biological Sciences, China Agricultural University, Beijing, China.
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
PLoS One. 2023 Aug 24;18(8):e0290556. doi: 10.1371/journal.pone.0290556. eCollection 2023.
Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants.
化学氮肥可以维持作物产量，但过度使用化学氮肥会导致经济成本增加和环境污染。减少氮肥使用的一种方法是将固氮酶生物合成途径转移到非豆科植物中。由nifH编码的铁蛋白和由nifD和nifK编码的钼铁蛋白是固氮酶的两个结构成分。由nifB编码的NifB是一种关键的成熟酶，它催化固氮酶铁钼辅因子生物合成中的第一个关键步骤，该辅因子结合并还原N2。nifB、nifH、nifD和nifK的表达对于培育能够固定大气中N2的植物至关重要。在本研究中，来自多粘芽孢杆菌WLY78的四个基因（nifB、nifH、nifD和nifK）通过Cre/LoxP重组系统组装到植物表达载体pCAMBIA1301中，得到重组表达载体pCAMBIA1301-nifBHDK。然后，利用农杆菌介导的转化方法将表达载体中携带的四个nif基因共同导入陆地棉R15中。通过PCR和RT-PCR筛选出T3代纯合转基因棉花株系B2、B5和B17。qRT-PCR结果表明，nifB、nifH、nifD和nifK在转基因棉花中以相似水平共表达。蛋白质免疫印迹分析表明，NifB、NifH、NifD和NifK在转基因棉花中共同产生。四个关键Nif蛋白（NifB、NifH、NifD和NifK）在棉花中的共表达代表了将固氮酶生物合成途径工程化到非豆科植物中的重要一步。
