斯氏假单胞菌A1501固氮新基因PST1305的功能分析

Analysis of a new nitrogen fixation gene in Pseudomonas stutzeri A1501

摘要:

摘要：【目的】研究斯氏假单胞菌A1501基因组“固氮岛”中PST1305基因在A1501生物固氮过程中所起的作用。【方法】利用同源重组与三亲接合的方法构建PST1305的非极性突变株。乙炔还原法测定固氮酶活。RT-PCR分析PST1305基因与其周围基因转录单元的关系，Real-Time PCR比较PST1305在最佳固氮与非固氮条件下表达水平的差异。【结果】突变株np1305的固氮酶活显著降低，功能互补菌株np1305Comp能基本恢复细胞的固氮作用。PST1305与其上游的nifB、fdxN、下游的nifQ等基因位于同一个转录单元，组成一个操纵子。基因芯片表明，PST1305基因在固氮比非固氮条件下表达量显著上调（约38.7倍），Real-Time PCR验证支持这一结果。【结论】PST1305基因参与固氮过程，其突变会影响固氮酶的活性，该基因可能通过参与A1501固氮酶电子传递或者固氮酶的氧保护过程影响固氮效率。

Abstract:

Abstract: [Objective] We studied the role of the nitrogen fixation gene PST1305 located within the nitrogen fixation island of Pseudomonas stutzeri A1501. [Methods] We constructed the mutant strain (np1305) by homologous recombination and triparental conjugation, and determined the nitrogenase activity by the acetylene reduction test. Through RT-PCR, we analyzed the transcriptional units of PST1305 gene and its nearby genes. Real-Time PCR was applied to compare the expression level of PST1305 gene between optimal and non-nitrogen fixating conditions. [Results] Compared to the wild type, the nitrogenase activity in mutant strain (np1305) was partially decreased, however, functional complementary strain (np1305Comp) could restore nitrogenase activity close to wild type level. PST1305 gene was co-transcribed with its upstream gene (nifB and fdxN) and downstream gene (nifQ, PST1303 and PST1302). In contrast to the nitrogen excess conditions, expression of PST1305 under nitrogen-fixing conditions was significantly upregulated for 38.7-fold. [Conclusion] Disruption of PST1305 exhibited a declined nitrogenase activity compared to the wild type A1501. PST1305 gene might participate in biological nitrogen fixation by involving in the electron transport or the oxygen protection mechanism of nitrogenase. These results suggested that PST1305 gene was a new gene required for optimal nitrogenase activity of Pseudomonas stutzeri A1501.