巴西固氮螺菌Yu62nifA基因克隆、测序及功能分析

用TD-PCR法克隆了巴西固氮螺菌(Azospirillun brasilense)Yu62的nifA基因.序列分析表明它与巴西固氮螺菌Sp7的nifA序列高度同源(96.5%),其编码的产物NifA蛋白与Sp7菌株NifA的氨基酸序列同源性为97.6%.该基因可以完全互补巴西固氮螺菌Sp7 nifA-突变株的Nif-表型.研究了NH4+和O2对Yu62 nifA基因的表达及NifA活性的影响.结果表明mfA基因在Yu62菌株中是部分组成型表达的,氨和氧不能完全阻遏其表达,在5mmol/LNH4Cl与微氧(0.5%O2)条件下表达最高;NifA蛋白在0.4%～0.5%O2时活性最高,氧分压降低和提高都使NifA活性下降,1mmol/L NH4Cl足以抑制NifA的活性.

CLONING SEQUENCING AND EXPRESSION PATTERN,FUNCTIONAL ANALYSIS OF nifA GENE IN AZOSPIRILLUM BRASILENSE Yu62

The nifA gene of Azospirillum brasilense Yu62 was cloned and sequenced. The expression of nifA gene was investigated in wild type strain Azospirillum brasilense Yu62. The results show that expression of nifA gene is not repressed by ammounium and oxygen completely. But the expression of Yu62 nifA gene is different from that of strain Sp7 nifA gene. Expression of Yu62 nifA seems more sensitive to oxygen than that of Sp7 nifA which shows the highest expression in condition of aerobic, while the Yu62 nifA gene shows the highest expression in the condition of microaerobic. The regulation of NifA protein activity by ammonia and oxygen was investigated. Results showed that the NifA protein is repressed by ammonia, 1 mmol/L NH4Cl can inhibit activity of NifA protein completely. Oxygen concentration affects activity of NifA protein. NifA protein is highly active in 0.4%-0.5% O2.