类芽孢杆菌固氮基因簇在大肠杆菌和枯草芽孢杆菌中的表达和调控研究

美国最近用体外固氮酶研究表明，生物固氮只有6个必需基因nifHDKBEN。但国内外尚没有证明6个固氮基因能否在非固氮菌，如大肠杆菌中完成生物固氮作用。我室从固氮类芽孢杆菌Paenibacillus sabinae和Paenibacillus beijingnica中各自克隆到1个由8个基因组成的固氮基因簇nifHBDKENXV，这2个固氮基因簇在大肠杆菌中有无固氮作用？固氮作用受铵和氧调控吗？为回答这些问题，本研究内容为：（1）完成P. beijingnica全基因组序列分析；（2）确定2个固氮基因簇转录起始位点和转录单元；（3）将2个固氮基因簇分别在自身启动子和lacZ启动子带动下，在大肠杆菌和枯草芽孢杆菌表达；（4）重组大肠杆菌和枯草芽孢杆菌固氮酶活性测定；（5）铵和氧对固氮基因在重组大肠杆菌和枯草芽孢杆菌的表达调控研究。结果将为构建高效固氮工程菌株和扩大寄主范围及转基因植物奠定基础

Nitrogen fixation has been studied extensively in various microorganisms. The well studied nitrogen-fixing microorganisms are Gram-negative bacteria including Klebesiela pneumoniae, Pseudomonas stutzeri, Azotobacter vinelandii, Azospirillum brasilense and Gram-positive bacteria including Heliobacterium chlorum.The organization of nitrogen fixation (nif) genes has great difference in Gram-negative and Gram-positive bacteria. In K. pneumoniae, 21 adjacent nif (nitrogen fixation) genes are organized in eight operons (nifJC, nifHDKTY, nifEN, nifUSVW, nifZM, nifF, nifLA, nifBQ) within ca. 24 kb of DNA. All nif genes are transcribed from σ54 promoters (-24/-12). The nifD and nifK genes specify the α and β subunits, respectively, of the molybdenum iron protein. The iron protein is encoded by nifH. The nifEN and nifB are required for the synthesis of FeMo-co factor of nitrogenase. NifA, the transcriptional activator, is required for expression of other nif genes. When the nif cluster is introduced to Escherichia coli, the E. coli has nitrogenase activity. However, the nitrogenase activity is affected by ammonium and oxygen.. Recent research shows that the minimal genes for nitrogen fixation are the 6 genes： nifH nifD nifK nifB nifE nifN.Our research results show that nitrogen fixation in Gram-positive Panebacillus sabinae T27 and Panebacillus beijingnica WLY1-18 is governed by a nif cluster consisting of 8 genes nifBHDKENXV.However, the identity between the 2 nif clusters is only 70%,suggesting that the 2 nif clusters might show difference in nitrogen-fixation..In this study, the work will be completed as follows: (1) to complete the genome sequence of P. beijingnica WLY1-18.(2)To determine the transcription origin and transcription unit of the nif cluster in P. beijingnica WLY 1-18和P. sabinae T27.(3)To clone the nif cluster of nifBHDKENXV from P. beijingnica WLY 1-18 and P. sabinae T27, and then the nif clusters will be under the control of nif-promoter and lacZ promoter. (4)To transfer the nif clusters to Escherichia coli and Bacillus subtilis, respectively. (5)To check the nitrogenase actitivy of the recombinant E. coli and B. subtilis.(6)To determine the nitrogenase level in E. coli and B. subtilis by Western blot. (7)To determine the effects of ammonium and oxygen on expression of nifH nifD nifK in the recombinant E. coli and B. subtilis by Quantitative Real-Time RT-PCR.