用酵母双杂合系统克隆固氮螺菌中的固氮负调控基因

The NifA protein activates the transcription of nif genes whose prducts are required for biological nitrogen fixation in a large number of diazotrophs including Azospirillum brasilense, whereas the regulation of NifA transcription and NifA activity differs from one organism to another. In Klebesiella pneumoniae and Azotobacter vinelandii, NifA activity is inhibited by a second regulatory protein NifL in the presence of O2 and / or combined N2. In members of the family Rhizobiaes and Azospirilum brasilense, NifL has not been detected. Yeast two-hybrid system is an effective method for studying the direct interaction between two proteins. It was demonstrated that NifA has direct interaction with NifL in Azotobacter vinelandii by using yeast two-hybrid system. It is still not known that there is NifL or not in Azospirillum brasilense. If there does exist a NifL in A. brasilense, the nifL gene encoding NifL could be selected out by using the method. Alternatively, any proteins that have direct interaction with NifA positively or negatively can be detected by using this method. In our research, the following works are finished: (1) nifA gene is cloned into two-hybrid plamid pGBD to generate pGBD-nifA plasmid. (2) AD fusion expressing library were constructed. .Genomic DNA was prepared from A. brasilense and partially digested with Sau3A. 500-3000bp DNA fragments were pooled and ligated to the three two-hybrid plasmid vectors pGAD-C1, pGAD-C2 and pGAD-C3 respectively, in order to make the library in reading frame with AD fusion. Ligations were transformed into Escherichia coli and a small aliquot was plated to determine the total number of primary transformants. Amplify the library to produce enough plasmid DNA for the yeast transformations. (3) Screen the AD fusion library: Introduce sequentially the pGBD-nifA and the AD library into Saccharomyces cerevisiae PJ69-4A. Selecte positive transformants by plating the transformation mixture on SD medium lacking Leu, Trp and Ade and by blue colonie of reporter gene lacZ's expression. (4) Sequence the fusion gene of positive clone .

利用酵母双杂合系统克隆巴西固氮螺菌中的固氮负调控基因，分析确定其功能，阐明该基因的蛋白与NifA相互作用的机理，从而进一步深入了解巴西固氮螺菌中固氮基因的转录调控机制。