| Firstly, the nifA gene was fused to the DNA-binding domain in the pGBD-C2 plasmid vector to generate a recombinant pGBD-nifA plasmid. It is used as the bait to screen Azospirillum brasilense Sp7 genomic plasmid library which was constructed by fusing 0.5-3kb fragments of A. brasilense Sp7 genomic DNA to the DNA-activation domain in the pGAD plasmid vectors. When cotransforming pGBD-NifA and AD fusion genomic library into Saccharomyces cerevisiae PJ69-4A, 109 candidates interacting with NifA had been selected by testing for the expression of the His3, ade2 and lacZ reporter genes. 11 interacting candidates proved to be the positives after isolating the mixed plasmids from yeast and recotransforming each purified AD/library plasmid and pGBD-nifA into S. cerevisiae PJ69-4A respectively. The genes of the 11 positives had been sequenced and 4 different gene fragments were obtained. By the analysis of Blastn (nucleitide level) and Blastp (amino acid level), some conversed domains in each protein sequence were detected, such as PAS domain, PAC domain and FhuE. To farther verify the interaction between each of the 4 positives and NifA protein, yeast two-hybrid system was carried out by exchanging the vectors for nifA and 4 positives respectively, and by mutagenesis of the 4 positives with shifted reading frame experiments. The research showed that their interactions in yeast two-hybrid system are still steady when the vector was exchanged, in contrast, the interaction was disappear when the reading frame of each positive had been changed.The four positives were subcloned into suicide plasmid so that gene mutant strains could be constructed via conjugation and homologous recombination. Some mutant strains carrying the destroyed genes of positives had been constructed and identified by PCR. The primary function of the four positive factors has been probed by nitrogenase activity at different ammonia concentrations in each mutant strain.
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