Construction Of Soybean Rhizobia Engineering Strain With NifA Gene Introduction And Its Effect On Nitrogen Fixation

Nitrogen fixation gene nifA in Rhizobium is the most important regulatory gene, and the proteins that it expresses can impel the expression of nitrogenase stractural genes nifH、nifD、nijK. Therefore, using Molecular biology and genetic engineering means to increase the number of extra copies of nifA gene and constructing efficient nitrogen-fixing Rhizobium strains can provide part of nitrogen that the plant need. after inoculation, and the strains can symbiotic of the legume also can prevent the plant be damaged by harsh environment.Homology-based cloning was used to clone nodulation gene nifA and PnifA-nifA with template of genomic DNA from Sinorhizobia fredii 15067 in this study. The results showed that its sequence had 96% similarity with nifA gene of Rhizobium sp. NGR234a plasmid, It was presumed that the gene was the nifA gene exsiting in Sinorhizobia fredii 15067, the nifA gene sequence contained 1 671bp, coding 556 amino acids. the promoter of nifA is between fixC and nifA. Utilization of DNA recombination technique, expression vector pTR-Plac-nifA and pTR-PnifA-nifA were constructed succesfully by using plasmid pTR102. Expression vectors pTR-Piac-nifA, pTR-PnifA-nifA and empty vector pTR102 were transformed into native Rhizobia japonicum and Sinorhizobia fredii 15067 with method of triparental hybridization. Four genetic engineering strains and strains containing empty vectors SFH(pTR-Plac-nifA), SFH(pTR-PnifA-nifA), SF(pTR-Plac-nifA), SF(pTR-PnifA-nifA), SFH(pTR102), SF(pTR102) were succesfully constructed by resistance screening and luminescent detection.We explore the stability of extraneous recombinant plasmid in transconjugant which uses luxAB as report gene. the result showed that retention of plasmid pTR-Plac-nifA, pTR-PnifA-nifA and pTR102 were 100% on authigenic conditions. and these vectors were found to be fairly stable in rhizobia. The results of fluorecent detection of nodules from soybean plants infected by engineering strains were 100% under symbiotic condition. Recombinant vectors pTR-PnifA-nifA, pTR-Plac-nifA and empty vector pTR102 were able to inherit steadily in both Rhizobia japonicum and Sinorhizobia fredii 15067 thigenic conditions, and these vectors were found to be fairly stable in rhizobia.By means of pot culture nodulation test, four genetic engineering strains were compared with native Rhizobia japonicum and Sinorhizobia fredii 15067, the results showed that after genetic engineering strains infecting soybean, there were differences in different degree in soybeen plant height, fresh weight, dry weight, nodule number and yield comparing with control group. Rhizobium genetic engineering strains SFH(pTR-Plac-nifA) and SFH(pTR-PnifA-nifA) compared with SFH, there was significantly different in fresh weight dry weight in native Rhizobia japonicum and the length of soybean and the number of nodule was extremely significantly different, and nodules of the soybean plants infected by SFH(pTR-Plac-nifA)or SFH(pTR-PnifA-nifA) increased 24.86%,25.97% respectly. Compared to soybean plants infected by Sinorhizobia fredii 15067, the soybean inffected by the SF(TR-Plac-nifA)or SF(pTR-PnifA-nifA)controlled by different promoter was very significantly different in the length of soybean and the mumber of nodule. Compared to soybean plants infected by Sinorhizobia fredii 15067, nodules of the soybean plants infected by SF (pTR-Plac-nifA)or SF(pTR-PnifA-nifA)increased by about 22.78%,23.33%. However, there was no difference between nifA in soybean controlled by different promter in all the Index. nifA gene with lac promoter to control and nifA gene with itself promoter was the same effect in the soybean.