Effects Of DctA, DctB And DctD Genes On Symbiotic Nitrogen Fixation Of Fast - Growing Soybean Rhizobia (Sinorhizobium Fredii)

There is a class of soybean Rhizobium in symbiotic nitrogen fixation microorganism.They form symbiotic nitrogen fixation system with host plants for biological nitrogen fixation which provide essential nitrogen elements for growth of host plants. Plants get nitrogen element is usually depended on absorption of ammonium salt, nitrate and other ionic compounds. A large amount of energy needs to be consumed in symbiotic nitrogen fixation of Rhizobium legume symbiosis system, however, thus plants provide bacteroids to change molecular nitrogen in air into photosynthetic products ammonia required. Many research results show that succinic, malic acid, fumaric acid and other dicarboxylate (dCAs) are carbon source and energy that symbiotic nitrogen fixation need and are directly supplied to the bacteroids by plants. And dCAs must pass through the cell membrane and peribacteroid membrane (PBM) the two barriers to enter the bacteroids cell. Studies suggest the existence of a common system transporting dCAs e.g. Dct transfer system and through this system plants and microbes can easily exchange energy. Transportation of dCAs is completed by dCAs transferase while dCAs transferase gene (dct) have been confirmed to be consisted of three genes dctA,dctB,dctD. dctA is structural gene coding dCAs transferase and its expression is regulated by two-component regulatory protein of dctB and dctD.Dct plays an important role in nodulation process of symbiotic nitrogen fixation in Rhizobium because it coded dCAs transferase gene. The nitrogen-fixing ability of existing Rhizobium is low therefore transferring it to Rhizobium will enhance the number of extra copies and lay a foundation for the further research on effect of nitrogen-fixing ability of Dct gene in Rhizobium.Homology-based cloning was used to clone three genes dctA, dctB, dctD with template of genomic DNA from Sinorhizobia fredii 15067 in this study. The results showed that their sequence had more than 95% similarity with three genes of Sinorhizobia fredii by the method of bioinformatic analysis. It was presumed that the gene was the dct gene exsiting in Sinorhizobia fredii 15067. Utilization of DNA recombination technique, dct gene was joined into downstream of lac promoter. And expression vector pTR-Plac-dctA, pTR-Plac-dctB and pTR-Plac-dctD were constructed successfully by using plasmid pTR102 marked by luxAB gene. Furthermore, the expression vectors were separately transformed into Sinorhizobia fredii 15067 and native Rhizobia japonicum with method of triparental hybridization, and six transgenic engineering strains SF (pTR-Plac-dctA), SF (pTR-Plac-dctB), SF (pTR-Plac-dctD), SFH (pTR-Plac-dctA), SFH (pTR-Plac-dctB), SFH (pTR-Plac-dctD) were successfully constructed.By means of pot experiment, the result showed that after soybean seedlings were respectively infected by six transgenic engineering strains there were differences in different degree in activity of nitrogenase, fresh weight, dry weight, soybean plant height, nodule number, et al. In which, compared with soybean plants infected by original strain and other transgenic engineering strains, the above indexes of SF (pTR-Plac-dctA) were especially obvious.The detection results of the genetic stability of plasmid indicated that the retention of six recombinant strains was 100% under authigenic conditions; The results of fluorecent detection of soybean nodules that were infected by transgenic engineering strains were 100% under symbiotic condition. They all could stably inherit in Sinorhizobia fredii 15067 and native Rhizobia japonicum.