The Function Of Receptor Protein TLPa1 And TLPa2 In Azorhizobium Caulinodans ORS571

Different from other rhizobia, Azorhizobium caulinodans ORS571 can grow and nitrogen fixation in the rhizosphere of symbiosis host? endogenous host and spontaneous state. Although the receptors for the signal system is more complicated, the study of how its receptor system sense signal has not been reported so far. It is severely limiting the widely use of this unique symbiotic nitrogen fixation system of rhizobia and its host due to lack the reports of how Azorhizobium caulinodans ORS571 induction plant root secretion. Through bioinformatics analysis, receptor proteins TLPal and TLPa2 located in the upstream and downstream of gene cluster, suggest that the two proteins are important chemokines receptors.The mutant strains was built by the principle of homologous recombination in this experiment. The upstream and downstream of target gene were inserted into the two multiple cloning sites of PCM351 plasmid respectively and then complete the knockout by exchange the target gene and Gen gene of PCM351 plasmid. We get complemented strains by using the recombinant plasmid of target gene and PBBR1MSC-2 plasmid transfer to the mutant strains.This experiment used the L3 semi solid plate method by adding different carbon source in the culture medium to observe the chemotaxis circle size of wild type, mutant strains and complemented strains. Congo red solid culture medium was used to observe the secretion of extracellular polysaccharide and secondary metabolites of different bacteria. The nitrogenase activity was determined by gas chromatograph through the principle of nitrogenase can react with acetylene to produce ethylene. We daubed on the sesbania stem with mixed strains by different proportion between the wild type strains and the mutant strains or soaked seed, then detected nodule competitive by calculating the proportion of the two strains from nodules.Through experiment, we found that the induction ability of the mutant strains ATLPal to citric acid, succinic acid, proline, tartaric acid, malic acid, glycerol and other carbon source are weakened compared with wild-type strains, and the chemotactic ability of complemened strains ?TLPal-com to the above carbon source could partially complement. The production of extracellular polysaccharides secreted between ATLPal and wild type has no difference, but the melanin of the secondary metabolite appeared earlier than the wild type. The nitrogenase activity of ?TLPal was significantly weaker than that of wild type, as well as the ?TLPal-com could complement part of the nitrogen fixation ability. In stems nodule competitive experiment, the competitiveness of ?TLPa1 was significantly worse than wild type. The mutant ?TLPa2 has no big difference compared with wild type in above experiments. Therefore, we speculate that chemotaxis receptors TLPa1 does not a specific receptors, but can sense a variety of carbon source, and can affect secondary metabolites, nitrogenase and nodular process. Chemotaxis receptor TLPa2 has no effect on above aspects, the gene is probably a redundant sequence though it located in downstream of the gene cluster.