Comparative Study Of Molecular Mechanisms Of Selectiveness Nodulation In Bradyrhizobium Diazoefficiens 4534 And 4222

Molecular mechanisms of selective nodulation for rhizobia have long been the focus of the research and a key factor to limit their potential of nitrogen fixation. Based on our extensive study in the past, we have got some basic knowledge of the process of selective nodulation and impact factors, and identified certain genes associated with rhizobial symbiosis, nitrogen fixation and selective nodulation. However, we still lack the systematic understanding of the relationship between these strains’genomic, genetic regulation, differences in gene expression, and gene interactions with the host. This study took two Bradyrhizobium diazoefficiens strains that demonstrated completely different nodulation abilities, strain 4534 being the most competitive and strain 4222 being the least competitive in nodulation as the research materials. Based on genomics, with the approach of comparative proteomic and comparative transcriptomic, we compared the protein and gene expression of these two B. diazoefficiens strains in response to soybean root exudates, in order to further explore the mechanisms of selective nodulation and the key impacting factors. The main results are as follows:1. The study designed and improved a preparation method to effectively promote soybean root exudates. One of the difficulties of rhizobial nod genes inducing test is how to get effective soybean rhizosphere secretion inducer, which is also the experimental basis for the following research. After repeated experiments, we designed an efficient preparation method of collecting soybean root exudates for selective nodulation, and select the influence of rhizosphere secretion to nod gene expression as an evaluation criterion. The comparative proteomic and transcriptomic results proved that this method is effective.2. The whole genome of B. diazoefficiens 4534 and B. diazoefficiens 4222 strains were sequenced. The results showed:two strains are composed of a single replicon each chromosome containing three ribosomal operons, with G+C content 65.0% and 64.9%, respectively; the average length of gene is 903 bp and 901 bp, and strain 4534 contains 8072 ORF, while strain 4222 contains 8233 ORF. But half unknown gene function in both genome makes our analysis of some important rhizobia genetic information difficult.3. To gain a better understanding of the molecular mechanisms of selective nodulation at the level of genes, the genomes of B. diazoefficiens 4534 and 5. diazoefficiens 4222 were compared. Differences between strain 4534 and strain 4222 in genomic characteristics were discovered. In strain 4534, more genes associated with proline metabolism, phosphate mannitol transport, quorum sensing and IAA metabolism were observed.4. To gain a better understanding of the molecular mechanism of competitiveness at the level of protein expression, we compared the proteomic responses of two B. diazoefficiens strains. The results showed:with the treatment of soybean rhizosphere secretion, the strain 4534 had 65 differential proteins while the strain 4222 had 29. So compared with the strain 4222, more amounts and categories of differential proteins were detected in the strain 4534. Based on the analysis of differential proteins, more proteins involved in S-adenosylmethionine synthetase (SAMS), PhyR-σEcG regulon, ABC-type transporters, flagellar protein, molecular chaperones, and redoxstate maintenance, as well as several hypothetical proteins made B. diazoefficiens stronger for selective nodulation. PhyR-σEcfG regulon and flagellar protein have recently been demonstrated to be involved in the competitiveness for nodulation in B. diazoefficiens. Our results also showed that soybean rhizosphere secretion was involved not only in nodulation-related gene expression, but also in up regulation of metabolic fitness proteins.5. The transcriptomic of B. diazoefficiens 4534 and B. diazoefficiens 4222 were analyzed. The results showed:with the treatment of soybean rhizosphere secretion, the strain 4534 had 457 differential genes, with 253 up-regulated and 204 down-regulated. The strain 4222 just had 293 differential genes, with 108 up-regulated and 185 down-regulated. Analysis of enriched GO functional pathways, biological metabolic pathways, and enriched KEGG pathways showed that with the treatment of soybean rhizosphere secretion, the differences of genes related to flagellum synthesis, signal stimulation and tropism were significant in strain 4534, and genes related to flagellum synthesis were changed in the strain 4222.