Study Mesorhizobium Huakuii7653R Bacteroids Differentiation And Nitrogen Fixation Mechanism Based On Global Transcriptome Analysis

Mesorhizobium huakuii7653R occurs either in nitrogen-fixing symbiosis with its host plant, Astragalus sinicus, or free-living in the soil. The M. huakuii7653R genome has recently been sequenced. To better understand the complex biochemical and developmental changes that occur in7653R during bacteroid differentiation, RNA-Seq and Microarrays were used to investigate the differential transcriptomes of7653R bacteroids and free-living cells.1. Comparative analysis of the methods of isolating bacteroids. The results showed that use physical method to get complete form of bacteroids for morphological observation or use Ambion MICROBEnrich kit to get high-quality bacteroids RNA. Using above methods, total RNA of bacteroids and free-living cells was extracted for RNA-Seq and Microarrays analysis.2. We constructed a protein-protein interaction (PPI) network for7653R using the method of orthologous.3. RNA-Seq and Microarrays were used to investigate the differential transcriptomes of7653R bacteroids and free-living cells. The results are as follows.(1) Both the two approaches identified about three thousand differentially expressed genes. Correlation between the two methods was high.(2) We have analyzed COG functional categories, biological metabolic pathways, enriched KEGG pathways and Gene set enrichment analysis using the both sources of data following an integrative strategy. These results all showed that free-living cells have a primary role in maintaining basal metabolism, whereas bacteroids have a primary role in nitrogen fixation. The most prominent up-regulation occurred in the symbiosis plasmids. The results suggested that the main energy metabolism is active while fatty acid metabolism is inactive in bacteroid and that most of genes relevant to cell cycle are down-regulated accordingly.(3) Integrated gene expression data into the7653R protein-protein interaction (PPI) network using Cytoscape. A highly inter-connected subnetwork, with function enrichment for nitrogen fixation, was found, and a set of hubs and previously uncharacterized genes participating in nitrogen fixation were identified.(4) In order to identify the subnetwork mediated by CtrA connected with the cell cycle and bacteroid differentiation, we dissected the7653R PPI network by using CtrA as a specific target. Then we got the CtrA subnetwork. The results described here provide a broader biological landscape and novel insights that elucidate rhizobial bacteroid differentiation, nitrogen fixation and related novel gene functions.4. Construction of mutants of MCHK-0866, one of the hub gene in nitrogen fixation subnetwork, and its adjacent gene MCHK-0867. Construction of single-gene mutation (MCHK-0866-or MCHK-0866-) and double-gene mutation vectors, using three parental hybridization to screen. Finally, obtain stable double-gene mutant and plant symbiotic phenotype detection was Fix-.5. Modification of transcription factor expression vector used to build the Bacterial one-hybrid random library. Pre-construction of random library of Bacterial one-hybrid with high transformation efficiency. Lay the foundation for constructing the Bacterial one-hybrid random library and analyzing the expression and regulation of important transcription factors in transcriptome results.