| Azorhizobium caulinodans interaction with the natural host Sesbania rostrata can cause stem nodules.According to the genomic analysis of bioinformatics,it was found that the symbiotic nitrogen fixation function was obtained through the evolutionary level of acquired evolution.The symbiotic island is mainly the nodulation related gene and does not contain nitrogen-fixing related genes.The biological nitrogen fixation between legumes and rhizobium is a representative of symbiotic nitrogen fixation,which plays a very important role.Therefore,it is particularly important to understand the mechanism of symbiotic nitrogen fixation.The aim of this study was to investigate the biological functions of related genes by using the CRISPR/Cas9 gene editing system for the site-specific knockout/knock-in of symbiotic nitrogen-fixing genes of Azorhizobium caulinodans,and to provide a study background on the symbiotic nitrogen fixation of non-legumes.The main contents and the research are as following:1.Using the CRISPR/Cas9 technology,the AZC＿2928 gene was successfully knocked out,and the AZC＿2928 gene deletion mutant of Azorhizobium caulinodans was established.2.A complement-mutant strain of AZC＿2928 of Azorhizobium caulinodans has been constructed by plasmid-mediated gene replenishment.3.A series of experiments were carried out on the mutants,revertants and wild-type strains of the Azorhizobium caulinodans: growth curve deteration,chemotaxis test,biofilm test,capillary quantitative test and plant infection test.The results showed that the loss of AZC＿2928 caused the growth rate of Azorhizobium caulinodans to slow down,affecting the basic metabolic function.The deletion mutants grew faster than the wild type at different concentrations of lysine,and were also affected by the concentration of lysine.The growth rate of the mutant mutant decreased with increasing lysine concentration.The biofilm formation of the mutant strain was higher than that of the wild type;the deletion mutant and the wild-type strain showed no significant difference in competition and motility,that is,the deletion of AZC＿2928 did not affect the competitiveness and motility of the strain;There was no significant difference in the nodulation status of the wild-type strain infecting Sesbania rostrata,indicating that the AZC＿2928 gene was not involved in the nodulation regulation of Sesbania rostrata.In general,the AZC＿2928 gene plays a negative regulatory role in the symbiotic nitrogen fixation process,which affects the biological nitrogen fixation by inhibiting the symbiotic nitrogen fixation process.4.Using the CRISPR/Cas9 technology,the nif U gene was successfully integrated into the chromosomal genome of E.coli DH10 B,obtained the genomically engineered E.coli DH10 B.5.Using CRISPR/Cas9 technology,study of various cloning methods for cloning nodulation gene clusters(87.6Kb)in Azorhizobium caulinodans. |
