Study On The Function Of SM0020₀4885 Gene Of Sinorhizobium Meliloti CCNWSX0020 In Symbiotic Nitrogen Fixation

In the early stage of nodulation between rhizobia and leguminosae symbiosis,rhizobia-infected plant roots must penetrate the host plant cell wall into the host cell.This process requires the relevant hydrolytic enzymes to degrade the cell wall to form channels for rhizobia to infiltrate.In this paper,an unknown functional gene SM0020₀4885 containing the ?-mannanase domain was found from Sinorhizobium meliloti CCNWSX0020 by means of bioinformatics.It is speculated that it may have the function of degrading plant cell wall.First,SM0020₀4885gene deletion mutants,gene overexpression strains,and replenishing strains were constructed,respectively.The constructed strains were inoculated together with the wild-type rhizobia and then inoculated into the medicago lupulina for pot experiment.The SM0020₀4885 gene was studied through a series of plant experiments.The function of the symbiotic nitrogen fixation system established between the rhizobia and the host plant and the effect of the target gene on the symbiotic nodulation of the host plant.Main results for this study were as follows:1.The SM0020₀4885 gene deletion mutant strains ?Man B2,overexpression strains and complemented strains are constructed.According to the S.meliloti CCNWSX0020 genomic sequence primers were designed to amplify the upstream and downstream fragments of the SM0020₀4885 gene to construct a gene knockout vector,and then the SM0020₀4885 gene deletion mutant ?Man B2 was successfully obtained by homologous recombination.The SM0020₀4885 gene overexpression vector was constructed using p RK415 plasmid,and the overexpression vector was introduced into the wild-type S.meliloti CCNWSX0020 using a triparental hybridization technique.The 04885 gene overexpression strain was successfully constructed.In addition,the 04885 gene fragment was also ligated into the broad-copy host low-copy expression vector p BBR1MCS-5,and then the target gene-recovered strain was successfully constructed by introducing the gene deletion mutant ?Man B2.2.To study the effect of SM0020₀4885 gene on the symbiotic system of rhizobia and host plants.Through the statistics and analysis of plant biomass,nitrogenase activity,number of nodulation,and the number of infestation events,the results showed that the deletion of the 04885 gene would affect the development of the host plant roots in the early stages of the plant infection with rhizobia.At the same time,the deletion of the 04885 gene impairs the ability of the symbiotic system to nodulate and fix nitrogen,creates abnormal discrete lines of infection in the root hairs of host plants,and causes abnormal root nodules in the roots of host plants.In addition,the deletion of the 04885 gene also resulted in delayed nodulation times and reduced numbers,reduced nitrogen fixation capacity,and ultimately reduced plant biomass,suggesting that the absence of the 04885 gene affects the establishment of an effective symbiosis system between the rhizobia and medicago lupulina.The overexpression of 04885 gene can enhance the nodulation capacity of symbiotic system,increase the number of nodule,increase the nitrogenase activity,and increase the plant biomass,indicating that the 04885 gene plays an important role on effective symbiosis between the rhizobia and host plants.In summary,experimental studies have demonstrated that the 04885 gene plays an important role in the establishment of an effective symbiotic system between rhizobia and host plants.Deletion of this gene impairs the nodulation capacity and nitrogen fixation capacity of host plants,causes plant biomass reduced.