Regulation Of Symbiotic Nitrogen Fixation Ability Of R. Yanglingense CCBAU 01603 By Transcription Factor AcrR

Rhizobium can establish an efficient symbiotic nitrogen fixation system with caragana leguminous plants,which can provide important nitrogen nutrition guarantee for windbreak and sand fixation,soil and water conservation in barren desert areas.R.yanglingense CCBAU 01603 is a rhizobium with the highest symbiotic nitrogen fixation efficiency with caragana.Taking this strain as the starting strain,after continuous transmutation,the gene encoding transcription factor acrR at site 161 was absent of base C,resulting in changes in the symbiotic phenotype of the evolved strain.This paper focused on studying the regulatory mechanism of acrR gene in the nosogenesis and nitrogen fixation process of R.yanglingense CCBAU 01603.Caragana microleaf was selected as the host plant and R.yanglingense CCBAU 01603,a highly efficient nitrogen-fixing rhizobium,was used as the test material to construct the deletion mutant of gene acrR.To explore the autogenetic phenotypes of mutant ΔacrR and the symbiotic phenotypes of plant height,plant weight,root weight and nodulation of host plants after inoculation,and to clarify the mechanism of transcriptional gene acrR regulating nodulation and nitrogen fixation ability of rhizobium R.yanglingense CCBAU 01603.The main results are as follows:The results showed that the recombinant plasmid pCM351-acrR and the gene deletion mutant ΔacrR were successfully constructed,and there was no significant difference in saline-alkali tolerance between them.The wild-type strain CCBAU 01603 secreted a large amount of extracellular polysaccharide,was intolerant to tetracycline,gentamicin and streptomycin,and had a narrow utilization range for a single carbon source.The mutant ΔacrR secreted less extracellular polysaccharide,was intolerant to chloramphenicol,and had a wide range of utilization of single carbon source.After the wild-type strain and mutant strain were inoculated with host Caragana microphylla in sterile vermiculite,the wild-type strain formed root nodules as ellipsoid with dark red color,while the mutant root nodules were relatively small ellipsoid or sphere with lighter and reddish color.However,the phenotypic changes of plant height,plant weight,root weight and nodulation of host inoculated by the two strains were not significant.The distribution of PHB particles in the root nodules formed by ΔacrR and the host was observed by immunohistochemical staining,and relatively mature PHB particles in the nitrogen-fixation zone were distributed sparsely,and PHB particles in the root nodules formed by ΔacrR were relatively few,suggesting that the accumulation of PHB particles in the root nodules was inhibited to a certain extent after the deletion of acrR gene.The low density of bacterioid is the direct cause of the decrease of nitrogen fixation efficiency.After 45 days of inoculation,the wild type ΔacrR and wild type R.yanglingense CCBAU 01603 were mixed inoculated with Caragana microphylla,and the wild typeΔacrR percentage of nodule was 0.It was speculated that the deletion of this gene seriously affected the competitive nodulation ability of rhizobia.Based on the above results,although the nodule formation process of the mutant and Caragana microphylla was not affected by the deletion of gene acrR,the symbiotic nitrogen fixation efficiency of the nodules formed was decreased,and the nodule morphology,myeloid development and PHB particle accumulation were all affected,and the nodular competition ability was relatively low.The results laid a foundation for further research on the molecular mechanism of transcription factor AcrR regulating nitrogen fixation,and provided a theoretical reference for the breeding of Caragana efficient nitrogen-fixing rhizobium.