A Preliminary Study On The Molecular Mechanism Of Glutamine Synthetase Gene GlnA Controlling Rhizosphere Colonization Of Enterobacter Ludwig AA4

The colonization of plant growth promoting rhizobacteria(PGPR)in the roots of host plants is a prerequisite for its growth-promoting and biocontrol functions,and it is also the key to the beneficial interaction of PGPR with the host plants.Therefore,studying the molecular mechanism of PGPR colonization in plant roots is of great significance for the development of agroforestry.AA4(Enterobacter ludwigii AA4)is a rhizosphere growth-promoting bacterium isolated from plant roots.In order to explore the molecular mechanism of glutamine synthetase gene glnA controlling the colonization of enterobacter ludwig AA4 on the plant root surface,in this study,enterobacter ludwig AA4 and arabidopsis thaliana were taken as the research subjects.firstly,the correlation between glutamine synthetase gene glnA gene and AA4 colonization on the plant root surface was verified.then the action mode of glnA gene controlling AA4 colonization was clarified through the determination of colonization-related phenotype.finally,the key genes of glnA gene controlling AA4 colonization on the plant root surface were identified through transcriptional analysis,and the colonization of glnA gene controlling strain AA4 on the plant root surface was preliminarily clarified The results of the study were as follows:(1)obtaining a glnA gene knockout mutant AA4 delta glnA of an enterobacter ludwig AA4 strain and a remedying strain AA4ΔglnA-c thereof through a homologous recombination technology,inoculating a wild-type AA4 and a mutant strain to an arabidopsis root system,and performing competitive colonization detection.The results showed that the colonization ability of AA4ΔglnA strain on the root surface of Arabidopsis thaliana was significantly lower than that of the wild type,indicating that the glnA gene was involved in the colonization of AA4 on the root surface of Arabidopsis thaliana.(2)The major colonization-related phenotypes of the AA4 and AA4ΔglnA strains were detected.The results showed that compared with the wild-type strain,AA4ΔglnA motility was decreased significantly,and the resistance to hydrogen peroxide was weakened.No significant changes were observed in the biofilm formation ability.Based on the existing research,it is speculated that glnA can affect the colonization of AA4 on the root surface of Arabidopsis thaliana by controlling its mobility.(3)qRT-PCR was used to analyze the transcription level of motilityrelated genes,and it was confirmed that glnA gene could promote the expression of flhD,flhC and fliA,the key colonization genes of strain AA4,at the transcription level.Subsequently,knockout mutants AA4ΔflhDC,AA4ΔflhD,AA4ΔflhC,AA4ΔfliA of motility-related genes were constructed.The test of their colonization abilities revealed that there existed obvious defects in the colonization abilities of AA4ΔflhDC,AA4ΔflhD,AA4ΔflhC and AA4ΔfliA.The colonization-related phenotypes of AA4ΔflhDC,AA4ΔflhD,AA4ΔflhC,and AA4ΔfliA strains were detected,and they were found to have completely lost mobility,slightly decreased biofilm formation ability,and no significant difference in hydrogen peroxide resistance compared with wild-type strains.All these results confirmed that glnA affected the colonization of AA4 by controlling the transcription of motility genes.In summary,the glutamine synthetase gene glnA can affect the expression of the motility gene of AA4 of Enterobacter ludwig and thus control the colonization ability of AA4 on the plant root surface.The expected experimental results will lay a theoretical basis for revealing the mechanism of the gene glnA controlling AA4 colonization on the arabidopsis root surface and provide a theoretical guide for the development and application of beneficial microbial preparations in the rhizosphere.