Regulation Network And Molecular Mechanism Research Of Pseudomonas Suringae MB03 Transcription Regulator InpR

Pseudomonas syringae is a common plant pathogen that has a wide range of hosts and is capable of infecting a wide variety of plants.It can be caused by brown spots on the leaves of plants,browning of buds,decay of trunks and branches,freezing damage of plants and collapse of fruits.The outbreak can cause serious economic losses.The main research object of this study is a strain of pseudomonas syringae MB03 with ice core activity isolated from this laboratory.The transcriptional regulation and regulation of a gene encoding a GntR family transcriptional regulator InpR in this strain was studied the molecular mechanism.When the InpR gene was interrupted,the growth was significantly slower than that of the wild strain,indicating that the InpR protein directly or indirectly regulates the growth and metabolism of MB03.Based on the phenotype,based on the transcriptome data,the genes with significant up-and-down regulation were screened,and the corresponding DNA probes were designed for in vitro EMSA experiments.The direct regulation of InpR on the genes VT47₁1440?amino acid permease?and VT47₂4100?acyl-CoA dehydrogenase?VT47₁7390?fumarate hydratase?was verified by experiments such as EMSA.RT-qPCR experiments on the above three genes revealed that the expression of the three genes in the mutant strain MB487 showed a significant down-regulation,indicating that InpR positively regulates the expression of these genes.In order to study the effect of small molecular substances on the regulation of InpR,the small molecule substance fumaric acid and related amino acids related to its function were selected from the above target genes.The effects of EMSA experiments were verified by in vitro experiments,and the small molecular substances lysine and Arginine can inhibit the binding of InpR to the target DNA and verify the regulatory relationship between InpR and the target gene.In order to further understand the molecular mechanism of its binding,provide a theoretical basis for its combined model,site-directed mutagenesis and expression purification of InpR protein by site prediction,in vitro EMSA to verify the binding activity of the mutated protein to the target sequence,found that the arginine at position 45 and 49 mutation lost the binding activity,indicating that the amino acids at these two sites are key amino acids for InpR binding to the target sequence.In addition,by comparing the DNA sequence of InpR binding,it was found to be in line with the motif sequence 5'-NTGGNNNAGNCCAN-3',which is a highly conserved incomplete palindromic sequence,which is combined with the GntR family transcriptional regulator binding motif reported in previous studies.There is a certain similarity,but not the same,it is a new model.A large number of DNA sequences conforming to this motif existed in the P.syringae MB03 genome,and a part of the sequence design probes were selected and verified by EMSA.Six promoters located in the gene region of this gene were found.Sequence,capable of direct interaction with InpR.These genes are involved in different physiological processes,suggesting that InpR may be a potential global transcriptional regulator.