Functional Analyses On The Transcriptional Regulator GL003331 Of Pseudomonas Syringae MB03

Pseudomonas syringae is a kind of common plant pathogenic bacteria while having ice nucleation activity, leading the plants frost damage lesions or the occurrence of apoptosis, it induces the forming of ice crystals in plant cells under relatively high temperature (-2°C to 5° C), and its main pathogenic factor is a secreted protein ice nucleoprotein (Ice nucleation protein, INP) produced by the bacteria. GntR is a transcription regulator of Pseudomonas syringae, belongs to GntR transcription regulator family. GntR genes generally locates near the genes that regulated by it. In this study, we studied the transcriptional regulatory function of a GntR transcription factor GL003331 of Pseudomonas syringae pv. syringae MB03 which is a high ice nucleation active strain isolated by ourselves, among GL003331’s transcriptional regulatory function to genes in its upstream and downstream and other genes in the whole genome.The gL003331 gene has been identified while MB03 genome sequencing, we designed primers to amplify gL003331 gene, ligated it with the E. coli expression vector pTrC-HisB to obtain a recombinant plasmid pMB437, pMB437 has been transferred into the E. coli expression strain JM109 and induced by IPTG, the expression product consistent with the theoretical molecular weight. GL003331 protein was purified using Ni-NTA, the purified GL003331 protein has a concentration of 0.983μg/μl.Promoter Analysis was performed by online software BPROM and BDGP, we found 11 areas that may be promoter regions near gL003331 gene. The purified GL003331 protein was used to screen these regions via EMSA to find the potential gene fragments that can react or combine with the regulator and found two such gene fragments.DNaseI footprint experiments shows the DNA binding region contains the palindromic sequences CTGGTCTAGTCCAG Mutations in the nucleotide binding sites and gel retardation assays shows that the mutated probe which has a mutation point in the palindromic sequence can still bind with GL003331 protein, and the mutated probe which has a mutation point out of the palindromic sequence lost the binding ability with GL003331.Chromatin co-immunoprecipitation (ChIP) experiments shows that GL003331 has binding activity with the target sequence in vivo. Identified other GL003331 target genes in vivo. The results show that GL003331 may be a GntR/HutC transcription regulator with extensive role, related to the regulation of ice nucleation activity, inorganic ions and macromolecules transmembrane transportation, nutrient metabolism signal transduction and other important biological functions.