Prokaryotic Expression And Characterizaction Of DNA-protein Interaction For GlnR Factor From Streptococcus Agalactiae

GlnR protein is a global transcriptional regulation factor, a member of MerRprotein family, and it can participate in the regulation of many genes relating tometabolic enzyme synthesis, secondary metabolism as well as transportation. It wasreported that the GlnR protein could recognize a specific inverted repeat sequences:5′-TGTNAN7TNACA-3′, and then binded with the DNA sequences to change thestructure of the gene fragment, sequentially affected the combination of RNApolymerase with the promoter, finally influenced the gene transcription.Moreover, a great number of studies had shown that glutamic acid and glutamineacted as the main nitrogen donors in bacterial cells, their metabolism was animportant part of nitrogen metabolism in bacterial cells. At present, researchers foundthe glnR gene and glnA gene(encoding the glutamine synthetase) in manybacterium(such as Bacillus subtilis, Streptococcus pneumoniae, Staphylococcusaureus, et al), and the two genes not only were linked together to compose the glnRAoperon but also offen suffered from the negative regulation of the GlnR factor.In recent years, already there had been a lot of reports about GlnR transcriptionregulatory factors of many kinds of bacterium, however, there had not been anysimilar reports associated with GlnR factor aiming at Streptococcus agalactiae athome and abroad. Thus, in order to confirm that whether the GlnR protein existe inStreptococcus agalactia, and whether it regulate the expression of glnRA operon ornot, this paper did the indirect verification for these issues via the interaction of GlnRprotein between with target DNA from S.agalactia, futhermore this study designedand completed the following experiment content:1. According to the glnR gene sequence of S. agalactiae NEM316published inGeneBank, a pair of specific primers were designed and synthesized, then the glnRgene of Streptococcus agalactiae from diseased tilapia in Guangxi, was amplified byPCR. And the result shew that ORF of the glnR gene is372bp, encoding124aminoacids.2. Successfully constructed recombinant plasmid pGEX-4T-glnR by fusionexpression vector pGEX-4T-3, and the target gene glnR was expressed favourably inE.coli JM109. And most recombinant proteins were in the form of soluble protein., Inaddition, this study optimized expression conditions when induced under37℃, theresult suggested that the optimal concentration of IPTG was3.0mmol/L, and the best induction time was5h. In addition, this paper purified GlnR protein by means ofaffinity chromatography method and thrombin.3. We screened the entire genome of S. agalactiae A909published in GeneBankfor the presence of putative DNA binding sites for GlnR protein using Genome2D.Then we designed a pair of specific primers and amplified the DNA fragment RA(containing the region of glnRA operon) by PCR using Streptococcus agalactiaeseparated from diseased tilapia in Guangxi. And the result pointed out that the DNAfragment RA is153bp, which included binding sites.4. Researched the interaction between GlnR protein with target DNA fragmentRA of Streptococcus agalactiae in vitro by EMSA (Electrophoretic Mobility ShiftAssay). Results of this paper demonstrated that GlnR factors of S.agalactia could bindwith the DNA fragment RA, this was preliminary confirmed that GlnR factors waslikely to play a negative regulatory role at the transcriptions of glnR and glnA gene inS.agalactiae, consequently effected the glutamic acid and glutamine metabolism ofS.agalactiae. And acting as the small molecules effect, glutamic acid maybe enhancedthe binding of GlnR-RA, while glutamine reduced the combining capacity of GlnRfactors with RA gene. Moreover, this study suggested that there is not only twobinding sites glnRAo1and glnRAo2in glnRA operon but also a binding site glnRAo3in the ORF of glnR gene of S.agalactiae.