The Comparison Of Methylation Difference Of The Acid-resistant GroE Operon Of The Fluoride-resistant Strain Of Streptococcus Mutans And Parental Strain

Objective:Dental caries are mainly about bacterial infection of tooth hard tissue diseases caused by a variety of factors.Caries are chronic progressive development, and destroy tooth hard tissue, result in the tooth color, shape, quality and other negative change, the process is not reversible.They are common oral diseases.WHO listed them as one of the three diseases serious threats to human health in the 70’s of the last century.Streptococcus pneumoniae is the strongest pathogenic bacteria in oral cavity[1-3].In recent years, fluoride as an effective method is widely used in the prevention of dental caries, resulting in the fluoride-resistant strain of Streptococcus mutans selective growth[4].At present,scholars are limitted to related acid tolerance genes,such as ffh、dfp、dlt C、dna K[6-8], and the strains have been successfully induced.Fluoride resistant strain of Streptococcus mutans are stronger than the parental strains of cariogenic ability[9,10].There is some connection with detection of mutation in the acid-resistant gene.DNA methylation is a common epigenetic phenomena,Which is a kind of gene modification catalyze by DNA methyl transferase,that to say,the methyl was transported from S-adenosylmethionine to DNA bases.A large number of studies show that DNA methylation can cause chromatin structure, DNA conformation, DNA stability and DNA and protein interaction mode changes, so as to control the gene expression[11].The DNA modification method does not change the gene sequence, but it regulates gene expression[12].At present, the whole genome sequence of UA159 strain and UA159-FR has been submitted to the Genbank database, and the accession numbers are AE014133 [13]and CP007016 [14].Gro EL-gro ES did not occur mutation, gro EL-gro ES is excited, the expression of Gro EL molecular chaperone, auxiliary protein assembly, folding,transport and degradating, and enhance its adaptability to acid.The mechanism is not clear.Further researches are needed on the correlation of genetic modification of gene methylation.This experiment compared fluoride-resistant strain of Streptococcus mutans with their parental strains gene methylation level which further reveal the fluoride-resistant strain of Streptococcus of cariogenic of mechanism, provide the theoretical basis for the future use of fluoride.Metheds:1.Induced the fluoride-resistant strain of Streptococcus(UA159-FR S.mutans).2.Culture of strain of Streptococcus strain(UA159 S.mutans) and fluoride-resistant strain of Streptococcus(UA159-FR S.mutans)3.Identification of UA159-FR 16 Sr DNA S.mutans strain.4.Extraction of whole genome of UA159 S.mutans and UA159-FR S.mutans5.Amplification of UA159-FR gro EL-gro ES target gene PCR S.mutans.6.Construction of the recombinant plasmid: UA159-FR PCR target gene S.mutans product and PMD18-T vector connection, then transformed into the E.coli DH5α.7.Lysis of Escherichia coli, extraction of recombinant plasmid,Identification of the recombinant plasmid through Pst I and HindⅢ enzyme digestion8.Detection of the sequence of gro EL-gro ES gene9.Compare S.mutans UA159 with S.mutans UA159-FR from whole genome on gene methylation.Results:1.Successful Induced the fluoride-resistant strain of Streptococcus.2.Successful culture S.mutans UA159 and S.mutans UA159-FR.3.Gro EL-gro ES was transformed into PMD18-T vector and successfully constructed the recombinant plasmid, and the recombinant plasmid was amplified in E.coli DH5α.4.Have finished sequencing and by means of BLAST,compared with gro EL-gro ES gene sequences,which is the target gene of Streptococcus mutans UA159 published in Gen Bank, it was found that there is no mutation.5.Have finished gene methylation assay. The results showed that Methylation levels of gro EL-gro ES gene and parental gene sequence were comparative without significant difference.Conclusion:Successfully built the recombinant plasmids of the acid-resistant genes gro ES-gro EL mutations of the fluoride-resistant strain of streptococcus mutans,identificated strains and detected the sequences,also tested the methylation levels.There is no mutation.And gene methylation level did not change.The increase of acid resistance of fluoride-resistant strain of streptococcus mutans was not related to the gro EL-gro ES gene mutation and methylation level.The results reveal the fluoride-resistant strain of streptococcus mutans of cariogenic mechanism to provide theoretical basis for clinical application,and to guide fluoride significance in the future.