Detection Of Mutation In The Acid-resistant DnaK Operon Of The Fluoride-resistant Strain Of Streptococcus Mutans

Objective：Genomics is assisted by the molecular biology technology, computertechnology,information network technology and other research methods, to researchbiological all genes, and explore the inherent law of life activities on the overall leveland its internal and external environment influence mechanism. The features includegenome mapping, nucleotide sequence analysis, gene mapping and functional analysisof all genes.Genomics impetus to the development of oral medicine is mainlymanifested in the genomics study of oral pathogenic microorganisms[1].Caries bacteria is mainly under multiple factors,leading to toothdemineralization inorganic, organic matter decomposition of dental hard tissue ofchronic destruction, is one of the common diseases of the oral cavity, and was listedas a World Health Organization one of the three diseases which harms humans.Dental caries is also known as bacterial infection of dental hard tissue diseases, andStreptococcus mutans is cariogenic ability the strongest caries pathogens[2-4].Cariogenicity of Streptococcus mutans primarily depends on its production of acidicand acid resistance[5]. In recent years, fluoride is a widely used anti-caries pathogenicagent, and long-term mass use of fluoride caries prevention may lead to fluoride-resistant strains. Research has shown that fluoride resista-nt strain of Streptococcusmutans is stronger than the parental strains of cariog-enic ability. At present,scholarsare limintted to related acid tolerance genes, such as ffh、dfp、dltC、dnaK[6-8], but forthe mutation of the fluoride resistant strain acid associated operon is not clear, andstill need further research. And the genome sequence of Streptococcus mutansUA159,UA159-FR have been finished, and the accession NO. AE014133[9]and NO.CP007016[10]. The experi-mental group has been successful in vitro fluoride-resistantstrains of Streptococ-cus mutans[11]. The dnaK operon of Streptococcus mutans iscomposed of hrcA-grpE-dnaK-dnaJ. This study is limited to the fluoride resistantstrain of acid resistant dnaK operon gene mutation detection, genome sequence offluoride-resistant strain of Streptococcus mutans dnaK operon in the molecular level,which can determine the gene mutation and protein mutation significance. The result can lay the foundation for the application of anti-caries, but also to further understandthe mechanism of fluoride-resistant cariogenic. This provides a theoretical andexperimental foundation for future clinical application of fluoride caries preventionmore rational, as well as the development of caries prevention drugs.Metheds:1. Vitro induction fluoride-resistant strains of Streptococcus mutans. Screeningand identification of fluoride-resistant Streptococcus mutans UA159-FR.2. Extraction of the whole genome of UA159-FR.3. Primers were designed from the most conservative region according to thepublished GenBank gene of Streptococcus mutans dnaK operon sequences. Thegenome of Streptococcus mutans fluoride-resistant strains was used to be templatesfor PCR to obtain the objective gene fragment. Using PCR product purification kit,recycling and purification PCR products. Products were sequenced by biochemicalcompany.4. Comparing fluoride-resistant dnaK operon of Streptococcus mutansUA159-FR and referenced strain UA159dnaK operon by using BLAST tool in theGenBank database.Results:1. Experimental cultured bacteria identified as Streptococcus mutans fluorideresistant strain UA159–FR.2. The dnaK operon structure genes sequencing of the fluoride resistant strain ofStreptococcus mutans UA159-FR has been completed.3. Comparing the dnaK operon structure genes sequencing of the fluorideresistant strain of Streptococcus mutans UA159-FR and genes of UA159.It is foundthat the fragment of hrcA-grpE gene mutation sites was943(T deletion), the fragmentof dnaK-dnaJ gene mutation sites was2128(A insertion),2298(C deletion).Conclusion: The dnaK operon structure genes sequencing of the fluoride resistant strain ofStreptococcus mutans UA159-FR has been completed. It is found that the fragment ofhrcA-grpE gene mutation sites was943(T deletion), the fragment of dnaK-dnaJ genemutation sites was2128(A insertion),2298(C deletion).Confirmed that the acidresistance of streptococcus mutans fluoride resistant strain increase has something todo with dnaK rig substruct-ure genetic changes. For the further study of streptococcusmutans fluoride resistant strains to caries virulence to lay the theoretical foundation.In Strepto-coccus mutans fluorine strains to caries resistance enhancement may alsowith operon acid regulatory mechanism and operon methylation has a certainrelationship. This for the future clinical methods more reasonable application offluoride, and for the development of caries disease prevention drugs provide certaintheoretical and experimental basis.