Detection Of Mutation In The Acid-resistant Gene GroES-groEL Of The Fluoride-resistant Strain Of Streptococcus Mutans

Objective:Dental caries,a chronic progressive infectious diseases, is breakdown of teeth dueto the activities of bacteria,combined with several risk fators.The bacteria mostresponsible for dental cavities are the mutans streptococci,most prominentlyStreptococcus mutans.They produce acid in the presence of fermentablecarbohydrates, and caries occur from bacterial acid demineralization.Fluoride is nowrecognized as the main factor responsible for the dramatic decline in caries prevalencethat has been observed worldwide.It helps prevent decay of a tooth by binding to thehydroxyapatite crystals in enamel,which will make enamel more resistant todemineralization and speed up the process of remineralization as well.Topical fluoridecan also provide antimicrobial action. However, excessive fluoride taken can causethe appearance of fluoride-resistant strains of streptococcus mutans.Domestic andinternational scholars have fully proof that fluoride-resistant strains of streptococcusmutans have stronger cariogenicity and resistance to fluoride.Heat shock response is aseries of physical and chemical reactions responding to heat,acid,H2O2,etc.It hadreported that groES-groEL gene of streptococcus mutans affects the metabolism ofcells and increases the tolerance to acid by translating the heat shock proteins, GroES-GroEL, which can mediate the folding, assembly, transport, and degradation of newor misfolding proteins. Our group have done the detection of mutation in theacid-resistant genes,such as dltC，Dgk，comD, ccpA etc,of the fluoride-resistantstrains of streptococcus mutans.The result indicates that there are mutations.Yet,thedetection of mutation in groES-groEL gene have not be reported.We try to detect themutation in the acid-resistant genes groES-groEL of the fluoride-resistant strains ofstreptococcus mutans this time, to improve the mechanism of acid tolerance responseof the fluoride-resistant strains of streptococcus mutans at the gene level,and provide the theoretical foundation to rational use of fluoride and the development of moreeffective methods to prevent and cure caries.Methods:1.Identifying the fluoride-resistant strain of Streptococcus mutans UA159.2.Extracting of the genome DNA of UA159-FR.3.Amplification and purification of the target genes.4.The construction of a recombinant plasmid: connect the PCR production withPMD-18T vector,and transform it into E.coli cells.5. Extracting and identifying the plasmid.6. Sending for sequencing.Results:1. Successfully identify UA159-FR.2.Successfully construct the recombinant plasmid.3.By using the BLAST tool to compare the homology of the obtained genessequences in the GenBank datebase.We found that the acid resistance related genegroEL has no difference compares with the data published in GenBank.While,genegroES gets one mutation,locating at1834963(Tâ†'C).Conclusion:Successfully built the recombinant plasmids of the acid-resistant genesgroES-groEL mutations of the fluoride-resistant strain of streptococcus mutans incompetent cell E.coli DH5α and indentify them by PCR and then send for genesequencing.The research is of great importance for preventing and controlling thecaries disease in the future.