| Objective:Dental caries,one of the most common human oral diseases,are mainly caused by Streptococcus mutans(S.mutans).And fluoride can effectively inhibit the growth of Streptococcus mutans that 1g/L fluoride can completely kills Streptococcus mutans.At present,fluoride has been widely used to prevent dental caries,but it also brought a new problem,that is,the emergence of fluoride-resistant Streptococcus mutans.Experiments showed that fluoride-resistant strains have stronger acid production capacity,acid resistance capacity and demineralization capacity,suggesting that fluoride-resistant strains have stronger cariogenic ability.There is still no clear conclusion about the reason for the changes of fluoride-resistant strains,especially fluoride resistance.Generally,we attribute to the changes of the bacterial genome.Our previous experiments,in which we detected the whole genome sequences of fluoride-resistant Streptococcus mutans,showed that twenty genes had changed compared with its parental strains.Four of these genes are probably associated with fluoride resistance,which are ciaH,eno,pykF and rpl.CiaH,eno,pykF and rpl respectively encodes histidine kinase,enolase,pyruvate kinase and transcriptional regulator,and they are closely related to the survival of bacteria,and its acid resistance,glycolysis and sugar transport process and fluoride resistance.Some researchers once cultivated Streptococcus mutans and its fluoride-resistant strains in low concentration fluoride environment to explore the difference between the two strains.In this experiment,the fluoride-resistant strains were cultivated in 1g/L fluoride concentration,then detected the expression levels of these genes and discuss the causes of fluoride resistance.Metheds:1.Recovered,cultured and identified Streptococcus mutans UA159 and its fluoride-resistant strains UA159-FR.2.The experiment set three groups,namely fluoride-resistant Streptococcus mutans subcultured in BHI containing 1g/L NaF or without NaF,and its parental strains subcultured in BHI without NaF.Measured the growth curves of the three groups.3.Designed and synthesized the primers of 16sRNA,ciaH,eno,pykF and rpl.4.Extracted total RNA of the three groups and reverse transcribed both in the logarithmic phase and stable phase.5.Detected the expression levels of ciaH,eno,pykF and rpl by qRT-PCR.Results:1.Successfully recovered,cultured and identified Streptococcus mutans UA159 and its fluoride-resistant strains UA159-FR.2.Draw the growth curves of the three groups of strains and determined 11h as the logarithmic phase,while 20h as the stable phase.3.Successfully designed and synthesized the primers of 16sRNA,ciaH,eno,pykF and rpl.4.Successfully extracted total RNA of the three groups and reverse transcribed both in the logarithmic and stable phases.5.qRT-PCR results showed that:(1)Of all three groups,the expression levels of ciaH,eno,pykF and rpl genes in the logarithmic phase were significantly higher than those in the stable phase(P<0.001).(2)The expression levels of ciaH,eno and pykF genes of fluoride-resistant Streptococcus mutans cultivated in fluoride-containing medium,compared with fluoride-free medium,were significantly increased both in the logarithmic phase and the stable phase(P<0.001).While the expression levels of rpl gene had no significant difference in the logarithmic phase(P>0.05),but increased in the stable phase(P<0.001).(3)Compared with its parental strains,the expression levels of eno,pykF and rpl genes of fluoride-resistant Streptococcus mutans,whether cultivated in fluoride-containing or fluoride-free medium,were significantly decreased(P<0.001).While the expression levels of ciaH gene had no significant difference in the logarithmic phase(P>0.05),but increased in the stable phase(P<0.01).Conclusion:Fluoride could increase the expression levels of ciaH,eno and pykF genes,which indicated that these genes were associated with the production of fluoride resistance in fluoride-resistant strains.The expression levels of eno,pykF and rpl genes were different between Streptococcus mutans and its fluoride-resistant strains,which indicated that there were differences in sugar transport and glycolysis between them.These results can help us explain the causes of fluoride resistance and provide a basis for studying the specific functions of these genes. |
