Development of mutant generation system of Streptococcus mutans, and analysis of the functional domain of glucosyltransferases using the mutant.

从变形链球菌-大肠杆菌穿梭载体pVA 838中克隆了变形链球菌红霉素耐药基因（resistance gene，resistance gene，resistance gene）。用Hind III和Cla I消化pVA 838，将其插入克隆载体pUC 19中，测序结果表明，该基因的必需部分约为800 bp，然后设计引物，在必需基因的末端添加限制性内切酶位点，经PCR扩增，获得了一个较小的（830 bp）红霉素抗性基因盒。用同样的方法从链球菌转座子Tn 1545中克隆到了aphA基因。对含有aphA基因的1.6kb插入片段的DNA序列进行了测定，结果表明，aphA基因的开放阅读框约为1000 bp。根据aphA基因的序列设计了PCR引物。通过PCR扩增，获得了一个较小的（1070 bp）卡那霉素抗性盒。此外，从Tn 1545中克隆了四环素抗性基因，通过随机突变的方法研究了细胞相关葡萄糖基转移酶的定位机制来自变形链球菌菌株MT 8148和GS-GTase的染色体DNA基因库随机转化变形链球菌，从Mitis-Salivarius琼脂中筛选红霉素抗性转化子，然后在大肠杆菌中表达CA-5。通过Western blot分析检测GTase，虽然测试了超过1000个突变体，但CA-GTase的定位没有改变。

The erythromycin resistant gene (erm) which works in Streptococcus mutans was cloned from Streptococcus-E.coli shuttle vector pVA838. pVA838 was digested with Hind III and Cla I, then inserted into a cloning vector pUC19.The DNA sequence of the insert was revealed that the essential part of the erm was app. 800 bp.Then PCR primers were designed to add restriction enzyme sites at the end of the essential gene.A smaller (830 bp) erythromycin resistant cassette was generated by PCR.The kanamycin resistant gene (aphA) was cloned from streptococcal transposon Tn 1545 by the similar procedure.DNA sequence of the 1.6 kb insert containing the aphA revealed that an open reading frame of the aphA was app. 1000 bp.From the sequence of the aphA gene, PCR primers were also synthesized.A smaller (1070 bp) kanamycin resistant cassette was produced by PCR.Furthermore, the tetracycline resistant gene was cloned from Tn 1545.On other hand, randomly mutagenesis was performed to investigate the localization mechanism of the cell-associated glucosyltransferase (CA-GTase).The chromosomal DNA gene banks from S.mutans strains MT8148 and GS-5 were generated with pVA891.The gene bank was randomly transformed into S.mutans.The erythromycin resistant transformants were screened from Mitis-Salivarius agar.Then expression of CA-GTase was examined by the Western blot analysis.Although over 1000 mutants were tested, the localization of the CA-GTase was not altered.