目的:Rothiaspp.正在成为与口腔健康相关的重要细菌,龙舌兰是最普遍的物种之一。然而,缺乏在遗传水平上研究这些特性的研究。本研究旨在建立一种适用于dentocariosa的基因改造平台。
方法:Rothiaspp.从健康志愿者收集的唾液样本中分离出来。随后,通过菌落形态学鉴定了牙菌属菌株,物种特异性聚合酶链反应(PCR),和16S核糖体RNA基因测序。然后用质粒pJRD215转化鉴定的菌株,并选择最有效的菌株。进行转座子插入诱变以研究遗传修饰的可能性。
结果:菌株表现出高转化能力,鉴定为DentocariosaLX16。该菌株进行了转座子插入诱变,并筛选了抗5-氟乳清酸的转座子。使用任意引物PCR确认插入位点,基因特异性PCR,还有Sanger测序.
结论:这项研究标志着第一次成功的对齿科的遗传修饰。在遗传水平上研究Dentocariosa可以提供对其在口腔微生物组中的作用的见解。
OBJECTIVE: Rothia spp. are emerging as significant bacteria associated with oral health, with Rothia dentocariosa being one of the most prevalent species. However, there is a lack of studies examining these properties at the genetic level. This study aimed to establish a genetic modification platform for R. dentocariosa.
METHODS: Rothia spp. were isolated from saliva samples collected from healthy volunteers. Subsequently, R. dentocariosa strains were identified through colony morphology, species-specific polymerase chain reaction (PCR), and 16S ribosomal RNA gene sequencing. The identified strains were then transformed with plasmid pJRD215, and the most efficient strain was selected. Transposon insertion mutagenesis was performed to investigate the possibility of genetic modifications.
RESULTS: A strain demonstrating high transforming ability, designated as R. dentocariosa LX16, was identified. This strain underwent transposon insertion mutagenesis and was screened for 5-fluoroorotic acid-resistant transposants. The insertion sites were confirmed using arbitrary primed PCR, gene-specific PCR, and Sanger sequencing.
CONCLUSIONS: This study marks the first successful genetic modification of R. dentocariosa. Investigating R. dentocariosa at the genetic level can provide insights into its role within the oral microbiome.