副血链球菌粘附素Fap1糖基化相关基因nss的功能研究-植物病理学专业论文.docxVIP

摘要 摘要 （3）被 Gtf1、Gtf2 和 Nss 共同修饰的重组 Fap1 蛋白出现了两个峰值，分 别为 N-乙酰葡萄糖胺（GlcNAc）和葡萄糖（Glucose），而在缺失 Nss，只有 Gtf1 、 Gtf2 修饰的重组 Fap1 蛋白中只出现一个峰值，即 N- 乙酰葡萄糖 （GlcNAc）。 结论：Nss 参与并调控 Fap1 糖基化的第二步；Nss 是一个葡萄糖基转移 酶。 关 键 词：Fap1，粘附素，糖基化，副血链球菌 论文类型： 基础研究 II Subject: The functional study of Fap1 glycosylation associated gene-nss in Streptococcus parasanguis Specialty: Phytopathology Name: Fan Zhu Supervisor: Meixian Zhou ABSTRACT Objectives ： Mature Fap1, a glycoprotein, is an adhesion of Streptococcus parasanguis FW213, which is critical to the formation of the most complicated human biofilm, dental plaque. Glycosylation of Fap1 is mediated by a gene cluster flanking the fap1 locus. Previous study suggested Gtf1 and Gtf2 located in the downstream of fap1 are responsible for the first step of Fap1 glycosylation, and catalyze the transfer of GlcNAc residues to the Fap1 backbone. However, to date nothing is known about the subsequent steps in Fap1 glycosylation. Here this study is to investigate the role of nss, located immediately upstream of fap1, in Fap1 glycosylation. Methods: (1) Construction of nss mutant and its complementary strain: An nss mutant was constructed using allelic replacement mutagenesis strategy with the start strain Streptococcus parasanguinis FW213. The full-length nss gene amplified from FW213 genomic DNA by PCR was cloned into E. coli-Streptococcal shuttle vector to generate the nss complementation plasmid. The plasmid was then transformed into the nss mutant to produce nss complementary strain. The Fap1 expression profile of the mutant was determined by Western Blotting analyses and BactELISA using Fap1-specific antibodies. Identification of nss function in E. coli glycosylation system: A series of plasmids containing different potential glycosyltransferases were co-transferred with two compatible plasmids: plasmid harboring Fap1 substrate, Fap1?RII and