利用框内缺失法构建变异链球菌srtA基因缺失株

doi:10.7518/hxkq.2017.06.005

华西口腔医学杂志 ›› 2017, Vol. 35 ›› Issue (6): 588-592.doi: 10.7518/hxkq.2017.06.005

利用框内缺失法构建变异链球菌srtA基因缺失株

陈璇(), 刘海霞, 彭显, 邹玲()

口腔疾病研究国家重点实验室国家口腔疾病临床医学研究中心,四川大学华西口腔医院牙体牙髓病科,成都 610041

收稿日期:2017-03-28 修回日期:2017-05-10 出版日期:2017-12-20 发布日期:2017-12-01
作者简介:
陈璇,硕士,E-mail:252132611@qq.com
基金资助:
国家自然科学基金（81570974）;四川省科技计划项目基金（2015JY0260）

Construction of srtA-deletion mutant of Streptococcus mutans by an in-frame deletion system

Xuan Chen(), Haixia Liu, Xian Peng, Ling. Zou()

State Key Laboratory of Oral Diseases & Dept. of Conservative Dentistry and Endodontics, National Clinical Research Center for Oral Diseases & West China Hospital of Stomatology, Sichuan University, Chengdu 610041, China

Received:2017-03-28 Revised:2017-05-10 Online:2017-12-20 Published:2017-12-01
Supported by:
Supported by: The National Natural Science Foundation of China (81570974);The Key Project of the Science and Technology Department of Sichuan Province (2015JY0260).

摘要/Abstract

摘要：

目的利用框内缺失法,通过IFDC2基因盒及融合聚合酶链反应（PCR）、同源重组技术构建变异链球菌UA159菌株srtA基因缺失株。方法 PCR扩增变异链球菌UA159菌株srtA基因上下游同源片段及IFDC2基因盒,将这些片段连接、转化入UA159,替换srtA基因同源片段,筛选、鉴定红霉素抗性克隆;PCR扩增、连接UA159 srtA基因上下游同源片段,将连接片段转化入前述红霉素抗性克隆中替换IFDC2同源片段,筛选、鉴定抗苯丙氨酸类似物p-chloro-phenylalanine（p-Cl-Phe）克隆。结果 PCR、琼脂糖凝胶电泳及DNA测序结果均证明srtA基因编码区被完全删除,上下游片段无缝连接,成功构建UA159 srtA基因缺失株。结论本研究成功构建了无标记的变异链球菌UA159 srtA基因缺失株,为进一步研究该基因在生物膜中的作用及其调控机制奠定了基础。

关键词: 变异链球菌, srtA基因缺失, 框内缺失法

Abstract:

Objective To construct srtA-gene deletion mutant of Streptococcus mutans (S. mutans) UA159 with IFDC2 cassette through overlapping polymerase chain reaction (PCR) and allelic homologous recombination. Methods First, the upstream and downstream fragments surrounding the srtA and IFDC2 cassette were PCR amplified and ligated through over-lapping PCR. The resulting amplicon was transformed into UA159, and positive transformants were selected on BHI plates containing erythromycin. Second, upstream and downstream fragments of srtA with overlap regions were generated by PCR and were overlapped to create upΔ-down amplicon. Then, the upΔ-down amplicon was transformed into the aforementioned positive transformants and selected on BHI plates containing p-Cl-Phe. Results The PCR analysis and DNA sequencing results indicated that the coding region of the srtA was completely deleted, and the upstream and downstream regions flanking the srtA were ligated seamlessly. Conclusion The markerless srtA-deletion mutant of S. mutans was constructed successfully, which laid a foundation for further study of its biological function and influence on the biofilm formation of S. mutans.

Key words: Streptococcus mutans, srtA-deletion mutant, in-frame deletion system

中图分类号:

Q781

陈璇, 刘海霞, 彭显, 邹玲. 利用框内缺失法构建变异链球菌srtA基因缺失株[J]. 华西口腔医学杂志, 2017, 35(6): 588-592.

Xuan Chen, Haixia Liu, Xian Peng, Ling. Zou. Construction of srtA-deletion mutant of Streptococcus mutans by an in-frame deletion system[J]. West China Journal of Stomatology, 2017, 35(6): 588-592.

图/表 4

参考文献 17

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引物名称	引物序列（5’-3’）	用途
upF	CTTGGTCAAGATGAGTTTCG	扩增up及upΔ
upR-IFDC2	GAGTGTTATTGTTGCTCGGTGCTAGCTACTCATTTTCAT	扩增up
ldhF	CCGAGCAACAATAACACTC	扩增IFDC2
ermR	GAAGCTGTCAGTAGTATACC	扩增IFDC2
dnF-IFDC2	GGTATACTACTGACAGCTTCAATAAGAAACTCGCTCTAACGAG	扩增dn
dnR	GGATCATCAGCAACATTCAT	扩增dn
updnR	CTCGTTAGAGCGAGTTTCTTATTTGCTAGCTACTCATTTTCAT	扩增upΔ
checkF	GTCACTCGAAGATGAAGACT	筛选阳性克隆
checkR	ACAGATGAATATAACTGCAA	筛选阳性克隆

利用框内缺失法构建变异链球菌srtA基因缺失株

Construction of srtA-deletion mutant of Streptococcus mutans by an in-frame deletion system

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