运用融合PCR技术敲除光滑念珠菌FLO8基因以及其对EPA黏附素家族表达的影响

doi:10.16150/j.1671-2870.2024.04.008

诊断学理论与实践 ›› 2024, Vol. 23 ›› Issue (04): 398-404.doi: 10.16150/j.1671-2870.2024.04.008

运用融合PCR技术敲除光滑念珠菌FLO8基因以及其对EPA黏附素家族表达的影响

赵珺涛¹^,², 袁捷²^,^#, 刘锦燕², 陈柯志¹^,², 项明洁¹^,²()

1.上海交通大学医学院附属瑞金医院检验科，上海 200025
2.上海交通大学医学院附属瑞金医院卢湾分院放免检验科，上海 200020

收稿日期:2023-08-22 接受日期:2023-11-05 出版日期:2024-08-25 发布日期:2024-08-25
通讯作者: 项明洁 E-mail: mjxiang123456@126.com
作者简介:第一联系人：#: 共同第一作者
基金资助:
国家自然科学基金(81871706);上海市自然科学基金(22ZR1439800);上海市医学重点专科建设项目(ZK2012A21);上海市卫生健康委员会课题基金(202240205);上海市卫生健康委员会课题基金(201840227);上海市卫生健康委员会课题基金(201740069);上海市黄浦区卫生和计划生育委员会课题(HKM201702)

Knocking out FLO8 gene of Candida glabrata and its effect on EPA family

ZHAO Juntao¹^,², YUAN Jie²^,^#, LIU Jinyan², CHEN Kezhi¹^,², XIANG Mingjie¹^,²()

1. Department of Clinical Laboratory, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Radioimmunology and ClinicalLaboratory, Ruijin HospitalLuwan Branch, Shanghai Jiao Tong University School of Medicine, Shanghai 200020, China

Received:2023-08-22 Accepted:2023-11-05 Published:2024-08-25 Online:2024-08-25

摘要/Abstract

摘要：

目的：构建光滑念珠菌FLO8基因敲除株，并分析FLO8敲除对光滑念珠菌上皮黏附素（epithelial adhesin, EPA)家族表达的影响。方法：使用融合PCR技术，以光滑念珠菌ATCC2001菌株基因组DNA、带有筛选标记诺尔斯菌素抗性基因（NAT）的质粒DNA为模板，构建敲除组件。采用醋酸锂转染法将敲除组件转染入ATCC2001中，从而获得flo8△菌株。使用实时荧光定量PCR检测菌株中EPA1、EPA6和EPA7基因的表达。结果：获得光滑念珠菌FLO8基因敲除株flo8△，该菌株的EPA1、EPA6和EPA7基因表达水平对明显低于ATCC2001株（P均<0.001）。结论：此法可便捷、有效地构建光滑念珠菌基因敲除菌株。敲除FLO8基因后，光滑念珠菌的EPA家族表达降低，为进一步研究光滑念珠菌毒力机制奠定基础。

关键词: 光滑念珠菌, 基因敲除, 黏附素, FLO8基因, 上皮黏附素家族

Abstract:

Objective To construct a FLO8 gene knockout strain of Candida glabrata and analyze the effect of FLO8 knockout on the expression of EPA family in Candida glabrata. Methods By fusion PCR technology, the knockout components were constructed with the genomic DNA of Candida glabrata ATCC2001 strain and the plasmid DNA with screening marker NAT as templates. The knockout components were transfected into Candida glabrataATCC2001 by lithium acetate transfection method to obtainflo8△strain. The expression of EPA1, EPA6 and EPA7 genes were detected by real-time quantitative PCR. Results The FLO8 gene knockout strain of Candida glabrata was efficiently constructed. The expression levels of EPA1, EPA6 and EPA7genes in flo8△strain were significantly lower than those in ATCC2001 strain (all P<0.001). Conclusions The gene knockout method permits rapid, easy and highly efficient generation of homozygous knockout mutations in Candida glabrata. The knockout of FLO8 gene reduced the expression of EPA family in Candida glabrata, which laid a foundation for further study on its virulence mechanism.

Key words: Candida glabrata, Gene knockout, Adhesin, FLO8 gene, Epithelial adhesin family

中图分类号:

R446.1
R519

赵珺涛, 袁捷, 刘锦燕, 陈柯志, 项明洁. 运用融合PCR技术敲除光滑念珠菌FLO8基因以及其对EPA黏附素家族表达的影响[J]. 诊断学理论与实践, 2024, 23(04): 398-404.

ZHAO Juntao, YUAN Jie, LIU Jinyan, CHEN Kezhi, XIANG Mingjie. Knocking out FLO8 gene of Candida glabrata and its effect on EPA family[J]. Journal of Diagnostics Concepts & Practice, 2024, 23(04): 398-404.

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参考文献 21

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Primer	Sequence (5’→3’)
Up F	GGGCTTCCCTTCGAGACTAC
Up R	cccggacagccgctaggaggtTTAACGCCCAGTGGAACCC
NAT F	acctcctagcggctgtccgggGTTGTAAAACGACGGCCAGT
NAT R	ctcgggactatcgggctacggAGGAAACAGCTATGACCATG
Down F	ccgtagcccgatagtcccgagTTTTAACCAACATGAATTCTCGC
Down R	TGGATAGGATTACCAGTATTTAAACAAC
FLO8-KO F	TTTTCAACACGACCACCAATCG
FLO8-KO R	GTTGACTTGAAGATCTCGTCCTC
T-FLO8 F	TCTCGTCTGCCCTCCCCTT
T-FLO8 R	GTTTCCGGCTGCACTGTTTCT
RT-EPA1 F	ACCGCAAGAAAATCCTCCTCC
RT-EPA1 R	TGGTGCTGATGATATTGATTTGTTG
RT-EPA6 F	GAAATCAGGATCGAATCCATG
RT-EPA6 R	GTGGTAATGTATCAAACAGCG
RT-EPA7 F	TGATTTACGGAAGAATGGTTCG
RT-EPA7 R	TTACCGGTAACACCATCAACT
RT-ACTIN F	TTCCAGCCTTCTACGTTTCC
RT-ACTIN R	TCTACCAGCAAGGTCGATTC

运用融合PCR技术敲除光滑念珠菌FLO8基因以及其对EPA黏附素家族表达的影响

Knocking out FLO8 gene of Candida glabrata and its effect on EPA family

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