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整合型CRISPR/Cas9系统构建白念珠菌MTQ2基因缺失株及其表型分析 被引量:1

Using an integrative CRISPR/Cas9 system to construct an MTQ2 deletion strain and analysis of its phenotypes in Candida albicans
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摘要 目的构建白念珠菌MTQ2基因缺失株,并检测该基因缺失对细胞生长及遗传毒性试剂耐受性的影响。方法采用整合型CRISPR/Cas9系统,将sgRNA克隆到pV1093质粒中,线性化后与含HIS1基因的修复DNA一起通过醋酸锂法转化白念珠菌野生型菌株SN148,于SD+NAT-His培养基筛选转化子,通过PCR验证基因型,利用平板表型试验检测MTQ2缺失对细胞生长及遗传毒性试剂耐受性的影响。结果成功构建了MTQ2纯合子缺失菌株。MTQ2缺失导致串珠样细胞比例(44.4%)较野生型菌株(2.5%)显著增加,细胞增殖能力变弱,生长缺陷明显。结论利用整合型CRISPR/Cas9系统构建了白念珠菌MTQ2纯合子缺失菌株,且发现MTQ2基因可能参与维持细胞的增殖。 Objective To construct a MTQ2 deletion strain of Candida albicans and study its function in regulating cell growth and tolerance to genotoxic reagents.Methods sgRNA for the MTQ2 gene was annealed and cloned into pV1093,generating pV1093-MTQ2,while repair DNA for MTQ2 was amplified from pFA-HA-HIS1.The linearized pV1093-MTQ2 and repair DNA were then transformed into C.albicans SN148 via the lithium acetate method,and the transformant was screened on SD+NAT-His medium.The genotype was verified using PCR,and the effect of MTQ2 deletion on cell growth and tolerance to genotoxic reagents was determined using a spot assay.Results A homozygous MTQ2 deletion strain was successfully constructed using an integrative CRISPR/Cas9 system,and it produced a unique band of 2.0 kb.Deletion of the MTQ2 gene results in weaker growth and a slight sensitivity to genotoxic stress.In addition,deletion of MTQ2 increased the ratio of connected cell forms(44.4%),which was higher than that in wild-type cells(2.5%).Conclusion An integrative CRISPR/Cas9 system was used to successfully construct a homozygous MTQ2 deletion strain,and that strain exhibited weaker growth.
作者 胡婧 冯金荣 HU Jing;FENG Jin-rong(Department of Pathogen Biology,Medical College,Nantong University,Nantong,Jiangsu,China 226001)
出处 《中国病原生物学杂志》 CSCD 北大核心 2020年第9期993-996,1004,共5页 Journal of Pathogen Biology
基金 江苏省南通市科技计划项目(No.JC2018052) 南通大学大学生创新训练计划项目(No.2019095) 江苏高校优势学科建设工程资助项目(No.PAPD)。
关键词 白念珠菌 MTQ2基因 CRISPR/Cas9 Candida albicans MTQ2 gene CRISPR/Cas9
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