白念珠菌SUMO结合酶UBC9基因缺失株的构建及其在形态调控中的作用研究  

Construction of a SUMO conjugating enzyme UBC9 deletion strain and investigation of its role in morphological regulation in Candida albicans

作　　者：单爱狄 冯金荣 SHAN Ai-di;FENG Jin-rong(Department of Pathogen Biology,Medical College,Nantong University,Nantong,Jiangsu 226001,China)

机构地区：[1]南通大学医学院病原生物学系,江苏南通226001

出　　处：《中国病原生物学杂志》2020年第7期800-804,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)。

摘　　要：目的构建白念珠菌UBC9基因缺失株,探索UBC9基因缺失对细胞形态转换和生物被膜形成的影响。方法采用瞬时CRISPR/Cas9基因敲除系统,分别于体外扩增Cas9基因、sgRNA以及修复DNA,通过醋酸锂法转化白念珠菌野生型菌株SN148,于SD-His选择培养基筛选转化子,通过菌落PCR验证基因型,构建UBC9双等位基因缺失株。分别检测UBC9缺失株在液体培养基中的细胞形态、固体YPD平板上的菌落形态和浸入生长情况以及生物被膜形成能力。结果PCR检测发现UBC9双等位基因缺失株中能扩出约2.8 kb的单一条带,UBC9基因缺失导致液体培养基中细胞伸长主要以菌丝态形式存在,固体培养基上菌落表面粗糙且深入到培养基内呈典型的浸入生长,并且生物被膜形成能力显著增高(A值为10.31,约为野生型的10.3倍)。结论利用CRISPR/Cas9系统快速构建了白念珠菌UBC9纯合子缺失株,且证明UBC9参与细胞的形态调控和生物被膜形成。Objective To construct a UBC9 deletion strain in Candida albicans and explore its effects on cell growth,morphological regulation,and biofilm formation.Methods A transient CRISPR/Cas9 system was used to delete UBC9 in C.albicans.sgRNA was constructed using fusion PCR with two independent DNA fragments containing a 20 bp sgRNA sequence as the template.The Cas9 gene was amplified from the pV1093 plasmid while repair DNA containing a HIS1 marker was amplified from a pFA-HA-HIS1 plasmid using PCR.The Cas9 gene sgRNA,and repair DNA were then transformed into the SN148 strain by the lithium acetate method.The transformants were selected on SD-His medium and directly verified using colony PCR.The morphology of the UBC9 deletion strain in liquid medium or on solid YPD plates as well as the biofilm formation was sequentially investigated.Results A UBC9 deletion strain was successfully constructed,and it produced a unique band of 2.8 kb when amplified with detection primers.The Phenotyping indicated that the UBC9 deletion strain exhibits strong filamentous growth on liquid YPD medium and invasive growth on solid YPD plates.The UBC9 deletion strain also exhibited an increased ability to form a biofilm that is 10.3 times greater than the ability of the wild-type strain.Conclusion A transient CRISPR/Cas9 system was used to successfully construct,a homozygous deletion strain for UBC9.UBC9 plays a negative role in filamentous growth and biofilm formation.

关 键 词：白念珠菌 CRISPR/Cas9 生物被膜 UBC9基因 

分 类 号：R379.4[医药卫生—病原生物学]