摘要
目的为了构建犬小孢子菌FSH1基因的RNA干扰重组质粒,并验证其干扰效率。方法以犬小孢子菌FSH1基因全长cDNA为模板,利用PCR技术扩增FSH1基因,将质粒pUC-PUT进行双酶切使其线性化,插入FSH1正向及反向干扰序列,获得中间载体pUC-PFUFT,将其及质粒pCB-309双酶切后连接,构建干扰载体pCB309-PFULFT。将其转化根癌农杆菌(Agrobacterium tumefaciens)EHA105,获得犬小孢子菌FSH1基因RNA干扰的转化体系。用实时定量PCR方法对犬小孢子菌FSH1基因干扰前后表达量进行鉴定。结果成功构建了FSH1基因的干扰载体pCB309-PFULFT,获得FSH1基因的干扰转化子;犬小孢子菌野生株干扰后FSH1基因mRNA相对表达量较干扰前下降约70%,标准株下降约60%。结论本试验成功构建了犬小孢子菌FSH1基因的干扰载体,为后续研究FSH1基因的功能及在头癣中的发病机制奠定了基础。
Objectives To construct a RNA interference plasmid targeting FSH1 gene and evaluate the interference efficiency.Methods The FSH1 gene was amplified by PCR from the FSH1 full length cDNA as the template.The plasmid pUC-PUT was linearized by double restriction and inserted the amplified FSH1 forward and reverse gene.The fusion plasmid pUC-PFUFT was constructed.Then the plasmid pCB-309 and pUC-PFUFT were linearized by double restriction and ligated to construct the RNA interference plasmid pCB309-PFULFT.The plasmid pCB309-PFULFT was transformed into Microsporum canis by Agrobacterium tumefanciens-mediated method.The mRNA expression of the FSH1 gene before and after interference was compared by qPCR analysis.Results The knockdown vector pCB309-PFUFT was constructed successfully.FSH1-i mutants were screened.qPCR analysis demonstrated that the degree of knockdown was stable and efficient,and the expression of the FSH1 gene in FSH1-i mutant were-70%and 60%lower compared with the wild-type and strandard strain,respectively.Conclusions The RNA interference plasmid targeting FSH1 gene in Microsporum canis was constructed successfully.These results provide a basis for future studies on the function and pathogenicity mechanism of FSH1 gene.
作者
张芙蓉
郭春梅
吴英
谭灿
杨国玲
ZHANG Fu-rong;GUO Chun-mei;WU ying;TAN Can;YANG Guo-ling(Department of Dermatology, Dalian Friendship Hospital, Dalian 116001, China;Department of Biotechnology, Dalian Medical University, Dalian 116044,China;Department of Dermatology, Chengdu Second People’s Hospital, Chengdu 610017, China;Department of Dermatology, the 1st Affiliated Hospital of Dalian Medical University, Dalian 116011, China)
出处
《中国真菌学杂志》
CSCD
2020年第6期354-358,共5页
Chinese Journal of Mycology
基金
国家自然科学基金(81071330)。
