关键词: 犬小孢子菌, RNA干扰, 基因，PQ-LRP, 基因沉默

Abstract: Chen Xinyi, Yang Guoling, Liu Jianwen, Liu Jinpeng, Zhang Fangfang, Zhao Xiaoxuan. Department of Dermatology and Venereology, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China Corresponding author: Yang Guoling, Email: yanggl@medmail.com.cn 【Abstract】 Objective To build a RNA interference vector for PQ-loop repeat protein （LRP） gene, and to evaluate the effect of the vector on the expression of PQ-LRP gene in Microsporum canis. Methods The PUC-PLULT and PCB309-PLULT vectors were constructed sequentially by using Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis, adding multiple cloning sites, and introducing the hygromycin-resistance gene. Microsporum canis was transformed with the PCB309-PLULT vector followed by a series of passages and hygromycin selection. Real-time quantitative PCR was performed to measure the expression of PQ-LRP gene in Microsporum canis before and after transformation. Results The intermediate vectors PUC-PLUT and PUC-PLULT were constructed and identified by PCR and gene sequencing. The 8 825-bp interference vector PCB309-PLULT was successfully built and confirmed by enzyme digestion. The optimum concentration of hygromycin for screening for Microsporum canis transformants was determined as 300 mg/L. The mRNA expression level of PQ-LRP was decreased by 61% in the transformants as compared with untransformed Microsporum canis （0.39 vs. 1.00）. Conclusion The constructed PCB309-PLULT interference vector can effectively inhibit the expression of PQ-LRP gene in Microsporum canis.

Key words: Microsporum canis, RNA interference, Genes, PQ-LRP, Gene silencing