RESUMO
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.