Construction And Transformation Of Three Paralogous CYP51s-GFP/RFP Expression Vector

Cytochrome P450 sterol 14a-demethylase(CYP51)is the key enzyme in fungal ergosterol biosynthesis,and is the target of azole fungicides.Many ascomycete fungi carry one or two copies of CYP51 genes,CYP51A and CYP51B,while in Fusarium spp.,there is a third CYP51 paralog,the Fusaria-specific CYP51C gene.The previous studies identified distinct functions of the three CYP51 genes of F.graminearum.CYP51B encodes the enzyme primarily responsible for sterol 14a-demethylation.CYP51A is an inducible gene by azoles and environmental stress to compensate for disruption of FgCYP51B function.FgCYP51C no longer functions as a sterol 14a-demethylase,but rather is required for full virulence on host wheat ears.However,the subcellular localization of the three paralogous CYP51 gene is not reported yet.In this study,by using restriction endonuclease digestion and ligation method,we constructed the CYP51 gene overexpression vector pYGTC,complementation vector pYK11C,and RFP fusion protein expression vector pKHR.CYP51A/CYP51A/CYP51C-GFP fusion protein expression vectors pKNTGA-1,pKNTGB ?pKNTGC2-1 were constructed as well.Genetic transformation of these vectors were proceed by PEG mediated protoplast transformation.