Cloning, Expression Of The Phytopathogenic Fungi CYP51 And Preparation Of Polyclonal Antibodies

Sterol 14ct-demethylase (other names: P450_14dm; CYP51) is the only member of the cytochrome P450 superfamily present in animals, plants, fungi and bacteria and considered to be the most ancient member of the superfamily. In all cases it catalyzes a three-step reaction of sterol 14α-demethylation. In fungi, the complex sterol 14α-demethylation reaction presents one of the key steps in sterol biosynthesis, an essential metabolic pathway producing ergosterol. The action mode of 14a-demethylase inhibitors (DMIs) involves selective inhibition of the fungal sterol 14a-demethylase over the plant enzyme activity during treatment. Inhibition of sterol 14a-demethylation by DMI fungicides result in the depletion of ergosterol, accumulation of abnormal sterol intermediates. Consequently the growth of fungi was arrested. This study reported that the Penicillium digitatum CYP51 cDNA and Ustilago maydis CYP51DNA were cloned from P. digitatum and U. maydis respectively. P. digitatum CYP51 cDNA was cloned by RT-PCR (reverse transcnption-polymerase chain reaction) and expressed in E. coli using the pET-28 expression system yielding recombinant protein (516 amino acid residues) with a molecular weight approximate 58 kDa. The recombinant protein was purified to electrophoretic homogeneity using the metal (Ni²⁺) chelate affinity chromatography. Rabbit polyclonal antibodies were prepared against the purified recombinant P. digitatum CYP51 protein. In addition, an incomplete CYP51 DNA sequence and a complete CYP51 DNA sequence of U. maydis were cloned from U. maydis. The recombinant expression vectors (pE30YHl and pE28YH2K) were constructed respectively. Cloning and expression of P. digitatum and U. maydis CYP51 will facilitate the further research on the structure and function of fungi CYP51 so as to design more specific DMI fungicides against fungi CYP51. This study provides an important tool for detailed enzymological and mechanistic studies so as to develop stronger affinitive and more specific DMI fungicides and for studying the resistance mechanisms of phytopathogenic fungi to certain DMI fungicides.