Cloning, Expression Of Ustilago Maydis CYP51 And Analysis By Combine Spectrum With Antiseptic

Sterol 14α-demethylase (other names: P450 14DM; CYP51) is the only member of the cytochrome P450 superfamily present in animals, plants, fungi ,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 14α-demethylase inhibitors (DMIs) involves selective inhibition of the fungal sterol 14α-demethylase over the plant enzyme activity during treatment. Inhibition of sterol 14α-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 Ustilago maydis CYP51 DNA were cloned and expressed in E. coli. The recombinant expression vectors (pETYH and pGEXKGYH) were constructed and the optimal expression conditions and different expression host which added the rare tRNAs were tried, but they could not express. Transmembrane structures of the Ustilago maydis CYP51 were analyzed by online software. Homology models of the CYP51 and the mutants were constructed to analyze the structures of among the N-terminal cleavage CYP51 protein by SYBYL7.0 software which was useful for further site-directed mutagenesis experiment. According to the analytics results, we constructed the recombinant expression vectors(pETYH-20,pGEXKGYH-20,pETYH-35,pGEXKGYH-35) and got the CYP51 mutants protein expressed in the pETYH-35, but other mutants could not express in E. coli.U.maydis CYP51 protein was purified and determined its biological activity based on P450-CO spectrometry which used to determining the cytochrome P450 characteristic absorption peak. U.maydis CYP51 protein concentration was 2.92mg/mL, the P450 concentration was 3.763pmol/mg. Spectrometry experiments were taken out to compute the inhibition constants according to Michaelis-Menten equation Hanes-Woolf method. In the research, there were four standard antiseptics and ten artifical triazoles antiseptics named XF series. The results were that all the combine constants of standard antiseptics were smaller than the artifical antiseptics'. Triadimenol got the score 0.057 and it was the smallest in the standard antiseptics while XF-113 got the score 0.307 and it was the smallest in the artifical antiseptics. The combination of new synthetic small molecules fungicide bioassay results on microbicides for the initial assessment. Analying the cloning and expression of and U.maydis CYP51 with bioinformatics method will facilitate the further research on the structure and function of fungi CYP51, so as to design more specific DMI fungicides against fungi CYP51. Eukaryotic expression vector pAUR123-YH of Saccharomyces cerevisiae was constructed by the conventional lithium acetate method and colony PCR was used to identificating the positive clones. 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.