Novel Inhibitors Screening Based On SDase And CYP51 From Magnaporthe Grisea

Rice blast disease, caused by the fungus Magnaporthe grisea, is one of the most severe and devastating diseases in rice production worldwide. Scytalone dehydratase (SDase) and Trihydroxynaphthalene reductase (3HNR) in the melanin biosynthetic pathway have attested to be important targets for development of rice blast fungicides.MBIs have low levels of resistance to drugs and off-target toxicity; MBIs are reported for the commercial fungicide targeted to 3HNR and SDase.But MBIs as non-direct antifungal inhibitors, sterilization effect is not obvious. Sterol 14a demethylase (CYP51) is an crucial enzyme in sterol biosynthesis that catalyzes the removal of the 14a-methyl group from the sterol nucleus, and a typical target for clinical and agricultural antifungal azoles.DMIs as direct antifungal inhibitors,although have high sterilization effect but also have high resistance to drugs.We hope to find a kind of antifungal inhibitors at the same time acting on SDase (3HNR) and CYP51.So that make the screening of antifungal inhibitors have advantages of MBIs but also have the advantages of DMIs,the antifungal inhibitors has the characteristic of fast and efficient resistance.This thesis were systemic researched by expression and purification technology?eny-ymatic analysis?spectral analysis and characterization techniques.For rice blast fungus MBIs and DMIs carried on the thorough research, on this basis,multi-targeted directed an-tifungal inhibitors were screened for SDase(3HNR) and CYP51.This article describes fo-llowing parts:First?Through the expression of recombinant protein,we gain the SDase and optim-um conditions for the expression of the enzyme. We use the technology of affinity chrom-atography to get the purified high activity enzyme.The study of the enzyme kinetics has been made,including the optimum Ph?temperature?the affect of the substrate.In the end, we established the enzyme activity test system.Second?Through the expression of recombinant protein,we gain the CYP51.we use the technology of differential centrifugation to get the purified enzyme.set up CYP51-CO differential spectrum method and inhibitor binding spectrum method.Thirdly?We carry out biological tests on enzyme level and thalli and then obtain the lead compounds which can be optimize in the future.