结合光谱法在DMIs类杀真菌剂筛选中的应用

Application of Binding Spectra in DMIs Fungicide Screening

摘要:

为建立一种快捷和准确的方法用于新型杀真菌剂的筛选，以外源表达的稻瘟菌羊毛甾醇14α-去甲基化酶为靶酶，以市售烯唑醇、戊唑醇、三唑醇、三唑酮为DMIs类杀真菌剂代表，分析了靶酶活性、靶酶纯度和靶酶浓度对二者结合光谱的影响，并与生物测试结果比较分析其可靠性。结果表明靶酶的高活性、无其他P450干扰和合适的靶酶浓度是获得准确结合光谱的必要条件。烯唑醇、戊唑醇、三唑醇、三唑酮与靶酶结合常数（K,/i>_d）分别为0.143μmol/L、0.24μmol/L、0.257μmol/L、0.307μmol/L，该结果与其对稻瘟菌生长抑制能力(120h-EC₅₀)显著相关，证明结合光谱法可作为一种简便可靠的DMIs类杀真菌剂筛选方法。

Abstract:

In order to establish a fast and accurate method for novel DMIs fungicide screening, lanosterol 14α-demethylase of Magnaporthe grisea expressed in E.coli was used as target enzyme and the DMI fungicides diniconazole, tebuconazole, triadimenol and triadimefon were used as representative fungicides，the effects of enzyme activity, enzyme purity and concentration on the binding spectra were investigated. The results showed that active enzyme, elimination of interference of other P450s and proper enzyme concentration were necessary for obtaining accurate binding spectra. The K_d values of diniconazole, tebuconazole, triadimenol and triadimefon were 0.143μmol/L, 0.24μmol/L, 0.257μmol/L and 0.307μmol/L respectively, which significantly correlated to their 120h-EC₅₀ values on the growth of Magnaporthe grisea. The results indicated that the binding spectra of fungicide and lanosterol 14α-demethylase can serve as a reliable and fast method for novel fungicide screening.