玉米黑粉菌cyp51基因结构分析与克隆表达

Analysis of Gene Structure, Cloning and Expression of cyp51 from Ustilago maydis

摘要:

通过生物信息学手段分析cyp51基因结构, 并根据GenBank登记的玉米黑粉菌cyp51 DNA序列, 设计cyp51引物和两对分别截短不同跨膜区的突变体引物, 构建了多种重组表达质粒及突变体重组表达质粒。选用不同宿主菌包括Escherichia coli BL21(DE3)、BL21(DE3) pLysS和Rosetta(DE3)诱导表达并优化条件。SDS-PAGE分析结果表明: 只有突变体pET32-YH-35能够在E. coli BL21(DE3)中高效表达(30oC, 0.5 mmol/L IPTG诱导)。通过与戊唑醇等4种商品化杀菌剂农药和14种XF系列农药先导化合物的紫外结合光谱分析表明: 重组蛋白具有生物学活性。其中一种XF系列化合物的结合常数接近商品化杀菌剂, 有可能开发为新的杀菌剂, 为设计开发新型高效抗真菌新药提供了理论依据。

Abstract:

The cyp51 primers and two pairs of mutant primers which removed different transmembrane region were designed based on Ustilago maydis cyp51 gene structure analysis. The full cyp51 DNA fragment as well as mutant cyp51 genes were amplified and cloned by using the total DNA from Ustilago maydis as template, then subcloned into different expression vectors. The recombinant expression plasmids were transformed into Escherichia coli BL21 (DE3), BL21 (DE3) pLysS and Rosetta (DE3) respectively. A series of experiments leads to the finding that only pET32-YH-35 could be highly expressed at the optimal condition of 30oC induced with 0.5 mmol/L IPTG. The expressed protein (CYP51) showed biological activity by spectra analysis of the protein binding to 4 standard fungicides and to 14 XF-synthetic fungicide compounds, and only one XF-synthetic fungicide compound (XF-113) was similar to standard fungicides in binding constant. This compound is promising to be a new effective antifungal drug. These results will facilitate the further study on the mechanism of pathogenic fungi CYP51 and pesticide molecules, and will provide a new idea for efficient design and development of new anti-fungal drugs.