禾谷镰孢菌Fusarium graminearum CYP51与DMIs类杀菌剂结合的分子机理研究

Fusarium head blight (FHB) was a destructive disease affecting global wheat production and the reduction in quantity and quality of grain had adversely impacted upon the agricultural economy of small-grain-producing regions. The disease was associated with several Fusarium spp., but Fusarium graminearum was the prevalent species in many regions. The resistance of Fusarium graminearum to carbendazim, which had been relied on to control FHB in China for many years, had appeared and become so serious that the fungicides had lost its value gradually. Management of FHB in some regions of China and many other countries hinged now on the performance of sterol demethylation inhibitors (DMIs), which inhibited the biosynthesis of ergosterol in fungi due to sterol 14a-demethylase (CYP51) binding. Genomic studies revealed the existence of three paralogous cyp51 genes in F. graminearum, but to date, it was not clear that which CYP51 was chose as the target of DMIs, and the binding domain on CYP51 of DMIs was unknown also. In this study, in order to identify the CYP51 subtype that DMIs targeted, spectral analysis was used to gain the binding constant (Kd) in vitro between CYP51 isotype and DMIs after the cyp51 genes were expressed and purified in Escherichia coli. The binding domain was forecasted by homology modeling and molecular docking. The results of the study would reveal CYP51 active site structure of DMIs binding, and lay the theoretical foundation for research and development of new fungicide with CYP51 as the target.

由禾谷镰孢菌复合种（Fusarium graminearum species complex）引起的小麦赤霉病为害全球粮食生产，其防治关系国计民生。真菌细胞色素P450甾醇14α脱甲基酶(P450-14DM，CYP51)是脱甲基酶抑制剂类杀菌剂（DMIs）的作用靶标。鉴于多菌灵抗性问题日趋严重，DMIs在小麦赤霉病防治药剂上所占份额越来越大。禾谷镰孢菌体内有三个同源的CYP51，目前尚无直接证据表明DMIs作用的CYP51亚型，对DMIs在CYP51上的结合区域也是未知。本项目拟采用原核表达、蛋白纯化、光谱分析技术研究禾谷镰孢菌三个CYP51同DMIs体外结合的差异，确认DMIs的作用靶标；采用同源模建及分子对接，预测DMIs在CYP51上的结合区域（特别是氨基酸残基的结合位点）。研究结果将阐明CYP51活性部位结构与DMIs结合的关系，为研发以CYP51为靶酶的新型杀菌剂奠定理论基础。

小麦赤霉病是由禾谷镰孢菌复合种（Fusarium graminearum species complex）引起的一种流行性病害，它不仅造成小麦产量上的损失，而且感病籽粒中的脱氧雪腐镰刀菌烯醇(DON)等毒素，对食品安全和人畜健康造成严重危害。我国对小麦赤霉病的防治一直采用小麦扬花灌浆期喷施多菌灵来进行化学防治，但目前在江浙地区，赤霉病菌已对多菌灵产生了严重的抗药性，且抗药性群体有向邻近省份扩散的趋势，生产中急需储备防治赤霉病的杀菌剂。真菌细胞色素P450即甾醇14α脱甲基酶(P450-14DM，CYP51)是脱甲基酶抑制剂类杀菌剂（DMIs）的作用靶标，而禾谷镰孢菌体内有三个同源的CYP51，即CYP51A、CYP51B和CYP51C。本项目在前期测定河南省小麦赤霉病菌对戊唑醇、多菌灵及咯菌腈敏感性的基础上，研究了戊唑醇对多菌灵敏感和抗药性菌株菌丝生长、孢子萌发、产孢及芽管伸长的影响；采用原核表达方法在大肠杆菌体内成功表达了N端跨膜区缺失的CYP51三个亚基的蛋白，并通过改变诱导条件获得了可溶性的融合蛋白；采用结合光谱法研究了戊唑醇同三个亚基的体外亲和常数。结果表明：河南省小麦赤霉病菌对戊唑醇、咯菌腈及多菌灵的敏感性基线为0.0414、0.0070及0.4637 μg/mL；所有测试菌株对三种药剂均表现为敏感；戊唑醇可导致多菌灵敏感和抗药性菌株的菌丝畸形、分枝增多，造成芽管畸形，但戊唑醇对产孢及孢子萌发速率影响较弱；N端跨膜区缺失CYP51在37度常诱导条件下，融合蛋白多以包涵体形式存在；通过改变诱导条件，OD600为0.2，诱导温度为20○C，IPTG浓度为0.01~0.05 mmol/L，诱导时间为12h，获得了可溶性蛋白，并通过亲和层析纯化；结合光谱分析表明：戊唑醇在体外同CYP51A有较强的亲和力，其次为CYP51C，而同CYP51B的体外亲和力较弱。上述研究结果阐明了禾谷镰孢菌CYP51亚基与戊唑醇结合的关系，为研发以CYP51为靶酶的新型杀菌剂奠定了理论基础。