柑橘霉菌农药靶标酶CYP51转录调控及其抗药性分子机理研究

The fungus diseases such as green mold are most major diseases in agriculture area, which are seriously affecting the harvest and quality of farm products. Recent years, fungi resistances to pesticides increase rapidly and become one of research hotspot in studying the resistant mechanism and effctively preventive treatments. This project will continue to study on the pesticide resistant mechanism about the sterol 14α-demethylase (CYP51) of fungi (focus on Penicillium digitatum and Penicillium italicum). CYP51 is the most important target enzyme of pesticide, which are inhibited effctively by sterol demethylation inhibitors (DMIs). The isolates of P.digitatum and P.italicum (including resistant strains and sensitive strains) will be colletcted from different producing area, and their CYP51 genes and promoters will be cloned. The diversity and the relationship between CYP51 (including promoter, enhancer, site-mutant) and the fungicide resistant will be analyzed using bioinfomatics methods. Transcriptional regulation elements such as SRE (sterol regulatory element) box located in the promoter region of CYP51 will be analyzed by biological information technology. The transcription factor such as Upc2 will be cloned and expressed, and its functions will be explored. The transcriptome of the typical resistant isolate of P.digitatum(F6) will be sequenced and some new resistance genes will be selected. The cytochrome b5 of P.digitatum will be cloned and the co-resistant mechanism between CYP51 and cytochrome b5 will be investigated. The homology modeling about PdCYP51 and PiCYP51 will be constructed based on multi-homologue modeling method, and their inhibit mechanism will be studied by molecular docking and site directed mutagenesis. 3D-QSAR pharmacophore will be constructed for virtual screening and the highly efficient lead compounds will be selectively synthesized. These results will provide a theoretical basis and research base for developing new fungicides against the specific target CYP51 of fungi.This study will be benefit to illustrate the resistant mechanism and to promote the development of the new pesticides with high-efficiency and low-toxicity.

真菌病害严重影响农业丰产和农产品质量，其抗药性机理十分复杂，已成为近年来的研究热点。本项目拟开展柑橘青绿霉菌农药靶标酶CYP51转录调控及其抗药性分子机理研究。采用基因工程和定点突变技术克隆多种青绿霉菌(高抗株/敏感株)靶标酶CYP51基因和启动子区，分析其多样性；阐明CYP51基因启动子结构、增强子和点突变与抗药性的关系。生物信息技术分析CYP51启动子区转录调控元件如SRE等，研究与相关转录因子Upc2相互作用及其调控机理。解析柑橘绿霉菌高抗株F6转录组，结合表达谱差异分析筛选新的靶标和抗性基因；研究CYP51与细胞色素b5共抗性机制。应用同源模建和分子对接技术研究绿霉菌靶标酶CYP51抑制机理；虚拟筛选并选择性合成高效抑制先导化合物。该研究对阐明绿霉菌抗性机理、促进高效低毒新型农药的研制具有十分重要的意义；将为设计研发以CYP51为特异性靶标的新型杀真菌剂农药提供理论依据和研究基础。

柑橘霉菌导致的青绿霉病是柑橘贮运期间发生的最严重病害，严重影响农产品质量。研究柑橘青绿霉菌农药靶标酶CYP51基因的转录调控，对阐明绿霉菌农药抗性分子机理，促进高效低毒安全的新型农药的研制有着非常重要的意义。我们按计划圆满完成了本项目的全部研究内容。项目执行期间，通过采集不同地区柑橘青绿霉菌，筛选分离到40多株咪酰胺抗性菌株；克隆分析了柑橘绿霉菌R/S株cyp51基因和启动子区与抗药性的关系。通过柑橘青绿霉菌农药靶标酶CYP51多样性分析，着重分析了cyp51B基因启动子特征、CYP51B点突变与抗药性的关系。结合PdCYP51转录调控分析，研究了绿霉菌PdCYP51A与Cytb5共同作用抗性机制。克隆了绿霉菌甾醇调控元件结合蛋白（SREBP）家族的转录因子（SreA）基因，并研究了其调控PdCYP51的作用机理。结果证明SreA蛋白是一个新的转录因子，可调控cyp51基因的表达，且与柑橘绿霉菌对农药咪鲜胺的抗性及其对柑橘类水果的侵染毒力有关。基于绿霉菌典型高抗株HSPd-F6的转录组解析，筛选出新的抗性相关基因。发现了一个新的MFS转运蛋白基因Pdmfs2；并完成了指状青霉MFS转运蛋白基因Pdmfs2的克隆及功能研究。结合分子模拟和定点突变技术研究了真菌靶标酶CYP51抑制机理，虚拟筛选并选择性合成了一批高效抑制先导化合物。结果发现5种化合物mf8、mf14、mf25、mf30和mf40抑制效果好，具有良好的应用前景。为设计研发以CYP51为特异性靶标的新型杀真菌剂农药奠定了良好基础。达到了预期目标。同时，增加了新的研究内容。我们首次在柑橘绿霉菌发现了新型真菌病毒PdV1，对该真菌病毒进行了较为深入的研究，并取得了重要突破。研究发现真菌病毒PdV1具有降低柑橘致病菌指状青霉的生长活力及减弱其对柑橘致病力的作用，显示出对柑橘绿霉病防控具有潜在的应用前景。有关指状青霉菌中的新型病毒研究结果已在Virology杂志发表，并被评为当期封面论文。.本项目取得的研究成果已在国际著名期刊 BMC Genomics、Biotechnol Lett.和Virology等杂志发表SCI学术论文7篇，其它国内外重要学术期刊上发表论文5篇。还有部分研究成果即将发表。该项目对培养研究生起到至关重要的作用，项目执行期间，先后有10多名研究生参加，其中已毕业硕士生6名。