元件ChTE1调控希金斯刺盘孢对咪鲜胺抗性的分子机理

Anthracnose disease, caused by Colletotrichum higginsianum Sacc., is a destructive disease on crucifers, including Brassica rapa var. parachinensis (Baily) Hanelt, in South China. Prochloraz, a sterol demethylation inhibitor (DMI), is the major fungicide that is widely used to control crucifer anthracnose disease. Results of our previous field investigations showed that resistance in C. higginsianum to prochloraz is beginning to emerge in South China. However, rare researches about molecular mechanisms of resistance of Colletotrichum species to DMIs were reported previously. Results of our previous study showed that ChCYP51A gene was over expressed in prochloraz-resistant isolates. Insertion of a novel transposable-like element, namely ChTE1, was found at the upstream of ChCYP51A gene in the prochloraz-resistant isolate but not in prochloraz-sensitive isolates. There were predicted promoters and transcription factor binding sites in the element ChTE1. These results indicated that ChTE1 might regulate the expression of ChCYP51A, which could cause prochloraz resistance of C. higginsianum. In this research, firstly, the biological significance of overexpression of ChCYP51A gene in prochloraz-resistant isolate of C. higginsianum will be evaluated. Secondly, biological functions of the element ChTE1 will be proved by analyzing characteristics of ChTE1-knocking out and ChTE1-knocking in transformants. Then, the promoter activity and active region of ChTE1 will be demonstrated genetically by expression of geneticin resistant gene NptII in prochloraz-sensitive isolate of C. higginsianum using full length or series of deletions of ChTE1 as promoter. Prochloraz-resistance-related transcription factors will be screened by yeast one-hybrid assays. The function of ChTE1 in regulation of ChCYP51A gene expression will be evaluated by verifying binging of the transcription factor with the core region of ChTE1. Finally, molecular mechanisms of resistance of Colletotrichum higginsianum to prochloraz regulated by the element ChTE1 will be demonstrated. This research will provide scientific basis for chemical control of anthracnose diseases.

希金斯刺盘孢侵染引起的炭疽病是华南地区十字花科蔬菜的重要病害，DMI杀菌剂咪鲜胺是防治该病害的主要药剂。田间监测发现希金斯刺盘孢已对咪鲜胺产生抗性，而前人对炭疽病菌DMI抗性产生的分子机理报道较少。我们研究发现抗性代表菌株的ChCYP51A基因存在超量表达，其启动子区插入了一个新的DNA转座子类似元件ChTE1，预测该元件含多个转录因子结合位点，推测其可能通过参与调控ChCYP51A表达引起咪鲜胺抗性。本项目首先对ChCYP51A基因超量表达，明确其超量表达的生物学意义；进而对ChTE1敲除和插入，探明其生物学功能；利用ChTE1调控NptII基因表达，探究其启动子活性与核心区；采用酵母单杂交筛选与核心区互作的转录因子，验证转录因子与核心区结合，从而解析ChTE1在ChCYP51A基因表达调控中的功能。最终阐明ChTE1调控希金斯刺盘孢对咪鲜胺抗性的分子机理，为炭疽病化学防治提供科学依据。

希金斯刺盘孢（Colletotrichum higginsianum）侵染引起的炭疽病是华南地区十字花科蔬菜的重要病害，生产上长期不科学使用化学农药导致病菌产生抗药性，严重威胁蔬菜生产安全和质量安全。本项目主要研究内容包括希金斯刺盘孢多重抗药性菌株分析、ChCYP51A基因超量表达的生物学意义研究、基因ChCYP51A、ChCYP51B及元件ChTE1的生物学功能研究、希金斯刺盘孢对咪鲜胺抗性产生的调控机理研究等方面。研究结果发现田间希金斯刺盘孢咪鲜胺抗性菌株具多重抗药性特征，希金斯刺盘孢咪鲜胺抗性产生受复杂机制调控，仅ChCYP51A基因过表达无法直接导致希金斯刺盘孢对咪鲜胺产生抗药性，ChCYP51A基因和ChTE1元件为希金斯刺盘孢菌丝生长必需基因，ChCYP51B基因与希金斯刺盘孢菌丝生长及其对咪鲜胺抗药性密切相关，跨膜转运蛋白和类固醇生物合成相关基因上调表达是导致希金斯刺盘孢对咪鲜胺抗性产生的关键分子机制。本研究为作物炭疽病化学防治及炭疽病菌抗药性治理提供科学依据。项目实施3年来，累计发表SCI论文2篇，授权国家发明专利1件和计算机软件著作权1项。