辣椒胶孢炭疽病菌Cytb基因对唑菌酯敏感性差异的调控机理

Chemical prevention is the main strategy to control Colletotrichum spp. which causing anthracnose of Capsicum annuum L.. The significant differentiation of sensitivity of C.g against Pyraoxystrobin was determined in preliminary research. The target genes of Pyraoxystrobin, Cytochrome b(Cytb) were cloned and analyzed. The alignment of the amino acid sequences revealed that the sensitivity differentiation may caused by the differences of six sites. However, the mechanism of differentiation sensitivity to Pyraoxystrobin which regulated by Cytb gene of C.g is still ambiguous. Therefore, we will use gene mutagenesis, homologous recombination and genetic transformation to construct mutant strains of C.g in this study. Combining with the analysis of biological characteristics and sensitivity differentiation beteween mutant strains, we will figure out the critical site of Cytb gene which impacting sensitivity decline. Meantime, we will illuminate the regulation mechanism on differentiation sensitivity to Pyraoxystrobin by Cytb gene of C.g by exploring the coordinated regulate relationship of the critical and other sites of Cytb gene to sensitivity. The expected result would provide theoretical basis for management of fungicide resistance and scientific medicate of Colletotrichum species to strobilurin fungicide in the field.

化学防治是辣椒炭疽病防控的主要方法。前期研究发现辣椒胶孢炭疽病菌（Colletotrichum gloeosporioides, C. g）对唑菌酯敏感性存在显著差异，作用靶标基因细胞色素b（Cytochrome b，Cytb）序列比对分析发现Cytb基因6个位点突变与敏感性差异存在关联，但对于其调控唑菌酯敏感性差异的作用机制不明确。因此，本项目以辣椒胶孢炭疽病菌（C. gloeosporioides）为研究对象，拟通过基因突变、同源重组敲除和遗传转化等技术，构建位点突变菌株，分析突变菌株的生物学特性和唑菌酯的敏感性差异，明确引起供试菌株敏感性下降的关键位点；分析关键位点与其他位点对敏感性的协同调控作用；阐明C. g Cytb基因对唑菌酯敏感性差异的调控机理。预期研究结果为田间辣椒炭疽病菌对甲氧基丙烯酸酯类杀菌剂抗药性治理和科学用药提供理论依据。

本研究以不同种辣椒炭疽病菌对唑菌酯的敏感性差异为基础，克隆并比对分析唑菌酯作用辣椒炭疽病菌靶标基因Cytb氨基酸序列，推测辣椒胶孢炭疽病菌C. gloeosporioides菌株的Cytb基因第29～31位、63位、93位、124～126位、130位和180位点可能与其对化学药剂的敏感性差异存在关联。首先利用PEG介导的原生质体转化方法把含GFP序列的DNA片段转入到辣椒炭疽病菌中，并采用继代遗传学和Southern blot法验证了GFP转化菌株的遗传稳定性，成功建立了高效、稳定的辣椒胶孢炭疽菌遗传转化体系。然后借助分子生物学、PEG介导的原生质体转化方法、基因定点突变等技术构建Cytb基因位点单点突变菌株。测定单点突变菌株生物学特性和对唑菌酯的敏感性差异，发现其对唑菌酯的敏感性减低，而且发现不同突变位点菌株对其敏感性的贡献不一样，其中氨基酸29~31位和124~126位的突变贡献最大，初步明确这两个氨基酸位点对其负责。研究结果旨在为田间辣椒炭疽病菌对甲氧基丙烯酸酯类杀菌剂抗药性治理和科学用药提供理论依据。