梨黑斑病菌遗传操作体系的建立与RFP标记转化子的致病性分析

由链格孢(Alternaria alternata)引起的梨黑斑病是我国梨生产上的主要病害之一,导致梨叶与果实产生黑斑症状及早期落叶,对我国梨产业的健康发展构成严重威胁。本实验在前期获得强致病性梨黑斑病菌菌株HN-5基础上,建立该病菌的遗传转化体系。通过优化原生质体制备过程以及转化体系发现梨黑斑病菌HN-5原生质体制备的最优条件为:菌丝在M R液体培养基中培养36 h;以0.7 M NaCl+0.8 M Mannitol为稳渗剂,配置复合细胞壁裂解酶(1%崩溃酶+1%裂解酶+1%纤维素酶+1%蜗牛酶),酶解菌丝3 h,原生质体的产量可达0.5×10~7个·mL~(-1)。通过PEG介导的原生质体遗传转化体系,将含有RFP基因的质粒p KD7转入链格孢菌HN-5中,转化效率为6个·μg~(-1)DNA。PCR检测和转化子荧光观察均表明RFP基因整合到了HN-5基因组中并成功表达。致病性分析发现RFP (Red Fluorescent Protein)标记转化子致病性未发生变化。本研究成功建立了PEG (Polyethylene glycol)介导的梨黑斑病菌遗传转化体系,构建了RFP标记菌株,为研究该病菌的致病机理奠定基础。

Established genetic transformation of Alternaria alternata and pathogenicity analysis of the RFP labeled transformants

Pear black spot disease is one of the major diseases of pear in China. It causes black spot symptoms and early defoliation, which seriously threatens pear production. Based on a previously obtained strain (HN-5) of Alternaria alternata with strong pathogenicity, we established a genetic transformation system of this fungus. We improved the conditions for increasing protoplast yield of HN-5 strain as below:the mycelia were cultured in MR medium for 36 hours, useing 0.7 M NaCl and 0.8 M Manitol as the osmotic stabilizer; the mycelia were digested with four enzymes mixture (1% lysing enzyme, 1% driselase, 1% cellulose, and 1% snailase) at 30℃ for 3 hours, and the protoplasts yeild was up to 0.5×10⁷·mL^-1. The plasmid pKD7 with RFP was transferred into HN-5 strain by PEG-mediated protoplast genetic transformation, and six transformants per μg DNA were obtained. Genome PCR validation and fluorescence analysis of the transformants confirmed that the RFP gene was successfully integrated and expressed into the HN-5 genome. There was no change in the pathogenicity of the RFP transformants. In this summary, we successfully established a PEG-mediated genetic transformation of A. alternata and generated the RFP transgenic strains, which laid the foundation for investigating the pathogenesis of this fungus.