一种优化的辣椒尖孢炭疽菌遗传转化体系

Optimization of PEG-mediated genetic transformation system of Colletotrichum acutatum HHDL02 from infected Capsicum annuum L.

由辣椒尖孢炭疽菌(Colletotrichum acutatum)侵染引起的炭疽病是辣椒生产中最具有破坏性的真菌病害,严重影响辣椒的品质和产量。本研究以辣椒尖孢炭疽病菌HHDL02为对象,采用1 mol·L^(-1)NH_(4)Cl 2%的酶裂解液裂解分生孢子萌发2 h的芽管,裂解2~3 h可以高效制备原生质体,结合PEG介导转化法成功将GFP基因导入,其转化菌株菌落形态、菌丝生长速率、分生孢子形态、孢子萌发、附着胞形成、产孢量和致病性与野生型菌株无明显差异,且后代荧光信号遗传稳定。PEG介导的原生质体转化适合辣椒尖孢炭疽菌遗传操作,有助于其致病机理的研究。

Pepper anthracnose, caused by Collectotrichum acutatum infection, is the most destructive fungal disease in pepper production, which seriously threatens yield and quality of pepper. The genetic transformation with higher efficiency for the pepper pathogen C. acutatum is still not available. Our research provided optimal conditions to obtain protoplasts with higher efficiency at 2% of the enzyme in 1 mol·L^(-1)NH_(4)Cl solution, 2 h of germination for the germ tube of conidium, and 2-3 h of incubation with application of the protoplasts, the GFP gene was successfully introduced into the wild type C. acutatum strain HHDL02 through modified PEG-mediated transformation. We further evaluated some of the transformants for colony morphology, growth rate, conidium morphology, conidial germination, appressorium formation, conidial production quantity and pathogenicity, which exhibited no obvious differences as compared to the wild type strains. In addition, visualizing the fluorescence signals of their progenies of the transformants suggested that the GFP gene integrated in C. acutatum strain HHDL02 was genetically stable. These results indicated that the protoplasts produced efficiently with the optimal approach from germ tube and the subsequent transformation mediated by PEG was suitable for the genetic operation of C. acutatum, and also helpful for the study on the mechanism of pathogenesis on pepper plants.