PEG介导原生质体转化构建链格孢荧光菌株及其侵染苹果花器特性

Construction of Alternaria alternata GFP-tagged strains based on PEG-mediated protoplast transformation and its infection characters to apple floral tissues

由链格孢侵染引起的苹果霉心病是一种普遍发生的苹果果实病害。该病菌从侵染到发病的周期较长,对其侵染规律较难开展研究,若借助荧光标记则有望有效解决这些问题。本研究基于PEG介导的菌丝体原生质体转化,将带有GFP基因的外源DNA随机插入链格孢菌基因组中,共获得150个转化子。选取菌落形态、生长速率、致病力均未受影响的转化子NC-1,在田间盛花期接种苹果花组织。48h后花瓣、花柱、花药和花丝发生褐变,出现水渍状病斑,4d后发病严重的花干枯脱落,20d后感病幼果霉变脱落,发病部位菌丝体在荧光显微镜下呈现明显的绿色荧光。试验结果表明,作者成功构建了PEG介导的链格孢菌丝体原生质体遗传转化体系,且获得的链格孢荧光菌株NC-1可用于田间侵染规律的研究。

Moldy core caused by Alternaria spp.is a common fruit disease of apples.The long period of pathogen makes difficulty in observing the exact pathway of infection.The fluorescent labeling is an effective way to solve this kind of problems.Exogenous DNA carrying the GFP gene was inserted into A.alternata genome based on PEG-mediated hyphal protoplast transformation,and a total of 150 transformants was obtained.The transformant NC-1,which had no significant variation in colony morphology,growth rate and pathogenicity,was inoculated on apple floral tissues at full-bloom stage in the field.After 48 h the petals,styles,anthers and filaments turned brown and water-stained lesions appeared.After 4 d,the floral tissues dried and the flowers dropped down.After 20 d,the young fruit became moldy and fell soon.The hyphae covering the lesions were observed obviously as green fluorescence under fluorescence microscope.The result indicated that the PEG-mediated A.alternata hyphal protoplast transformation was successfully constructed,and the fluorescent strain NC-1 could be used for the study of wild infection mechanism of apple moldy core.