猕猴桃间座壳菌Diaporthe actinidiae的绿色荧光蛋白基因标记及侵染过程  

Green fluorescent protein gene markers and infection of Diaporthe actinidiae

作　　者：冯丹丹 邓蕾 苏琦雅 刘德江[2] 钟彩虹[1] 李黎[1] FENG Dandan;DENG Lei;SU Qiya;LIU Dejiang;ZHONG Caihong;LI Li(CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture,CAS Engineering Laboratory for Kiwifruit Industrial Technology,CAS Innovation Academy for Seed Design,Wuhan Botanical Garden,Chinese Academy of Sciences,Wuhan 430074,Hubei,China;College of Life Sciences,Jiamusi University,Jiamusi 154007,Heilongjiang,China)

机构地区：[1]中国科学院植物种质创新与特色农业重点实验室中国科学院猕猴桃产业技术工程实验室中国科学院种子创新研究院中国科学院武汉植物园,湖北武汉430074 [2]佳木斯大学生命科学学院,黑龙江佳木斯154007

出　　处：《菌物学报》2022年第3期450-458,共9页Mycosystema

基　　金：国家重点研发计划项目(2019YFD1000200);国家自然科学基金青年科学基金(31701974,31901980);长江猕猴桃产业技术研究项目(CJZX20210102)。

摘　　要：果实软腐病是猕猴桃贮藏期间最严重的真菌病害,猕猴桃间座壳Diaporthe actinidiae是该病检出率最高且致病力最强的病原菌。该病菌从花前期开始侵染,至果实贮藏期才表现症状,侵染至发病周期较长,可借助荧光标记对其侵染过程进行研究。本研究采用PEG介导原生质体转化的技术,运用携带GFP及潮霉素抗性基因的双元载体pCAMBgfp对D.actinidiae菌株进行遗传转化。首先,对转化子进行潮霉素及绿色荧光筛选,随后进行菌落的生物学特征观察、荧光稳定性验证及致病力检测,最终得到3株菌丝形态、生长速度及致病力与野生型菌株一致的稳定荧光菌株。将KFDA-17转化子接种于贮藏期猕猴桃果实‘金梅’,1 d后果实出现水渍症状,随后出现软腐症状且逐步加重;发病部分菌丝体在荧光显微镜下呈现明显的绿色荧光,可清晰观察侵染进程。本研究成功构建了PEG介导的间座壳菌丝体的原生质体遗传转化体系,且KFDA-17荧光菌株可用于侵染过程研究。Fruit soft rot is the most serious fungal disease of kiwifruit during storage period.Diaporthe actinidiae is the pathogen with the highest detection rate and the strongest pathogenicity.The pathogen invaded before florescence and showed symptoms until fruit storage.The long infection process can be studied with the help of fluorescent markers.In this study,PEG mediated protoplast transformation of D.actinidiae strain was performed by applying recombinant vector pCAMBgfp carrying GFP and hygromycin gene.The transformants were screened by hygromycin and GFP fluorescence,and then the colony morphology,stability of fluorescence and pathogenicity were analyzed.Three positive transformants showing no significant difference from wild strain in colony morphology,growth rate and pathogenicity were screened out.The transformant KFDA-17 was inoculated to kiwifruit‘Jinyan’during storage period.After inoculation,the fruit appeared watery symptoms in 1 d,and soft rot appeared and gradually accelerated.Mycelium showed obviously green fluorescent under fluorescence microscope,and the infection process can be clearly observed.The result proved that the PEG-mediated protoplast transformation of D.actinidiae was successfully constructed,and the obtained fluorescent strains KFDA-17 could be used for the study of infection process.

关 键 词：猕猴桃软腐病 荧光蛋白 遗传转化 转化子 致病力 

分 类 号：S436.634.1[农业科学—农业昆虫与害虫防治]