Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts
Transformation of Sclerotinia sclerotiorum with the green fluorescent protein gene and fluorescence of hyphae in four inoculated hosts
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DOI：
10.1111/j.1365-3059.2009.02022.x
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发表时间：
2009-06-01
期刊：
影响因子：
2.7
通讯作者：
Nelson, B. D.
中科院分区：
文献类型：
--
作者：
de Silva, A. P.;Bolton, M. D.;Nelson, B. D.
为了获得一个用于观察和研究核盘菌与其寄主互作的遗传标记，用pCT 74和gGFP构建体转化了核盘菌（Sclerotinia sclerotiorum）菌株ND 30和ND 21，这两个构建体都含有绿色荧光蛋白（GFP）和潮霉素B磷酸转移酶基因。使用聚乙二醇介导的原生质体转化获得推定的转化体。鉴定了7个稳定的绿色荧光蛋白转化体，并对其体外和植物体内荧光、致病性和宿主组织定殖进行了评价。实时定量聚合酶链反应检测到转化体中gfp基因的单拷贝。直接从菌丝体和从菌丝体提取的蛋白质中定量荧光。7个转化子（4个来自ND30，3个来自ND21）对干豆、油菜、大豆和向日葵具有致病性。然而，取决于宿主，与野生型相比，三个转化体在形成的病变的长度上显著不同（P = 0.05）。菌丝在植物组织中发出荧光，可与植物细胞清楚区分。用荧光显微镜可以清楚地看到组织的感染和定殖。转化体在体外和植物中GFP表达的强度不同。
To obtain a genetic marker to observe and study the interaction of Sclerotinia sclerotiorum with its hosts, isolates ND30 and ND21 were transformed using pCT74 and gGFP constructs, both containing genes for the green fluorescent protein (GFP) and hygromycin B phosphotransferase. Putative transformants were obtained using polyethylene glycol-mediated transformation of protoplasts. Seven stable gfp transformants were identified and evaluated for fluorescence in vitro and in planta, pathogenicity and colonization of host tissues. Real-time quantitative polymerase chain reaction detected a single copy of the gfp gene in transformants. Fluorescence was quantified directly from mycelium and protein extracted from hyphae. The seven transformants (four from ND30 and three from ND21) were pathogenic on dry bean, canola, soybean and sunflower. However, depending on the host, three transformants differed significantly (P = 0.05) in the length of lesions formed compared to the wild-type. Hyphae fluoresced in plant tissue and could clearly be distinguished from plant cells. Infection and colonization of tissues were clearly visible with a fluorescent microscope. Transformants differed in the intensity of GFP expression both in vitro and in planta.