借助ITS序列用GFP标记巴西橡胶树棒孢霉落叶病病原菌

GFP as a Marker in Corynespora cassiicola from Hevea brasiliensis Using ITS Sequence

由多主棒孢菌(Corynespora cassiicola)侵染引起的巴西橡胶树棒孢霉落叶病是影响橡胶树产胶量的主要病害之一。为了开展该病原菌在活体叶片中的病理学研究,本实验将绿色荧光蛋白gfp基因表达盒插入了多主棒孢菌经定点突变后的ITS序列中,构建了重组质粒p ITGH,并经PEG介导转化了原生质体,获得了GFP标记的多主棒孢菌转化子,所获得的转化子经过连续7次转接培养后仍能在分生孢子、分生孢子梗、芽管和菌丝中稳定地发出强烈的绿色荧光,其生长特性和致病性与野生菌株无明显差异。

Corynespora leaf fall（CLF） disease, caused by the fungal pathogen Corynespora cassiicola, is a major disease of rubber tree（Hevea brasiliensis） that affects the production of natural rubber. The objective of this paper was to provide desired materials for studing pathogenesis on living leaves. The green fluorescent protein（GFP）-expression vector p ITGH was constructed by inserting a gfp cassette into r DNA-ITS of site-directed mutagenesis.C. cassiicola protoplasts were transformed by p ITGH using a PEG-mediated method. A GFP-tagged transformat was gained. The transformation was efficient, and GFP expression remained stable for at least seven subcultures with fluorescence clearly visible in both the hyphae and spores. The transformed isolate also retained its pathogenicity and growth pattern, both similar to those of wild type. This result would be useful for understand biology, observing infection process, predicting the CLF disease and evaluating resistance of H. brasiliensis to C.cassiicola by means of leaf colonization studies.