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PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola
被引:44
作者:
Amey, RC
Athey-Pollard, A
Burns, C
Mills, PR
Bailey, A
Foster, GD
机构:
[1] Univ Bristol, Sch Biol Sci, Bristol BS8 1UG, Avon, England
[2] Hort Res Int, Warwick CV35 9EF, England
来源:
MYCOLOGICAL RESEARCH
|
2002年
/
106卷
关键词:
D O I:
10.1017/S0953756201005251
中图分类号:
Q93 [微生物学];
学科分类号:
071005 ;
100705 ;
摘要:
Verticillium funigicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (beta-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per mug DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at -80 degreesC in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V.fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.
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页码:4 / 11
页数:8
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