Kemppainen Minna, Circosta Ariana, Tagu Denis, Martin Francis, Pardo Alejandro G
Programa de Investigación en Interacciones Biológicas, Universidad Nacional de Quilmes, Roque Sáenz Peña 180, B1876BXD, Bernal, Provincia de Buenos Aires, Argentina.
UMR INRA-UHP 1136 Interactions Arbres/Micro-organismes, Centre INRA de Nancy, 54280, Champenoux, France.
Mycorrhiza. 2005 Dec;16(1):19-22. doi: 10.1007/s00572-005-0008-7. Epub 2005 Nov 11.
The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.
为了改变共生调节基因的表达并在外生菌根担子菌双色蜡蘑Laccaria bicolor S238N中开展插入诱变，需要开发一种高效的转化系统。通过根癌农杆菌介导的基因转移对该真菌的营养菌丝体进行转化。选择标记是在双孢蘑菇gpd启动子和CaMV 35S终止子控制下的大肠杆菌潮霉素抗性基因（hph），作为T-DNA的一部分。hph的PCR扩增和Southern印迹分析表明，潮霉素抗性转化体的基因组包含该盒式结构。后者证明大多是单拷贝的，并且转基因的一部分随机整合到真菌基因组中。根癌农杆菌介导的基因转移应有助于双色蜡蘑未来插入诱变、靶向基因破坏和RNA干扰技术的发展。
