Romaine C Peter, Schlagnhaufer Carl
Department of Plant Pathology, The Pennsylvania State University, University Park, PA, USA.
Methods Mol Biol. 2006;344:453-63. doi: 10.1385/1-59745-131-2:453.
We have devised an easy and effective genetic transformation method for the preeminent edible mushroom, Agaricus bisporus. Our method exploits the T-DNA transfer mechanism in Agrobacterium tumefaciens and relies on the reproductive fruiting body as the recipient tissue. The use of fruiting body explants, particularly the gill, provided high-frequency transformation, overcoming the inefficacy of Agrobacterium-based methods targeting fungal spores or vegetative mycelium. The protocol entails incubation of A. tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin. Between 7 and 28 d on the selection medium, upwards of 95% of the gill explants generate hygromycin-resistant colonies. About 75% of the mushroom transformants show a single-copy of the hygromycin-resistant gene integrated at random sites in the genome.
我们为著名的食用蘑菇双孢蘑菇设计了一种简便有效的遗传转化方法。我们的方法利用了根癌农杆菌中的T-DNA转移机制，并以生殖子实体作为受体组织。使用子实体外植体，特别是菌褶，可实现高频转化，克服了基于农杆菌靶向真菌孢子或营养菌丝体方法的低效问题。该方案包括用乙酰丁香酮（一种启动基因转移机制的信号分子）孵育根癌农杆菌3小时，将诱导的细菌与菌褶外植体共培养3天，以及基于对潮霉素的遗传抗性筛选转化体。在选择培养基上培养7至28天之间，超过95%的菌褶外植体产生潮霉素抗性菌落。约75%的蘑菇转化体显示潮霉素抗性基因的单拷贝随机整合在基因组中的位点上。
