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A colonized millet grain method for Agrobacterium-mediated transformation of the button mushroom Agaricus bisporus

文献类型： 外文期刊

作者： Liu, Jianyu 1 ; Song, Chunyan 1 ; Li, Qiaozhen 1 ; Xu, Zhen 1 ; Zhang, Dan 2 ; Zhang, Meiyan; Tan, Qi; Shang, Xiaod 1 ;

作者机构： 1.Shanghai Acad Agr Sci, Natl Res Ctr Edible Fungi Biotechnol & Engn, Key Lab Appl Mycol Resources & Utilizat,Minist Ag, Shanghai Key Lab Agr Genet & Breeding,Inst Edible, 1000 Jinqi Rd, Shanghai 201403, Peoples R China

2.Shanghai Acad Agr Sci, Natl Res Ctr Edible Fungi Biotechnol & Engn, Key Lab Appl Mycol Resources & Utilizat,Minist Ag, Shanghai Key Lab Agr Genet &

关键词： Agaricus bisporus; Agrobacterium-mediated transformation; Foxtail millet, hygromycin B; Enhanced green fluorescent protein

期刊名称：JOURNAL OF MICROBIOLOGICAL METHODS （ 影响因子：2.363； 五年影响因子：2.24 ）

ISSN： 0167-7012

年卷期： 2018 年 152 卷

页码：

收录情况： SCI

摘要： Lack of an efficient transformation system has hampered the molecular breeding of Agaricus bisporus. Here, we describe a highly efficient Agrobacterium-mediated transformation system for A. bisporus using foxtail millet (Setaria italica L. Beauv) grains as cultivation and infection media. Mycelium-millet complexes were prepared for co-culture and treated with ultrasonication for 10 s to improve infection efficiency. After a 72-h culture period, the newly grown mycelium-surrounded millet grain was transferred to selection medium supplemented with 200 mu g/mL cefotaxime and 15 mu g/mL hygromycin B (hyg) to screen positive transformants. Putative transformants were analyzed for the presence of the hyg gene by polymerase chain reaction and Southern blotting. Expression of eGFP in A. bisporus transformants was detected by fluorescence imaging, and the D-glucuronidase (GUS) protein was detected by histochemical staining. Our protocol resulted in an average 53.85% transformation frequency, and over 85% of the transformants tested remained mitotically stable, even after five successive rounds of subculturing. A feasible method for A. bisporus mushroom transformation using foxtail millet as an innovative culture medium was developed, which will benefit future functional genetic studies of this mushroom.

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