title: An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.
abstract: Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species.
authors.affiliation: College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China; National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China.
authors.forename: Jin jing
authors.initials: Jj
authors.lastname: Zhang
authors.affiliation: College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Shi
authors.affiliation: National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China; National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China.
authors.forename: Yun qi
authors.initials: Yq
authors.lastname: Sun
authors.affiliation: College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China.
authors.forename: Ming wen
authors.initials: Mw
authors.lastname: Zhao
authors.affiliation: College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China.
authors.forename: Ang
authors.initials: A
authors.lastname: Ren
authors.affiliation: National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China.
authors.forename: Ming jie
authors.initials: Mj
authors.lastname: Chen
authors.affiliation: National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wang
authors.affiliation: College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China; National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China. Electronic address: feng_zy@yahoo.com.
authors.forename: Zhi yong
authors.initials: Zy
authors.lastname: Feng
mesh_terms: D000363:Agaricales / Q000235:genetics*; D016960:Agrobacterium tumefaciens / Q000235:genetics*; D015139:Blotting, Southern; D003470:Culture Media / Q000737:chemistry; D015870:Gene Expression; D005827:Genetics, Microbial / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D061848:Optical Imaging; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011523:Protoplasts / Q000502:physiology; D012641:Selection, Genetic; D013696:Temperature; D014170:Transformation, Genetic
chemical_list: D003470:Culture Media; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase
keywords: Agrobacterium-mediated transformation; Hypsizygus marmoreus; Promoter; gpd