Agrobacterium tumefaciens mediated fused egfp-hph gene expression under the control of gpd promoter in Pleurotus ostreatus.
A transformation system for the basidiomycete Pleurotus ostreatus was established using agrobacterium-mediated infection. Following P. ostreatus glyceraldehyde-3-phosphate dehydrogenase gene analysis, its promoter region including two introns was used as cis-regulatory element to drive expression of enhanced green fluorescent protein (eGFP). As a selection marker, the hygromycin phosphotransferase (hph) gene cassette was used in the binary vector pPEH. Mycelia without pretreatment were found to be the most efficient recipients in transformation experiments while fruiting body tissue or basidiospores showed lower transformation rates. A transformation efficiency of 75% was achieved. After subculturing, putative transformants were screened by PCR and Southern blot analysis showing the expected ectopic integration of the transforming DNA. At the same time, the promotor region was shown to drive expression of selection marker as well as eGFP that could be visualized, which will be helpful for future investigation using Agrobacterium tumefaciens mediated transformation for functional characterization of genes in the mushroom forming basidioymcete P. ostreatus.
Ding
Yi
Y
College of Food Science and Technology, Huazhong Agricultural University, 1 Lion Hill Road, Wuhan 430070, China.
Liang
Shen
S
Lei
Jinghang
J
Chen
Liguo
L
Kothe
Erika
E
Ma
Aimin
A
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D020076:Pleurotus / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase