Isolation of GPD promoter from Tremella fuciformis and driving expression of EGFP gene.
Glyceraldehyde-3-phosphate dehydrogenase (GPD) cDNA was cloned by RT-PCR using total RNA from Tremella fuciformis as template with a pair of degenerate primers. Then, a 500-bp 5'-upstream promoter region of the gene encoding GPD from T. fuciformis genomic DNA was isolated by thermal asymmetric interlaced PCR. The cloned promoter was fused to 5'-upstream of enhanced green fluorescent protein gene to construct T. fuciformis expression vector pCB-TEGFP with hygromycin gene as a selectable marker. Electroporation was performed to transfer plasmid DNA of pCB-TEGFP into yeast-like conidia from T. fuciformis. Molecular evidence, including PCR analysis, fluorescence detection, fluorescence spectra assay, and SDS-PAGE, indicated that the EGFP gene had been integrated into the genome of transgenic T. fuciformis strains and was expressed successfully. The results also showed that this promoter could be used to carry out regulated expression of heterologous gene products in T. fuciformis.
Sun
Shu-Jing
SJ
Mycological Research Center, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, People's Republic of China.
Chen
Dong-Xing
DX
Xie
Bao-Gui
BG
Hu
Fang-Ping
FP
Zheng
Jin-Gui
JG
D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics*; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D020218:Response Elements / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013172:Spores, Fungal / Q000235:genetics / Q000378:metabolism; D016385:TATA Box / Q000235:genetics; D014170:Transformation, Genetic
D018076:DNA, Complementary; D005656:Fungal Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases