Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus.
Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Pogpd) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Pogpd-P2 fragment (795 bp) drove enhanced green fluorescence protein (egfp) gene expression in P. ostreatus much more efficiently than full-length Pogpd-P1. Further truncating Pogpd-P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between -356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application.
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Chaomin
C
Yin
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Liesheng
L
Zheng
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Jihong
J
Zhu
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Liguo
L
Chen
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China Key Laboratory of Agro-Microbial Resources and Utilization, Ministry of Agriculture, Wuhan 430070, China aiminma@mail.hzau.edu.cn.
Aimin
A
Ma
D015153:Blotting, Western; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis*
D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
Agrobacterium tumefaciens; oyster mushroom; transformation