Enhancement of protein expression in Hypsizygus marumoreus by multiple Agrobacterium tumefaciens-mediated transformation
Date Issued
2012
Date
2012
Author(s)
Cheng, Chih-Jui
Abstract
Mushroom molecular farming recently attracts tremendous attention because of its application pontentials and the advantages over plant molecular farming. Agrobacterium tumefaciens-mediated transformation (ATMT) is commonly used in mushroom transformation but its application was limited due to the low heterologous gene expression. In this study, Hypsizygus marumoreus was chosen as the expression host and was transformed by multiple ATMT in order to enhance heterologous protein expression. For the first step of multiple ATMT, A. tumefaciens harboring p0390-Cbx-Hiegfp, a Ti-plasmid contains carboxin resistance gene (cbxr) and enhanced green fluorescent protein (egfp), was used for transformation. The single transformants were re-transformed by A. tumefaciens harboring p0390-AH-Aiegfp, which contains hygromycin phosphotransferase (hph) and egfp, or re-transformed by A. tumefaciens harboring p0390-Cbx-Hiegfp. Finally, EGFP was analyzed by ELISA and the copy number of egfp was determined by real-time PCR. This study demonstrated that the heterologous gene expression was enhanced by multiple ATMT in H. marumoreus. The highest EGFP production in two-vector double transformants was 418.83 ng/g TSP (total soluble protein), which increased up to 5.5 folds in comparison with its parental single transformant. In one-vector double transformants, the highest EGFP production was 319.58 ng/g TSP, which raised to 4.25 folds and the transgene copy number accordingly increased, too. Multiple ATMT described in this study provides a new approach in improvement of the heterologous gene expression.
Subjects
molecular farming
Agrobacterium tumefaciens-mediated transformation
Hypsizygus marumoreus
multiple transformation
Type
thesis
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