Development of Highly Efficient Transformation System of Yeast-Like Conidia of Tremella fuciformis |
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Authors: | GUO Li-qiong LIU Er-xian WANG Jie LIN Jun-fang |
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Institution: | 1. Department of Bioengineering, College of Food Science, South China Agricultural University, Guangzhou 510640, P.R.China;Institute of Biomass Research, South China Agricultural University, Guangzhou 510640, P.R.China 2. Department of Bioengineering, College of Food Science, South China Agricultural University, Guangzhou 510640, P.R.China |
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Abstract: | Tremella fuciformis is one of higher basidiomycetes. Its basidiospore can reproduce yeast-like conidia, which is also called the blastospore by budding. The yeast-like conidia of T. fuciformis is monokaryotic and easy to culture by submerged fermentation similar to yeast. Thus, it is a good recipient cell for exogenous gene expression. In this study, the expression plasmid pAN7-1 (containing promoter gpd-An derived from Aspergillus nidulans and selectable marker gene hph conferring resistance to hygromycin B) and plasmid pLg-hph (containing promoter gpd-Le derived from Lentinula edodes and selectable marker gene hph) were transformed into the yeast-like conidia of T. fuciformis by PEG-mediated protoplast transformation, respectively. The primary putative transformants were selected by the sandwich screening method with the selective medium containing 50 μg mL-1 hygromycin. The putative transformantswere obtained from the primary putative transformants transferred on PDSA plates containing 100 μg mL-1 hygromycin for second round selection. Experimental results showed that the optimal concentration of PEG 4000 for mediating protoplast transformation was 25%. PCR and Southern blotting confirmed that the selectable marker gene hph was integrated effectively into the genome of the yeast-like conidia of T. fuciformis with plasmid pLg-hph transformation.Its transformation efficiency was 110 transformants per μg DNA, and the hph gene was integrated into the genome of some yeast-like conidia with plasmid pAN7-1 transformation. However, its transformation efficiency was only 9 transformants per μg DNA. The presence of hph gene in the genome of transformants after 5 generations of sub-culturing on PDSB medium was confirmed by PCR, suggesting that the foreign gene hph was stable during subculture. |
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Keywords: | Tremellafuciformis PEG-mediated protoplast transformation yeast-like conidia hph gene promoter |
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