A reliable transformation method and heterologous expression of β-glucuronidase in Lentinula edodes
Resource
Journal of Microbiological Methods 72(2):111-115
Journal
Journal of Microbiological Methods
Journal Volume
72
Journal Issue
2
Pages
111-115
Date Issued
2008-02
Date
2008-02
Author(s)
Kuo, C.-Y.
Abstract
A simple and reliable mushroom transformation procedure based on electroporation of basidiospores or mycelial fragments was developed. This method eliminated the problem of protoplast preparation, the transformation efficiency were 30-150 transformants per mug DNA and the hygromycin resistant marker gene and gus were expressed in Lentinula edodes successfully. No false positive antibiotic-resistant cultures were detected by PCR amplification and the beta-glucuronidase (GUS) expression was maintained stable during mitotic cell division without selection pressure for more than 6 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. Using the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter with the first intron of gpd gene, the average GUS activity in L. edodes reached 144.6+/-3.9 U mg(-1) soluble protein, while only 30.1+/-0.7 U mg(-1) soluble protein was detected for those without the intron. The percentage of GUS in total soluble protein was 5.67x10(-4) (0.06%) for the transformant with the highest GUS activity. This rapid and convenient electroporation procedure offers a new approach for the genetic manipulation and tool to tag genes of important edible mushroom species.
Subjects
Electroporation
β-glucuronidase
Heterologous expression
Lentinula edodes
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