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 μg 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 β-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 5.67 × 10 − 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.
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