A reliable transformation method and heterologous expression of beta-glucuronidase in Lentinula edodes.
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 5.67 x 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.
Kuo
Chun-Yi
CY
Institute of Microbiology and Biochemistry, National Taiwan University, No. 1 Section 4 Roosevelt Road, Taipei 106, Taiwan.
Huang
Ching-Tsan
CT
D015139:Blotting, Southern; D004271:DNA, Fungal / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D025282:Mycelium / Q000235:genetics; D011401:Promoter Regions, Genetic; D020942:Shiitake Mushrooms / Q000201:enzymology / Q000235:genetics*; D013170:Spores / Q000235:genetics; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005966:Glucuronidase