Format

Send to

Choose Destination
Microbiol Res. 2014 Sep-Oct;169(9-10):741-8. doi: 10.1016/j.micres.2014.01.004. Epub 2014 Feb 8.

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus.

Author information

1
College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China; National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China.
2
College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China.
3
National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China.
4
College of Life Science, Nanjing Agricultural University, No. 1, Weigang Road, XuanWu District, Nanjing 210095, China; National Research Center for Edible Fungi Biotechnology and Engineering, Key Laboratory of Applied Mycological Resources and Utilization, Ministry of Agriculture; Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, No. 1000, Jinqi Road, FengXian District, Shanghai 201403, China. Electronic address: feng_zy@yahoo.com.

Abstract

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species.

KEYWORDS:

Agrobacterium-mediated transformation; Hypsizygus marmoreus; Promoter; gpd

PMID:
24612605
DOI:
10.1016/j.micres.2014.01.004
[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center