The Efficient Transformation of Pleurotus ostreatus using REMI Method

Abstract

Restriction enzyme-mediated integration (REMI) was used to transform uracil auxotrophs of Pleurotus ostreatus to prototrophy. When protoplasts of Pleurotus ostreatus were treated by the reaction mixture containing 10 units of BamHI, the frequency of REMI was about 64 transformants per 1 μg of DNA. This efficiency was increased by 14.2 times compared with that of the conventional PEG transformation. The optimal condition for REMI of P. ostreatus was achieved when 1 μg of linearized pTRura3-2 DNA was added into 1×10⁷ protoplasts along with 10 units BamHI. Southern blot analysis revealed that about 50% of transformants examined were caused by REMI event and 30% carried single copy insertion at the genome. This suggested that the REMI method might be a useful tool for efficient transformation and tagging mutagenesis of P. ostreatus.

Keywords::

• Pleurotus ostreatus
• Restriction enzyme-mediated integration (REMI)
• Uracil auxotrophic marker