Current Technologies and Related Issues for Mushroom Transformation

Mushroom transformation requires a series of experimental steps, including generation of host strains with a desirable selective marker, design of vector DNA, removal of host cell wall, introduction of foreign DNA across the cell membrane, and integration into host genomic DNA or maintenance of an autonomous vector DNA inside the host cell. This review introduces limitations and obstacles related to transformation technologies along with possible solutions. Current methods for cell wall removal and cell membrane permeabilization are summarized together with details of two popular technologies, Agrobacterium tumefaciens-mediated transformation and restriction enzyme-mediated integration.

The Efficient Transformation of Pleurotus ostreatus using REMI Method

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 microg 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 microg of linearized pTRura3-2 DNA was added into 1x10(7) 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.

Genetic Transformation of Candida glycerinogenes by REMI and Electroporation / 微生物学通报

In order to isolate genes related with the osmoadaptation and glycerol metabolism of Candida glycerinogenes, a transformation system based on the dominant selectable marker Zeocin and restriction enzyme-mediated integration (REMI) was established. Effects of seven restriction enzymes on transformation efficiency of C.glycerinogenes were tested. Transformation conditions were optimized in the presence of Hind III. Under the optimal conditions of OD_ 600 ≈1.3, voltage of 1.5 kV, 2.0?10~9 competent cells/mL, 100 units of Hind III added, the transformation efficiency was up to 129 trnaformants/?g DNA. 58% of transformants were stable on nonselective medium. These results suggest that REMI technique would be beneficial to the genetic transformation of C.glycerinogenes.