Evaluation of agrobacterium-mediated transformation of Agaricus bisporus using a range of promoters linked to hygromycin resistance.
There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trp2 promoters. The Coprinus cinereus beta-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with AGL-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.
Burns
C
C
School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK.
Leach
K M
KM
Elliott
T J
TJ
Challen
M P
MP
Foster
G D
GD
Bailey
A
A
D000364:Agaricus / Q000235:genetics*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001233:Aspergillus nidulans / Q000235:genetics; D003302:Coprinus / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D006921:Hygromycin B / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic
D000900:Anti-Bacterial Agents; D006921:Hygromycin B