Agrobacterium rhizogenes-mediated transformation of a high oil-producing fil a m en to us fungusUmbelopsis isabellina

Abstract

The Agrobacterium rhizogenes-mediated transformation procedure was developed by using the hygromycin B phosphotransferase gene (hph) as a selective marker for the oil-producing fungus Umbelopsis isabellina. Different conditions were combined to increase the transformation efficiency. The highest efficiency was obtained by using A. rhizogenes strain R105 and a vector with zygomycete promoter. Southern blot analysis demonstrated that 71% of transformants contained random integrations of T-DNA sequences under optimal conditions. We randomly selected 115 positive transformants resistant to hygromycin to analyze the amount of total fatty acid and gamma-linolenic acid (GLA). Six transformants produced a higher amount of total fatty acids than the wild strain, and one transformant also produced a higher level of GLA than the wild strain in gas chromatography analysis. This is the first report about using A. rhizogenes strain R105 and germinated conidia to transform successfully the recalcitrant zygomycetes and to obtain transformants with a stable phenotype.

Agrobacterium tumefaciens-Mediated Transformation

“…The combination of A. tumefaciens strain and binary plasmids used can also affect the transformation frequency signifi cantly (Yamada et al 2009 ;Wei et al 2010 ); however, no single combination has been proven optimal for a larger number of fungal species.…”

A silver bullet in a golden age of functional genomics: the impact of Agrobacterium-mediated transformation of fungi

“…[ 206 – 209 ], which may reflect transient transformation, gene silencing or a foreign DNA surveillance system, or loss over time during passaging of these species that have coenocytic hyphae (without septa) and multinucleate spores. Umbelopsis isabellina is an interesting case, wherein standard A. tumefaciens strains used to transform fungi were compared with a strain of A. rhizogenes , which is another Agrobacterium species able to transform plants [ 210 ]. In this case, the latter bacterial species yielded higher numbers of transformants.…”

Candida parapsilosis produces prostaglandins from exogenous arachidonic acid and OLE2 is not required for their synthesis

“…After washing with PBS, yeast cells were lyophilized. The sample preparation was carried out according to Wei et al 33 with minor modifications. Approximately 100 mg of a lyophilized sample was supplemented with 100 mg heptadecanoic acid (Sigma, H3500) as internal standard (1000 mg/ml).…”