Transformation of Rhodosporidium toruloides |
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Authors: | M Tully H J Gilbert |
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Institution: | 2. Therapeutic Products Laboratory, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire U.K. Tel. (0980)610391;1. Department of Agricultural Biochemistry and Nutrition, The University of Newcastle upon Tyne, Newcastle, NE 1 7RU U.K. Tel. (0632)328511 ext. 2939 |
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Abstract: | Rhodosporidium toruloides protoplasts could be transformed, in the presence of polyethylene glycol (PEG), at frequencies of approx. 1 X 10(3) transformants/micrograms of DNA. The plasmid used, pHG2, which contains the phenylalanine ammonia-lyase (PAL)-coding gene (PAL) of R. toruloides, could replicate as an unstable plasmid in the yeast, or could integrate at the PAL locus to give stable transformants. Plasmids that function in R. toruloides were constructed using either the PAL gene or LEU2 gene of Saccharomyces cerevisiae as dominant selectable markers. R. toruloides transformed with pHG8, which contains both genes, coinherited the two markers. It is also shown that the 2mu replicon of S. cerevisiae does not function in R. toruloides; neither is the PAL gene expressed in S. cerevisiae. |
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Keywords: | Protoplasts yeast vectors autonomous replication sequence phenylalanine ammonia-lyase recombinant DNA Ap ampicillin bp base pair(s) EtBr ethidium bromide kb 1000 bp Km kanamycin PAL phenylalanine ammonia-lyase gene coding for PAL PEG polyethylene glycol 4000 resistance resistant sensitive sensitivity Tc tetracycline YEPD yeast extract (1 %) bactopeptone (2%) and glucose (2%) |
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