Design and cloning of a synthetic gene for the flounder antifreeze protein and its expression in plant cells.
A synthetic gene coding for the winter flounder antifreeze protein (AFP) has been constructed. A new strategy for the synthesis has been employed such that one strand of the duplex was chemically synthesized and the other was produced enzymatically by chain extension. The chemically synthesized blocks were constructed so that the second strand was self-priming. The resulting DNA fragment was incorporated into the vector, pGCS1, which contained a translational fusion of the sequence encoding AFP and the N terminus of cat (encoding chloramphenicol acetyltransferase, CAT), under the control of the cauliflower mosaic virus 35S promoter. This plasmid was introduced into protoplasts of corn (var. Black Mexican Sweet) by electroporation. Production of the fusion peptide was monitored by CAT assay and Western blotting with antisera to AFP and CAT.
Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan.
F
F
Georges
M
M
Saleem
A J
AJ
Cutler
D000818:Animals; D021301:Antifreeze Proteins; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003001:Cloning, Molecular; D005432:Flounder / Q000235:genetics*; D005615:Freezing; D015870:Gene Expression; D005796:Genes; D006023:Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D011523:Protoplasts / Q000378:metabolism; D014158:Transcription, Genetic
D021301:Antifreeze Proteins; D006023:Glycoproteins; D015500:Chloramphenicol O-Acetyltransferase