Synthetic cryIIIA gene from Bacillus thuringiensis improved for high expression in plants.
A 1974 bp synthetic gene was constructed from chemically synthesized oligonucleotides in order to improve transgenic protein expression of the cryIIIA gene from Bacillus thuringiensis var. tenebrionis in transgenic tobacco. The crystal toxin genes (cry) from B. thuringiensis are difficult to express in plants even when under the control of efficient plant regulatory sequences. We identified and eliminated five classes of sequence found throughout the cryIIIA gene that mimic eukaryotic processing signals and which may be responsible for the low levels of transcription and translation. Furthermore, the GC content of the gene was raised from 36% to 49% and the codon usage was changed to be more plant-like. When the synthetic gene was placed behind the cauliflower mosaic virus 35S promoter and the alfalfa mosaic virus translational enhancer, up to 0.6% of the total protein in transgenic tobacco plants was cryIIIA as measured from immunoblot analysis. Bioassay data using potato beetle larvae confirmed this estimate.
Sutton
D W
DW
Plant Genetic Engineering Laboratory, New Mexico State University, Las Cruces 88003.
Havstad
P K
PK
Kemp
J D
JD
D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000633:toxicity; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay; D015152:Blotting, Northern; D003001:Cloning, Molecular / Q000379:methods; D001517:Coleoptera / Q000187:drug effects; D004731:Endotoxins; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D006460:Hemolysin Proteins; D007814:Larva; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014026:Nicotiana / Q000235:genetics*; D014158:Transcription, Genetic
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D012333:RNA, Messenger; C030956:insecticidal crystal protein, Bacillus Thuringiensis