Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants.
The expression of the modified gene for a truncated form of the cryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein from Bacillus thuringiensis var. kurstaki (B.t.k.) HD73, under control of the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit ats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that the ats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase in cryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the same cryIA(c) gene. Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein. Furthermore, the CaMV-En35S promoter can be used with the Arabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein. While subcellular fractionation revealed that the truncated CryIA(c) protein fused to the ats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast. The results reported here provide new insight into the role of 5' untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.
Wong
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EY
Monsanto Company, St. Louis, MO 63167.
Hironaka
C M
CM
Fischhoff
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DA
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics; D001483:Base Sequence; D004731:Endotoxins; D005822:Genetic Vectors; D006460:Hemolysin Proteins; D007915:Lepidoptera; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D030821:Plants, Genetically Modified; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D012333:RNA, Messenger; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D012273:Ribulose-Bisphosphate Carboxylase