The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants

Abstract

A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants. The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t. cry genes. We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs. For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs. We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts. CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene. Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter. Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays. Egg masses which produced ca. 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes. No CPB larvae developed past the second instar on any of these plants. Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae. We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.

Genetic Transformation, Recovery, and Characterization of Fertile Soybean Transgenic for a Synthetic Bacillus thuringiensis cryIAc Gene

“…Since plants do not have many of the tRNAs required by the bacterial gene codons, transgenic plants often express these native genes at low levels and do not kill insects that feed on them (Adang et al, 1993). For this reason, there has been much interest in the use of "codon-optimized" synthetic Bt genes for transgenic plant insect control (Perlak et al, 1990;Adang et al, 1993;Koziel et al, 1993). For example, the synthetic Bt crylII transcript constituted more than 0.1% of the total mRNA in transgenic potato, compared with undetectable amounts using the native gene (Adang et al, 1993).…”

Actividad biológica de <i>Bacillus thuringiensis</i> sobre la polilla guatemalteca de la papa, <i>Tecia solanivora</i> Povolny (Lepidoptera: Gelechiidae)

“…Insecto (Adang et al, 1993). En el mismo año se introdujo el gen cry3A en la variedad de papa Russet Burbank para protegerla del mismo coleóptero plaga (Perlak et al 1993).…”

A plant-based oral vaccine to protect against systemic intoxication by Shiga toxin type 2

“…First, the bacterial Stx2 toxoid-encoding sequences were modified, or plant-optimized (indicated by subscript PO), to eliminate sequences that might be recognized as eukaryotic mRNA termination sites, splice sites, or polyadenylation sequences in NT-1 cells and thus alter antigen expression (18)(19)(20). Three such silent mutations were introduced into the toxoid A subunit gene to yield stxA 2PO .…”