Specific sequence modifications of a cry3B endotoxin gene result in high levels of expression and insect resistance

Solanum melongena (eggplant) cv. Picentia and the wild species Solanum integrifolium were transformed with both a wild type (wt) and four mutagenized versions of Bacillus thuringiensis (Bt) gene Bt43 belonging to the cry3 class. The Bt gene was partly modified in its nucleotide sequence by replacing four target regions (W: +1 to +170; X: +592 to +1057; Y: +1203 to +1376; Z: +1376 to +1984) with synthetic fragments obtained by polymerase chain reaction amplification of crude oligonucleotides. The synthetic Bt genes were designed to avoid, in their modified regions, sequences such as ATTTA sequence, polyadenylation sequences and splicing sites, which might destabilize the messenger RNA. Furthermore, the codon usage was improved for a better expression in the plant system. The amino acid composition was not altered. Four versions of the modified Bt gene were obtained, BtE, BtF, BtH and BtI, with a nucleotide subtitution percentage of 8.2, 8.6, 14, and 16%, respectively, in comparison to the wt gene Bt43. Modified versions contained different subsets of substituted regions: BtE-W + Z, BtF - Y + Z, BtH-X + Y + Z, BtI - W + X + Y + Z. In the final modified version (BtI), overall guanine+cytosine was increased from the 34.1% of the wt gene to 45.5%, and most of the destabilizing sequences were eliminated. Transgenic plants obtained with the more modified versions, BtH and BtI, were fully resistant to Leptinotarsa decemlineata Say first- and third-instar larvae, while Bt43 wt, BtE and BtF genotypes did not cause mortality and did not affect larval development.