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 mu tagenized versions of Bacillus thuringiensis (Bt) gene Bt43 belonging to the cry3 class. The Bt gene was partly modified in its nucleotide s equence by replacing four target regions (W: +1 to +170; X: +592 to +1 057; Y: +1203 to +1376; Z: +1376 to +1984) with synthetic fragments ob tained by polymerase chain reaction amplification of crude oligonucleo tides. The synthetic Bt genes were designed to avoid, in their modifie d regions, sequences such as ATTTA sequence, polyadenylation sequences and splicing sites, which might destabilize the messenger RNA. Furthe rmore, the codon usage was improved for a better expression in the pla nt system. The amino acid composition was not altered. Four versions o f the modified Bt gene were obtained, BtE, BtF, BtH and BtI, with a nu cleotide substitution percentage of 8.2, 8.6, 14, and 16%, respectivel y, in comparison to the wt gene Bt43. Modified versions contained diff erent subsets of substituted regions: BtE - W+Z, BtF - Y+Z, BtH - X+YZ, 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 res istant to Leptinotarsa decemlineata Say first- and third- instar larva e, while Bt43 wt, BtE and BtF genotypes did not cause mortality and di d not affect larval development.