ENHANCED EXPRESSION OF THE BACILLUS-THURINGIENSIS CRY9AA2 GENE IN TRANSGENIC PLANTS BY NUCLEOTIDE-SEQUENCE MODIFICATION CONFERS RESISTANCE TO POTATO-TUBER MOTH

The Bacillus thuringiensis cry9Aa2 gene encodes a 129 kDa protein with insecticidal activity against Lepidoptera, including the larvae of po tato tuber moth (Phthorimaea operculella). The insecticidal moiety of Cry9Aa2 resides within the N-terminal 665 amino acids. Site-directed m utagenesis was used to modify a truncated version of the gene (cry9Aa2 (T) nucleotides 1-1995), removing motifs likely to be deleterious to f ull-length transcription and transcript stability in plants. The codon usage of the gene was also altered to that more similar to the codon bias of dicotyledonous plane genes. The native gene and three modified versions of cry9Aa2T, with incremental modifications from the 5' end, were each transformed into Nicotiana tabacum, under the control of th e CaMV 35S promoter. Plants transformed with the native gene did not s how resistance to potato tuber moth larvae. In contrast, significant l evels of larval mortality and reductions in larval growth and leaf dam age were observed on many of the plants transformed with the modified genes. The cry9Aa2(T) mRNA was barely detectable in plants transformed with the native gene, whereas significant accumulation of full-length cryoAa2(T) transcript was seen in plants transformed with modified ge nes. Modifications in the 5'-terminal 693 nucleotides of cry9Aa2(T) ha d the most significant effect on increasing the steady-state levels of cry mRNA. Transcription initiation rates of both the native and modif ied cry9Aa2T genes were similar, suggesting that the lack of native tr anscript accumulation was a consequence of transcript instability and that the sequence modifications had significantly improved the stabili ty of the cry9Aa2(T) transcript. This improvement in steady-state full -length transcript levels resulted in expression of the insecticidal g ene in N. tabacum to levels which conferred significant resistance to potato tuber moth larvae.