Enhanced expression of the shape 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 potato tuber moth (Phthorimaea operculella). The insecticidal moiety of Cry9Aa2 resides within the N-terminal 665 amino acids. Site-directed mutagenesis was used to modify a truncated version of the gene (cry9Aa2T nucleotides 1–1995), removing motifs likely to be deleterious to full-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 plant 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 the CaMV 35S promoter. Plants transformed with the native gene did not show resistance to potato tuber moth larvae. In contrast, significant levels of larval mortality and reductions in larval growth and leaf damage were observed on many of the plants transformed with the modified genes. The cry9Aa2T mRNA was barely detectable in plants transformed with the native gene, whereas significant accumulation of full-length cry9Aa2T transcript was seen in plants transformed with modified genes. Modifications in the 5'-terminal 693 nucleotides of cry9Aa2T had the most significant effect on increasing the steady-state levels of cry mRNA. Transcription initiation rates of both the native and modified cry9Aa2T genes were similar, suggesting that the lack of native transcript accumulation was a consequence of transcript instability and that the sequence modifications had significantly improved the stability of the cry9Aa2T transcript. This improvement in steady-state full-length transcript levels resulted in expression of the insecticidal gene in N. tabacum to levels which conferred significant resistance to potato tuber moth larvae. Abbreviations: CaMV, cauliflower mosaic virus; IPTG, isoprophylthio-β-galactoside; PCR, polymerase chain reaction; poly(A), polyadenylation; PTM, potato tuber moth; UTR, untranslated region.