Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study

Abstract

Abstract Background As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1B M and Cry1C M ; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing Cry M genes, and to remove or alter sequences that might adversely affect their activity in plants. Results To assess the insecticidal efficacy of the Cry1B M /Cry1C M genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1B M /Cry1C M expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1B M /Cry1C M expression. Protein accumulation for Cry1C M ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1B M /Cry1C M genes, with a similar range of Cry1C M transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1B M /Cry1C M genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. Conclusions Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1 M genes in GM crop plants in the future.

Keywords

• Bacillus thuringiensis
• Insecticidal gene
• Bt toxin
• Gene stacking
• Diamondback moth
• Cry1B