Expression of modified Cry1Ac gene of Bacillus thuringiensis in transgenic tobacco plants

Several mutations were introduced into the Cry1Ac toxin gene, resulting in four variants with altered sequences that were responsible for low expression of the toxin in transgenic plants. These variants were as follows: V1, with modified three A/T-rich regions, including the first signal of transcription termination; V2, with modified five putative polyadenylation signals (polyadenylation signals PAS) and the second signal of transcription termination; V3, with four initial AUUUA motifs; V4, with modification of six PASs, four AUUUA motifs, as well as the first and the second signals of transcription termination. The modified variants and the initial WT gene were cloned into the binary vector pBI121 and introduced into tobacco plants (Nicotiana tabacum) by Agrobacterium tumefaciens-mediated transformation. The presence of transgenes in the tobacco plants was confirmed by the polymerase chain reaction (PCR) method. The expression of particular variants of the Cry1Ac gene in tobacco was assayed using Western blotting with antibodies against the domain II of the Cry1Ac toxin. The average expression of WT was estimated to be 0.0025% of soluble proteins, and the expression levels of modified variants were 0.004%, 0.0098%, 0.0125%, and 0.0043% for V1, V2, V3, and V4, respectively. In this article we described the construction of a variant of the Cry1Ac gene (V3) with 12 point mutations leading to an average level of expression in transgenic plants five times higher than that observed in the case of the WT gene. Our results have shown for the first time that the modification of AUUUA sequences has a significant effect on the expression of the Cry1Ac gene in transgenic plants.