Vacuolar Targeting of Cry1Ac and its Effects on Expression and Stability in Tobacco.

Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes.Increasing heterologous expression of delta endotoxins of Bacillus thuringiensis in transgenic plants is being actively pursued as a means to increase their efficacy and to delay insect resistance. To examine if vacuoles could be used as alternate localization sites of delta endotoxins we developed binary vectors with a chimeric vacuole targeting signals and verified its localization efficiency by creating GFP fusions of Cry1Ac. Transgenic tobacco plants expressing Cry1Ac localized either to cytosol and vacuoles were generated and confirmed by PCR, QPCR and ELISA. Comparative protein expression analysis by quantitative ELISA showed that maximum, percentage total soluble protein of Cry1Ac was 0.64 and 1% in cytosol and vacuole targeted plants, respectively. However, detailed protein expression analysis showed that there are no significant differences in expression of Cry1Ac between cytosol and vacuole targeted plants. These results were further corroborated by immunoblot analysis as well as insect bioassays. Nevertheless, our study demonstrated that delta endotoxins could be targeted to vacuoles and expressed successfully which is of importance when gene stacking is being pursed where alternate localization sites are employed for different genes

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Not AvailableWeed infestation is one of the major biotic stress factors that is responsible for yield loss in direct-seeded rice (DSR). Herbicide-resistant rice has potential to improve the efficiency of weed management under DSR. Hence, the popular indica rice cultivar IR64, was genetically modified using Agrobacterium-mediated transformation with a codon-optimized CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, with N-terminal chloroplast targeting peptide from Petunia hybrida. Integration of the transgenes in the selected rice plants was confirmed by Southern hybridization and expression by Northern and herbicide tolerance assays. Transgenic plants showed EPSPS enzyme activity even at high concentrations of glyphosate, compared to untransformed control plants. T 0, T 1 and T 2 lines were tested by herbicide bioassay and it was confirmed that the transgenic rice could tolerate up to 1 % of commercial Roundup, which is five times more in dose used to kill weeds under field condition. All together, the transgenic rice plants developed in the present study could be used efficiently to overcome weed menace.Not Availabl