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Constructs for expressing GNA lectin, PHA-E lectin and Bt-toxin in transgenic rice

The aim was to produce and verify constructs for expression of snowdrop lectin (GNA), Phaseolus vulgaris agglutinin E-form (PHA-E) and Bacillus thuringiensis toxin (Bt) in the rice (Oryza sativa L.) cultivars ASD16, EYI105, and Xiushui 11 respectively.

Two different methods of plant transformation were used to produce transgenic rice plants, namely particle bombardment (GNA or PHA-E expressing rice) and Agrobacterium mediated transformation (Bt).

For generation of GNA expressing rice, both the gna coding sequence (from plasmid pLECGNA2) and the promoter region were used to produce the ubi-gna plasmid for subsequent transformation. Similarly, for generation of PHA-E expressing rice, both the pha-e coding sequence and the promoter region were used to produce the ubi-pha-e plasmid. Plants containing the gene construct for expression of either GNA or PHA-E were generated by particle gun bombardment of mature embryos. In addition to the expression pasmid, the pWRG1515 plasmid (containing both the hygromicine–resistance gene, hpt, and the reporter gene gusA) was also used. Following co-transformation, calli were transferred to a proliferation medium to allow plantlet formation and transformants selected on the basis of hygromycin resistance. Putative transformants were screened for the presence of the transgene by PCR.

Bt (cry1Ab) expressing rice was generated by Agrobacterium mediated transformation. The T-DNA region of the binary vector pKUB was used for transformation. In addition to the cry1Ab synthetic insecticidal protein gene, the transformation cassette also included a hygromycin phosphotransferase gene, a neomycin phosphotransferase gene, the termination signal of nopaline synthase, the maize ubiquitin promoter, and the coding region for the gus gene.

After selection of putative transformants (based upon hygromycin resistance and/or gus assay) transgene expression in the resulting plants was confirmed by immuno-assay using Western blot analysis. Immunodotblot assay was additionally employed to quantify transgene expression in rice tissues, including seeds. Bt rice showed the lowest levels of transgene expression in seeds when compared to the other rice tissues. In contrast, GNA and PHA-E rice plants exhibited the highest level of expression in the seeds, compared to other tissues.

More information on the project can be found at: http://www.entransfood.com/RTDprojects/SAFOTEST/safotest.html

Reported by

University of Newcastle
University of Newcastle upon Tyne
NE1 7RU Newcastle upon Tyne
United Kingdom
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