Expression of a Bacillus thuringiensis cry1B synthetic gene protects Mediterranean rice against the striped stem borer
Cry1Ba endotoxin, which was shown to exhibit a tenfold lower lethal concentration 50 (LC50) than Cry1Ac in a Striped Stem Borer (SSB) diet incorporation assay. The 1.950-bp synthetic cry1B gene, possessing an overall GC content of 58 %, was cloned under the control of the maize ubiquitin promoter first intron and first exon regions. The resulting vector, designated as pUbi-cry1B, was transferred to two commercial Mediterranean cultivars of rice, Ariete and Senia, using microprojectile acceleration-mediated transformation. Thirty-two and 47 T0 events were generated in cvs. Ariete and Senia, respectively. Southern blot and immunoblot analyses allowed the identification of 7 Senia and 1 Ariete events harbouring both an intact gene cassette and expressing Cry1B at a level ranging from 0.01% to 0.4% of the total soluble proteins. Three Senia and 1 Ariete events were found to be protected against second instar SSB larvae in whole plant feeding assays, exhibiting 90–100% mortality 7 days after infestation. Spatial and temporal variation in transgene expression was further examined in resistant event 64 of cv. Ariete. Stable accumulation of Cry1B, representing 0.4% of the total soluble proteins, was observed over the T2 to T4 generations in leaf tissue 20, 40, 70 and 90 days after germination in both young and old leaves and in internodes. Ariete event 64 was found to be fully protected from attacks of third and fourth instar SSB larvae over subsequent generations.
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