Expression of hybrid cry3aM–licBM2 genes in transgenic potatoes (Solanum tuberosum)
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The full-modified Bacillus thuringiensis cry3a (cry3aM) gene was designed and synthesized for effective expression in plants. A plant expression vector pC29RBCS-leader-cry3aM–licBM2 was constructed for potato transformation. In this vector, the cry3aM sequence was fused in reading frame with a new reporter gene (licBM2) and a leader sequence for the rbcs gene. The reporter gene encoded thermostable lichenase and the leader sequence encoded a signal peptide for transporting protein product to chloroplasts. The vector contained the light-inducible promoter for rbcs gene isolated from Arabidopsis thaliana. Transgenic plants were obtained by Agrobacterium mediated transformation using microtuber explants. Transgenic plantlets were selected by kanamycin resistance and confirmed as transgenic by PCR with specific primers, evaluation of lichenase activity, and bioassay of Colorado potato beetle neonate larvae. Promoter activity assays under light induction (kinetic analysis) using lichenase activity and bioassay both showed high and stable expression of hybrid genes in transgenic plantlets. Furthermore, the presence of lichenase as a reporter protein in the composition of hybrid protein was shown to facilitate selection and analysis of the expression level of hybrid genes in transgenic plants.
KeywordsBacillus thuringiensis β-1,3-1,4 Glucanase Clostridium thermocellum Colorado potato beetle Lichenase Full modified cry3a gene Reporter gene Transgenic potato Yeasts
Colorado potato beetle
Full modified cry3a gene
3,5 Dinitrosalicylic acid
Rib-1,5-bisphospate carboxylase gene
We thank A. Shevelev (GNII, Moscow, Russia) for providing the Bt strain. This study was supported by the program of the Russian Academy of Sciences “Dynamics of Plant, Animal, and Human Gene Pools” (Project no. 24P-IOG-08–2004) and a scholarship from the Organization for Agricultural Research and Education of Iran.
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