Expression of hybrid cry3aM–licBM2 genes in transgenic potatoes (Solanum tuberosum)
- 87 Downloads
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.
- Bohorova N, Frutos R, Royer M, Estaсol P, Pacheco M, Rascуn Q, McLean S, Hoisington D (2001) Novel synthetic Bacillus thuringiensis cry1B gene and the cry1B–cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize. Theor Appl Genet 103:817–826CrossRefGoogle Scholar
- Chang HH, Chan MT (1991) Improvement of potato transformation efficiency by Agrobacterium in the presence of silver thiosulfate. Bot Bul Acad Sin 32:63–70Google Scholar
- Crickmore N, Zeigler DR, Feitelson J, Schnepf E, Van Riel J (1998) Revision of the nomenclature for the Bt pesticidal crystal proteins. Micro Mol Biol Rev 807–813Google Scholar
- Komakhin RA, Abdeeva IA, Salehi Jouzani GR, Goldenkova IV,Zhuchenko AA (2005) Thermostable lichenase as a translational reporter. Russ J Genet 41(1):40–47Google Scholar
- Perlak FJ, McPherson SA, Fuchs RL, Macintosh SC, Dean DA, Fischhoff DA (1988) Expression of Bacillus thuringiensis protein in transgenic plants. In: Roberts DW, Granados RR (eds) Proceedings of a conference on Biotechnology, Biological Pesticides and Novel Plant-Pest Resistance for Insect Pest Management. Boyce Thompson Institute for Plant Research, Ithaca, pp 77–81Google Scholar
- Piruzian ES, Goldenkova IV, Musiychuk KA, Abdeev RM, Volkova LV, Kobets NS (2000) New reporter system based on termostable lichenase for plant gene expression regulation. Russ J Plant Physiol 47(3):382–389Google Scholar
- Salehi Jouzani GR, Goldenkova IV (2005) A new reporter gene technology: opportunities and perspectives. Iran J Biotechnol 3(1):1–15Google Scholar