Plant Cell, Tissue and Organ Culture

, Volume 92, Issue 3, pp 321–325 | Cite as

Expression of hybrid cry3aM–licBM2 genes in transgenic potatoes (Solanum tuberosum)

  • Gholamreza Salehi JouzaniEmail author
  • Irina V. Goldenkova
  • Eleonora S. Piruzian
Research note


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.


Bacillus 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


Bacillus thuringiensis


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.


  1. Adang MJ, Brody MS, Cardineau G, Eagan N, Roush RT, Shewmaker CK, Jones A, Oakes JV, McBride KE (1993) The reconstruction and expression of a Bt cry3A gene in protoplasts and potato plants. Plant Mol Biol 21:1131–1145PubMedCrossRefGoogle Scholar
  2. Alliotte T, Huang Zhu L, Van Montagu M, Inze D (1988) Plant expression vectors with origin of replication of the W-type plasmid Sa. Plasmid 19:251–254PubMedCrossRefGoogle Scholar
  3. 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
  4. Bradford MM (1976) A rapid sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. 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
  6. 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
  7. 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
  8. Leroy T, Herry AM., Royer M, Altosaar I, Frutos R, Duris D, Philippe R (2000) Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner. Plant Cell Rep 19:382–389CrossRefGoogle Scholar
  9. 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
  10. Perlak FJ, Stone TB, Muskopf YM, Petersen LJ, Parker GB, McPherson SA, Wyman J, Love S, Reed G, Biever D, Fischhoff DA (1993) Genetically improved potatoes: protection from damage by Colorado potato beetles. Plant Mol Biol 22:313–321PubMedCrossRefGoogle Scholar
  11. 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
  12. Piruzian ES, Goldenkova IV, Musiychuk KA, Kobets NS, Arman IP, Bobrysheva IV, Chekhuta IA, Glazkova D (2002) A reporter system for prokaryotic and eukaryotic cells based on thermostable Clostridium thermosellum lichenase. Mol Genet Genomics 266:778–786PubMedCrossRefGoogle Scholar
  13. Salehi Jouzani GR, Goldenkova IV (2005) A new reporter gene technology: opportunities and perspectives. Iran J Biotechnol 3(1):1–15Google Scholar
  14. Salehi Jouzani GR, Komakhin RA, Piruzian ES (2005) Comparative study of the expression of the native, modified, and hybrid cry3a genes of Bacillus thuringiensis in prokaryotic and eukaryotic cells. Russ J Genet 41(2):171–177CrossRefGoogle Scholar
  15. Wood TM, Bhat KM (1988) Methods for measuring cellulase activities. Meth Enzymol 160:87–112CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Gholamreza Salehi Jouzani
    • 1
    Email author
  • Irina V. Goldenkova
    • 2
  • Eleonora S. Piruzian
    • 2
  1. 1.Agricultural Biotechnology Research Institute of Iran (ABRII)KarajIran
  2. 2.Functional Genomics DepartmentVavilov Institute of General Genetics, Russian Academy of SciencesMoscowRussia

Personalised recommendations