Molecular Biology Reports

, 37:1011 | Cite as

Codon optimization of cry1Ab gene for hyper expression in plant organelles

  • Rasheda Jabeen
  • Muhammad Sarwar Khan
  • Yusuf Zafar
  • Tehmina Anjum
Article

Abstract

With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome, following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well.

Keywords

Bacillus thuringiensis Chloroplast Codon optimization Transgenes Gene 

References

  1. 1.
    Golds T, Maliga P, Koop HU (1993) Stable plastid transformation in PEG treated protoplasts of Nicotiana tabacum. Bio/Tech 11:95–97CrossRefGoogle Scholar
  2. 2.
    Kota M, Daniel H, Varma S, Garctynski F, Gould F, Moar WJ (1999) Over expression of the Bacillus thuringiensis Cry2A protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proceed Natl Acad Sci USA 96:1840–1845CrossRefGoogle Scholar
  3. 3.
    Kumar S, Chandra A, Pandey KC (2008) Bacillus thuringiensis (Bt) transgenic crop: an environment friendly insect-pest management strategy. J Environ Biol 29(5):641–653PubMedGoogle Scholar
  4. 4.
    Li WB, Zarka KA, Douches DS, Coombs JJ, Pett WL, Grafius EJ (1999) Coexpression of potato PVYo coat protein and cryV-Bt genes in potato. J Am Soc Hortic Sci 124(3):218–223Google Scholar
  5. 5.
    Lichtfouse E, Navarrete M, Debaeke P, Souchere V, Alberola C, Menassieu J (2009) Agronomy for sustainable agriculture: a review. Agron Sustain Dev 29(1):1–6CrossRefGoogle Scholar
  6. 6.
    Lin CH, Chen YY, Tzeng CC, Tsay HS, Chen LJ (2003) Expression of Bacillus thuringiensis cry1c gene in plastid confers high insecticidal efficiency against tobacco cutworm-a Spodoptera insect. Bot Bull Acad Sin 44:199–210Google Scholar
  7. 7.
    Losey JE, Rayor LS, Lyons PC (1999) Transgenic pollen harms monarch larvae. Nature 399:214CrossRefPubMedGoogle Scholar
  8. 8.
    McBride KE, Svab Z, Schaaf DJ, Hogan PS, Stalker DM, Maliga P (1995) Amplification of a chimeric Bacillus gene in chloroplasts leads to an extra ordunary level of an insecticidal protein in tobacco. Biotechnology 13:362–365CrossRefPubMedGoogle Scholar
  9. 9.
    Phillips T. (2008) Regulation of transcription and gene expression in eukaryotes. Nat Educ 1(1)Google Scholar
  10. 10.
    Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Sanford JC (1990) Biolistic plant transformation. Physiol Plant 79:206–209CrossRefGoogle Scholar
  11. 11.
    Saxena D, Flores S, Stotzky G (1999) Insecticidal toxin in root exudates from Bt corn. Nature 402:480PubMedGoogle Scholar
  12. 12.
    Schwember AR (2008) An update on genetically modified crops. Ciencia e Investigacion Agraria 35(3):231–250Google Scholar
  13. 13.
    Sharma HC, Sharma KK, Seetharame N, Ortiz R (2000) Prospects for using transgenic resistance to insects in crop improvement. Mol Biol Genet 3:217–239Google Scholar
  14. 14.
    Spychalla J, Bevan MW (1993) Agrobacterium mediated transformation of potato stem and tuber tissues, regeneration and PCR screening for transformation. Plant Tissue Culture Man 11:1–18Google Scholar
  15. 15.
    Staub JM, Maliaga P (1993) Accumulation of D1 polypeptide in tobacco plastids is regulated via the untranslated region of the psbA mRNA. EMBO J 12:601–606PubMedGoogle Scholar
  16. 16.
    Svab Z, Maliga P (1993) High frequency of plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90:913–917CrossRefPubMedGoogle Scholar
  17. 17.
    Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530CrossRefPubMedGoogle Scholar
  18. 18.
    Wong EY, Hironaka CM, Fischhoff DA (1992) Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants. Plant Mol Biol 20:81–93CrossRefPubMedGoogle Scholar
  19. 19.
    Zhang ZL, Ren YG, Shen YX, Shan S, Fan GC, Qian WuXF, KX ShenGF (2000) Expression of Bacillus thuringiensis crystal gene in the chloroplast of tobacco. Yi Chuan Xue Bao 27:270–277PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Rasheda Jabeen
    • 1
  • Muhammad Sarwar Khan
    • 2
  • Yusuf Zafar
    • 3
  • Tehmina Anjum
    • 1
  1. 1.Institute of Mycology and Plant PathologyUniversity of the PunjabLahorePakistan
  2. 2.National Institute for Biotechnology and Genetic EngineeringFaisalabadPakistan
  3. 3.National Institute for Genomics and Advanced BiotechnologyIslamabadPakistan

Personalised recommendations