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Area of Science:

  • Microbiology
  • Molecular Biology
  • Entomology

Background:

  • Bacillus thuringiensis (Bt) is a soil bacterium widely used as a biopesticide.
  • Genetic engineering offers potential to enhance Bt efficacy and spectrum of activity.
  • Development of efficient gene transfer methods is crucial for creating improved Bt strains.

Purpose of the Study:

  • To develop a recombinant Bacillus thuringiensis strain expressing both chitinase and Cry2Aa insecticidal proteins.
  • To evaluate the efficacy of a novel triple recombineering technique for gene insertion.
  • To assess the insecticidal activity of the engineered strain against Helicoverpa armigera.

Main Methods:

  • A triple recombineering technique using plasmid pHT315 was employed to construct pHTEC.
  • The pHTEC construct, containing chitinase and cry2Aa genes, was used to transform B. thuringiensis strains HD73 and Cry-B.
  • Recombination efficacy was optimized by comparing RecE/RecT and Redα/Redβ systems with single-strand DNA.

Main Results:

  • Recombinant B. thuringiensis strains successfully expressed Cry2Aa as cubic crystals and soluble chitinase.
  • The engineered HD73 (pHTEC) strain exhibited a sevenfold increase in toxicity against Helicoverpa armigera larvae compared to the control (HD73 (pHT315)).
  • The triple recombineering protocol demonstrated efficient gene insertion and strain development.

Conclusions:

  • The developed triple recombineering method is effective for engineering Bacillus thuringiensis with multiple genes.
  • The co-expression of chitinase and Cry2Aa significantly enhances insecticidal activity against Helicoverpa armigera.
  • This engineered strain holds promise for improved biological control of lepidopteran pests.