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Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals

Nature Biotechnology volume 19, pages 71–74 (2001)Cite this article

Abstract

In nuclear transgenic plants, expression of multiple genes requires introduction of individual genes and time-consuming subsequent backcrosses to reconstitute multi-subunit proteins or pathways, a problem that is compounded by variable expression levels. In order to accomplish expression of multiple genes in a single transformation event, we have introduced several genes into the chromoplast genome. We confirmed stable integration of the cry2Aa2 operon by PCR and Southern blot analyses in T0 and T1 transgenic plants. Foreign protein accumulated at 45.3% of the total soluble protein in mature leaves and remained stable even in old bleached leaves (46.1%), thereby increasing the efficacy and safety of transgenic plants throughout the growing season. This represents the highest level of foreign gene expression reported in transgenic plants to date. Insects that are normally difficult to control (10-day old cotton bollworm, beet armyworm) were killed 100% after consuming transgenic leaves. Electron micrographs showed the presence of the insecticidal protein folded into cuboidal crystals. Formation of crystals of foreign proteins (due to hyperexpression and folding by the putative chaperonin, ORF 2) provides a simple method of purification by centrifugation and enhances stability by protection from cellular proteases. Demonstration of expression of an operon in transgenic plants paves the way to engineering new pathways in plants in a single transformation event.

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Figure 1: Chloroplast expression vector and PCR analysis.
Figure 2: Southern blot analysis of T0 and T1 generations.
Figure 3: 10% SDS–PAGE gel stained with R-250 Coomassie blue.
Figure 4: Protein quantification by ELISA in young, mature, and old transgenic leaves.
Figure 5: Insect bioassays.
Figure 6: Transmission electron micrographs.

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Acknowledgements

We thank F. Gould (North Carolina State University) for providing Heliothis virescens. This study was supported in part by the USDA-NRICGP grants 95-37500-2664, 97-35504-5297, and 98-35300-6973 to H.D.

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Authors and Affiliations

  1. Department of Molecular Biology and Microbiology and Center for Discovery of Drugs and Diagnostics, University of Central Florida, 12722 Research Parkway, Orlando, 32826-3227, FL

    Brandy De Cosa, Seung-Bum Lee & Henry Daniell

  2. Departments of Entomology and Plant Pathology, Auburn University, Auburn, 36849, AL

    William Moar

  3. Biological Electron Microscopy Imaging Facility, Auburn University, Auburn, 36849, AL

    Michael Miller

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  1. Brandy De Cosa
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Correspondence to Henry Daniell.

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Cosa, B., Moar, W., Lee, SB. et al. Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19, 71–74 (2001). https://doi.org/10.1038/83559

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