Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals - PubMed (original) (raw)
Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals
B De Cosa et al. Nat Biotechnol. 2001 Jan.
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 T(0) and T(1) 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.
Figures
Figure 1
Chloroplast expression vector and PCR analysis. (A) pLD-BD _Cry2_Aa2 operon (9.8 kb) with PCR primer binding sites and expected fragment sizes. PCR analysis of untransformed and putative chloroplast transformants using two primer sets: (B) 1P1M and (C) 3P3M. Lane 1, 1 kb ladder; lane 2, untransformed; lanes 3–7, pLD-BD Cry2Aa2 operon putative transformants; lane 8, pLD-BD Cry2Aa2 operon plasmid DNA.
Figure 2
Southern blot analysis of T0 and T1 generations. Lane 1, 1 kb ladder; lane 2, untransformed; lanes 3–7, T0 transgenic lines; lanes 8 and 9, T1 transgenic lines.
Figure 3
10% SDS–PAGE gel stained with R-250 Coomassie blue. Loaded protein concentrations are provided in parentheses. Lane 1, prestained protein standard; lane 2, partially purified Cry2Aa2 protein from E. coli (5 μg); lane 3, single gene-derived Cry2Aa2 pellet extract solubilized in 50 mM NaOH (22.4 μg); lane 4, single gene-derived Cry2Aa2 supernatant (66.5 μg); lane 5, operon-derived Cry2Aa2 pellet extract solubilized in 50 mM NaOH (22.9 μg); lane 6, operon-derived Cry2Aa2 supernatant (58.6 μg); lane 7, untransformed tobacco pellet extract solubilized in 50 mM NaOH (29.8 μg); lane 8, untransformed tobacco supernatant (30.4 μg). Colored compounds observed in the supernatant of transgenic plants interfered with the DC Bio-Rad protein assays.
Figure 4
Protein quantification by ELISA in young, mature, and old transgenic leaves. (A) Single gene-derived Cry2Aa2 expression shown as a percentage of total soluble protein. (B) Operon-derived Cry2Aa2 expression shown as a percentage of total soluble protein.
Figure 5
Insect bioassays. (A, D, G) Untransformed tobacco leaves; (B, E, H) single gene-derived Cry2Aa2 transformed leaves; (C, F, I) operon-derived Cry2Aa2 transformed leaves. (A–C) Bioassays with Heliothis virescens; (D–F) bioassays with Helicoverpa zea; (G–I) bioassays with Spodoptera exigua. All leaf samples for each replicate were from the same leaf. Two samples were evaluated per treatment, and observed daily for mortality and leaf damage for five days. Treatments were replicated three times. Insects were tested at 5 or 10 days old (see text for details).
Figure 6
Transmission electron micrographs. Operon-derived Cry2Aa2 leaf sections in young (A), mature (B, D), and old, bleached leaf (C). (E) Single gene-derived Cry2Aa2 mature leaf; (F) mature untransformed leaf.
Similar articles
- Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects.
Kota M, Daniell H, Varma S, Garczynski SF, Gould F, Moar WJ. Kota M, et al. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1840-5. doi: 10.1073/pnas.96.5.1840. Proc Natl Acad Sci U S A. 1999. PMID: 10051556 Free PMC article. - Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.).
Zheng SJ, Henken B, de Maagd RA, Purwito A, Krens FA, Kik C. Zheng SJ, et al. Transgenic Res. 2005 Jun;14(3):261-72. doi: 10.1007/s11248-005-0109-2. Transgenic Res. 2005. PMID: 16145834 - Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer.
Singsit C, Adang MJ, Lynch RE, Anderson WF, Wang A, Cardineau G, Ozias-Akins P. Singsit C, et al. Transgenic Res. 1997 Mar;6(2):169-76. doi: 10.1023/a:1018481805928. Transgenic Res. 1997. PMID: 9090064 - Monitoring and adaptive resistance management in Australia for Bt-cotton: current status and future challenges.
Downes S, Mahon R, Olsen K. Downes S, et al. J Invertebr Pathol. 2007 Jul;95(3):208-13. doi: 10.1016/j.jip.2007.03.010. Epub 2007 Mar 25. J Invertebr Pathol. 2007. PMID: 17470372 Review. - Problems that can limit the expression of foreign genes in plants: lessons to be learned from B.t. toxin genes.
Diehn SH, De Rocher EJ, Green PJ. Diehn SH, et al. Genet Eng (N Y). 1996;18:83-99. doi: 10.1007/978-1-4899-1766-9_6. Genet Eng (N Y). 1996. PMID: 8785128 Review. No abstract available.
Cited by
- Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).
Hossain MJ, Bakhsh A, Joyia FA, Aksoy E, Gökçe NZÖ, Khan MS. Hossain MJ, et al. Transgenic Res. 2023 Dec;32(6):497-512. doi: 10.1007/s11248-023-00366-6. Epub 2023 Sep 14. Transgenic Res. 2023. PMID: 37707659 - The first chloroplast sequence of Rosa davurica Pall. var. Davurica.
Xia Y, Wu H, Li S. Xia Y, et al. Mitochondrial DNA B Resour. 2023 Jun 13;8(6):668-672. doi: 10.1080/23802359.2023.2220431. eCollection 2023. Mitochondrial DNA B Resour. 2023. PMID: 37325773 Free PMC article. - Multidimensional futuristic approaches to address the pandemics beyond COVID-19.
Kotwal SB, Orekondey N, Saradadevi GP, Priyadarshini N, Puppala NV, Bhushan M, Motamarry S, Kumar R, Mohannath G, Dey RJ. Kotwal SB, et al. Heliyon. 2023 Jun;9(6):e17148. doi: 10.1016/j.heliyon.2023.e17148. Epub 2023 Jun 11. Heliyon. 2023. PMID: 37325452 Free PMC article. Review. - Optimising expression and extraction of recombinant proteins in plants.
Coates RJ, Young MT, Scofield S. Coates RJ, et al. Front Plant Sci. 2022 Dec 8;13:1074531. doi: 10.3389/fpls.2022.1074531. eCollection 2022. Front Plant Sci. 2022. PMID: 36570881 Free PMC article. Review. - Transgene insertion into the plastid genome alters expression of adjacent native chloroplast genes at the transcriptional and translational levels.
Ghandour R, Gao Y, Laskowski J, Barahimipour R, Ruf S, Bock R, Zoschke R. Ghandour R, et al. Plant Biotechnol J. 2023 Apr;21(4):711-725. doi: 10.1111/pbi.13985. Epub 2023 Jan 17. Plant Biotechnol J. 2023. PMID: 36529916 Free PMC article.
References
- Bogorad L. Engineering chloroplasts: an alternative site for foreign genes, proteins, reactions, and products. Trends Biotechnol. 2000;18:257–263. - PubMed
- Ma J, et al. Generation and assembly of secretory antibodies in plants. Science. 1995;268:716–719. - PubMed
- Ye X, et al. Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science. 2000;287:303–305. - PubMed
- Daniell H. New tools for chloroplast genetic engineering. Nat Biotechnol. 1999;17:855–856. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources