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

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.

Figure 1

Chloroplast expression vector and PCR analysis. (A) pLD-BD Cry2Aa2 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.