Aug 3, 2007

Research Projects

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Production of a Bt toxin standard (Cry3Bb1) and development of methods of detection

(2004 – 2008) Department of Viticulture and Enology, Neustadt an der Weinstraße

Topic

The aim is to centrally

• produce a standardised Bt toxin (Cry3Bb1)
• develop and establish a uniform method of measurement to record the amount of Bt toxin produced in the maize plants

for the project group working on diabrotica-resistant Bt maize.

A Bt toxin standard and a uniform method to quantitatively detect the toxin are both required in order to be able to compare the results from the different subprojects.

The Bt toxin standard produced will be made available to the other subprojects in the group.

Experiment description

Expressing and purifying the Cry3Bb1 toxin

The first step was to produce a Bt toxin standard. This was achieved by isolating the Cry3Bb1 gene from the transgenic maize Mon88017 and introducing it into E. coli to produce the Bt protein.

In November 2005 Monsanto also supplied an expression plasmid with the Cry3Bb1 gene.

Purification of the Cry3Bb1 protein is currently being optimised further. The stability and shelf life of the toxin are also being examined.

Characterising the toxin standard

The Bt toxins produced by bacteria and plants are characterised immunologically and also by molecular weight to ensure that the toxin standard is identical to the protein found in the plants.

As parameters for biological activity, bioassays (to determine the dose at which 50 percent of the test subjects die, referred to as LD50) are conducted on various leaf beetles (Coleoptera) in cooperation with the Institute for Biological Control at the Federal Biological Research Centre for Agriculture and Forestry (BBA) in Darmstadt.

Developing a method of detecting Cry3Bb1

At present there is no commercial quantitative method of measurement for Cry3Bb1. One of the existing qualitative methods of detection has been optimised and established as a quantitative method. In addition, a new quantitative detection method is being developed.

Detecting Cry3Bb1 in plant material

Plant material (roots, stems, lower and upper leaves, anthers, cobs and kernels) is taken from the group’s release site at four growth stages (BBCH 20, 30, 60 and 80) and analysed for Cry3Bb1 expression.

The fresh and dry weights of the samples are measured.

Results

Expression and purification of the Cry3Bb1 toxin

Up to the beginning of 2007 the project partners were supplied with a total of 67.2 milligrams of toxin. Since 2007 it has been possible to produce the toxin with a degree of purity of 85 per cent.

Characterising the toxin standard

Both of the available Cry3Bb1 genes were integrated into E. coli and the Cry3Bb1 protein was produced. With both genes, a Bt protein of the same size (77 kilodaltons) was isolated from E.coli.

Developing a detection method for Cry3Bb1

With the ELISA detection method that has been optimised for quantitative detection, a detection limit of 0.2 nanograms was achieved. This test was used for measuring Cry3Bb1 expression in the field.

With the new detection method being developed it is already possible to detect Cry3Bb1 from extracts of Bt maize ears, mixed maize samples and from E. coli using Western Blot.

Detecting Cry3Bb1 in plant material

The Cry3Bb1 content of 306 samples was analysed in 2005, and of 342 samples in 2006.

The Cry3Bb1 levels in the upper leaves were higher than in the roots, lower leaves, anthers and ears (23 - 34 µg/g fresh weight). The ears contained the lowest concentrations of Cry3Bb1 toxin (6.8 µg/g fresh weight). The anthers contained almost as much Cry3Bb1 toxin as the upper leaves and 1.5 times as much as the silks. The Cry3Bb1 levels fell towards the later development stages. This becomes clear when the toxin levels are based on dry weight. The variations in toxin level between individual plants were not high.