Jun 8, 2010

Research Projects

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Producing a Bt protein standard and optimising detection methods

(2008 – 2011) Dienstleistungszentrum Ländlicher Raum (DLR) Rheinpfalz, Department of Phytomedicine, 67435 Neustadt an der Weinstraße

Topic

For the research group of biosafety projects investigating the genetically modified Bt maize cultivar MON89034xMON88017, which produces three different Bt proteins, this project will

• produce standardised Bt proteins and
• develop and establish a standard measuring method for determining the amount of the Bt proteins formed in the maize plants.

Bt protein standards and a standard method of detecting Bt toxin quantities are necessary to allow the results of the individual projects within the group to be compared.

The Bt protein standard produced will be made available to the projects within the research group.

Experiment description

Producing the Bt proteins

To be able to produce larger quantities of the Bt proteins, the genes that carry the relevant information will be isolated and inserted into Escherichia coli bacteria. If the bacteria are cultivated under suitable conditions, they produce the Bt proteins.

The Bt maize will be grown for four to six weeks in the greenhouse. Then the young leaves will be harvested from the plants and the DNA will be isolated from them. The genes for the Bt proteins will be selectively amplified from the total DNA of the maize plants using a polymerase chain reaction (PCR). Every gene will then be inserted into a plasmid, which will be inserted into bacteria.

The bacteria will be cultivated and the Bt proteins they produce will be isolated and purified. The concentration and degree of purity will then be measured. The conditions for cultivation, isolation, purification and storage must be optimised for each Bt protein. In addition, the proteins produced in E.coli need to be characterised immunologically to ensure that they are comparable with the proteins from the genetically modified plants.

A bacterial strain that produces one of the three Bt proteins is already available from the last project.

Monitoring the bioactivity of the Bt proteins produced

The bioactivity, i.e. the toxicity, of the Bt proteins produced in E.coli has to be tested on the target organisms. Since the Western corn rootworm is still a quarantine pest in Germany, it is replaced in this experiment by the related Colorado potato beetle. A breeding programme for Colorado potato beetles will first have to be established. There is already abreeding programme for the European corn borer at the DLR.

Different concentrations of the Bt proteins will be applied to the food source of newly emerged European corn borer or Colorado potato beetle larvae. After seven days, the number of larvae that die at each concentration level will be counted to calculate the mortality rate.

To check whether the Bt proteins have acumulative effect, these experiments will also be carried out with combinations of two proteins. These tests will also serve as a basis for research into non-target organisms in other projects within the group.

Measuring the Bt protein concentration in different plant parts

The concentration of the Bt proteins in plant tissue can be measured using the ELISA detection method. Measurement methods will have to be developed and optimised for two of the three Bt proteins produced by the maize variety under investigation.

The Bt protein levels in various different plant parts (root, stem, lower and upper leaves, pollen, silks and ears) will be measured at three growth stages. The fresh and dry weights of the samples will be measured as a reference value. The project will investigate the amount of variation between individual plants and the extent to which the Bt protein levels in the different parts of a plant correlate.

Results

Producing the Bt proteins

Ten plants from the Bt maize line MON89034xMON88017 and ten from the isogenic line were grown in the greenhouse. DNA of the three Cry genes was isolated from the young leaves, amplified and sequenced.

Isogenic Line of the three Bt proteins: plasmids were produced with the Bt genes and inserted into E.coli bacteria. Work is currently focusing on optimising the expression conditions and protein purification.

Monitoring the bioactivity of the Bt proteins produced

Preliminary bioactivity experiments were conducted for the Cry1 and Cry2 proteins using European corn borer larvae, and for the Cry3 protein using Colorado potato beetle larvae. Further bioassays are planned once the toxin purification process is complete.

Measuring the Bt protein concentration in different plant parts

In 2008, at the start of the longitudinal growth phase and during the flowering period, a total of 816 root, leaf and stem samples and other plant parts were collected from the maize trial field and stored at -80 ºC. The ELISA detection method will be used to measure the levels of the three Cry proteins in the different plant parts.