Project summary
The key idea of this research project is to eliminate coeliac toxicity from wheat proteins while maintaining their functional properties. The most important functional property of wheat proteins refers to the capability to form extensible and elastic films. This is a basic prerequisite for the production of baked goods from fermentative leavened doughs with porous crumbs.
To confirm the working hyothesis that only gliadins and not glutenins cause
coeliac disease, initially pure glutenin will be produced in yeast cells by
genetic modification. With the same approach it shall be proven that the elastic
functions of gluten are caused solely by glutenins whereas the viscous properties
can be maintained by other proteins, e.g. by maize proteins.
If contrary to the expectations both glutenins and gliadins are shown to be
linked with coeliac disease, then the working hypothesis must be dismissed.
In this case, the glutenins will be genetically modified in such a way that
they retain their functional properties but loose their coeliac toxicity.
The result of the coeliac toxicity testing for glutenins is a prerequisite
for the future strategy. Eight working groups will work on the following steps
in detail:
1. expression of wheat-glutenin in yeast
2. transformation of maize with wheat-glutenins
3. inactivation of gliadins in wheat
4. elimination of the immunoreaction by immunomodulation of the intestinal system
5. development, physiological evaluation of nutrition and investigation of commercialisation
of products from genetically engineered varietes.
According to project planning the first four steps are in progress. For organizational reasons work on approaches 1 - 3 started in the spring 2000 (April 1, May 1). Work on approach 4 started as scheduled in February 2000. Below are the working groups results so far been in shortened form.
1. Expression of wheat-glutenin in yeast
A gliadin-gene from the wheat storage proteins was totally and partly integrated
into transformation vectors and cloned. The produced gliadin was accumulated
in the yeast cells resp. secreted into the medium. Currently optimization of
expression and purification of the expressed protein is under way.
Genes of LMW-glutenin without regions which trigger coeliac disease were isolated,
integrated into transformation vectors and cloned. The investigation of the
transgene yeast has begun.
2. Transformation of maize with wheat storage protein genes
In wheat the glutenin-proteins form the major part of storage proteins.
It is necessary that the expression of the storage proteins in maize is regulated
by strong promoters. Currently, the integration of different reporter gene constructs
with genes of the HMW-glutenin subunit and promoters into maize will be achieved
using the biolistic co-transformation method. The Basta-resistence-gene was
used as marker gene. Several lines of Basta resistent plants could be produced.
With certainty one line has integrated the glutenin-gene.
3 Inactivation of gliadins in wheat
The objective of this sub-project is the targeted inactivation of all gliadin
genes in wheat, which code for the coeliac specific proteins. The transformation
of wheat protoplasts out of wheat cell cultures with a defective pat-gene (Glufosinate-herbicide
resistence) which is active after mutagenesis and with the nptII-gene (resistence
to Canamycin- and Canamycinanalogs) was started. For the later transformation
of wheat cell cultures with agrobacteria, lines were transferred with the marker
gene construct and antibiotic-resistent lines were cloned.
4. Elimination of the immunoreaction by immunomodulation of the intestinal
system
Within the study of immunomodulation of the intestinal immune system a simplified
method for isolation of gluten sensitive T-cells was developed, which showed
a remarkable increase of reproduciability of the obtained results. In continuation
of these works, it is planned to produce clones of gluten sensitive T-cells.
A first selection of synthetic peptides of the N-domain of A-gliadin was testet
in vivo and in vitro for coeliac toxicity
In further tests, the results will be ensured statistically. There are first
signs that all synthetic peptides tested so far are non toxic in comparison
to the peptotryptic gliadin
5. Development, physiological evaluation of nutrition and investigation
of commercialisation of products from genetically engineered varietes.
According to the working plan the studies have not started yet.