Application of the thioredoxin system to produce hypoallergenic wheat (Triticum aestivum L.)

The cytosolic NADP-thioredoxin system (NTS), composed of thioredoxin h, NADP-thioredoxin reductase, and NADPH, plays an important role in plants. There is also evidence that the system may be used for the improvement of foods, including a lowering of their allergenicity. To this end two of the protein components of the cytosolic NTS were overproduced in the endosperm of stably transformed wheat and certain properties of the grain were determined. Using an efficient in vitro tissue culture system, a recalcitrant Californian wheat cultivar, Yecora Rojo, was successfully transformed with the DNA vectors containing thioredoxin h or NADP-thioredoxin reductase under the control of a B1-hordein promoter with a signal peptide for targeting to the protein body. More than forty transgenic wheat lines were produced and identified by PCR analysis.; The gene for cytosolic glucose 6-phosphate dehydrogenase was isolated from a barley cDNA library in order to attempt to overproduce the third component of the NTS, NADPH. The open reading frame of the isolated gene consists of 1530-bp nucleotides, corresponding to a deduced sequence of 509 amino acids and a Mr of 58.5 kDa. The gene shares a high degree of homology with the counterparts in other plants. The genomic sequence includes 14 introns, the number and position of which were completely conserved with the Arabidopsis counterpart. The DNA vectors containing the isolated glucose 6-phosphate dehydrogenase gene were constructed for future study.; The transgenic wheat plants transformed with thioredoxin h and/or NADP-thioredoxin reductase showed an integration of multiple copies of transgene(s) in their genome as shown by DNA hybridization analysis. When thioredoxin h and NADP-thioredoxin reductase were overproduced in the transgenic wheat grains either singly or in combinations, the overproduced protein was biochemically active and the level of expression varied among the different transgenic lines by a factor of 2 to 20 relative to that of controls. A mitigation of allergenicity in grain from three independent transgenic lines enriched with thioredoxin h was observed using an atopic dog colony. The gliadin fraction from all three transgenic lines was less allergenic than that from control null segregant lines. This result provides promising evidence that the thioredoxin system can be applied for the in vivo production of hypoallergenic wheat using genetic engineering technologies.