Using Proteomics in Transgenic Wheat to Assess the Effects of an RNA Interference Construct that Targets a Family of Seed Storage Proteins.

RNA interference (RNAi) is an approach that uses genetic transformation to introduce DNA constructions aimed at decreasing the expression levels of endogenous genes. We used this strategy with the goal of reducing the levels of secalins, a class of rye seed storage proteins found in wheat varieties that carry a translocation between a rye chromosome and the short arm of the wheat 1B chromosome. The presence of secalins is a possible cause of dough handling difficulties experienced by bakers that use flours from wheats carrying this translocation chromosome. The design of RNAi vectors is especially problematic for genes that encode wheat and rye seed storage proteins because bread wheat contains three large genomes and most families of seed storage proteins are encoded by several closely related genes. For this reason, we were concerned about off-target effects of our RNAi construction, which included 500 bp from the coding region of a rye omega secalin gene. Proteomics provided a sensitive method to assess the effects of our RNAi vector on the seed proteins of two independent transgenic events, compared to their non-transformed parent. Both transgenic seed proteomes exhibited significant decreases in the levels of rye omega secalins and of closely related wheat omega gliadins, as expected, as well as in the levels of some wheat gamma gliadins. The levels of some wheat alpha gliadins were increased in the transgenic wheat seeds. The challenges encountered in identifying individual members of various wheat and rye seed storage protein families will be discussed.