|Hurkman Ii, William|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: July 1, 2009
Publication Date: September 1, 2010
Citation: Blechl, A.E., Lin, J.W., Altenbach, S.B., Hurkman Ii, W.J., Sloane, S.M., Tanaka, C.K., Vensel, W.H. 2010. Applying Genetic Engineering to Understand the Contributions of Various Seed Proteins to Wheat Flour Properties. Xth International Gluten Workshop, September 7-9, 2009, Clermont-Ferrand, France. Technical Abstract: The contributions of individual seed proteins and protein families to wheat dough visco-elasticity can be uncovered by comparison of the end-use properties of flours prepared from the seeds of closely related genotypes that differ only or mainly in the presence/absence of those proteins. Genetic engineering allows us to make these changes in a purposeful way by introducing new genes into wheat that encode native proteins or variants with altered compositions. Such an approach has already yielded insights into the differing contributions of adding or raising the levels of native high-molecular-weight (HMW-) glutenin subunits to dough mixing properties. These experiments have shown that subunit Dx5 contributes to dough strength in ways that are quantitatively and qualitatively different from the contributions of subunits Ax1 or Dy10. In the former comparison, the difference could be due to the presence of an extra cysteine in Dx5 compared to all other x-type subunits. To explore this hypothesis, we made changes in the cysteine composition of subunit Dx5. We mutagenized the native 1Dx5 gene to convert the codons for the 4th and/or 5th cysteines to serine codons. These altered genes were introduced into “Bobwhite”, a bread wheat cultivar whose seeds contain subunits Ax2*, Bx7, By9, Dx5 and Dy10. The introduced Dx5 subunits have a “c-myc” triplet at their C-termini. Addition of this epitope allows the transgene-encoded subunits to be distinguished from native Dx5 with antisera. As a control, we also introduced Dx5 genes that had the c-myc tag but no changes in cysteine composition. Interestingly, the c-myc-tagged Dx5 subunits lacking either cysteine 5 or cysteines 4 and 5 were distributed similarly to c-myc-tagged wild-type Dx5 between the soluble and insoluble fractions of flours extracted with 50% 1-propanol. Results of further comparisons among transgenic flours carrying these Dx5 variants will be reported. We have also applied the genetic engineering strategy to assess the contributions of rye proteins to the properties of doughs from cultivars such as “Bobwhite” that contain the 1BL.1RS translocation from “Petkus” rye (Secale cereale). This translocation includes at least part of the Sec-1 locus, which encodes the omega and 40K gamma secalins. An RNA interference (RNAi) vector was constructed with the promoter from the 1Dy10 gene driving expression of a double-stranded RNA covering 500 bp of the coding region of an omega secalin gene isolated from “Bobwhite”. Introduction of the RNAi construct into “Bobwhite” resulted in decreases of at least two 40-50kDa seed protein bands that could be distinguished by SDS-PAGE of 50%-propanol-soluble extracts. Proteomic analyses showed that a transformant homozygous for the RNAi transgene exhibited losses of eight 2-D gel spots identified as secalins and four spots identified as wheat omega gliadins. Some, but not all, spots identified as wheat gamma gliadins showed decreased levels in the transformant compared to the parent. The effects of these protein changes on the small-scale mixing properties of doughs made from the transgenic flour will be reported. These studies show that genetic transformation is a useful way to change the levels of seed proteins or protein families and to allow comparisons of the effects of these changes in isogenic backgrounds.