Date of Award
Doctor of Philosophy
Paul M. Scott
We have developed a method for detecting a transgene and its protein product in maize endosperm that allows the kernel to be germinated after analysis. Our method involves first sampling the endosperm with a hand-held rotary grinder. This tissue is then serially extracted for phenotypic analysis by SDS-PAGE with visualization by total protein staining or immuno-blot detection and genotypic analysis by PCR reactions to detect the transgene. This method is useful for screening transgenic kernels in breeding experiments. We have produced transgenic maize plants containing a wheat Glu-1Dx5 gene encoding the high molecular weight glutenin subunit 1Dx5. Analysis by SDS-PAGE showed that a protein similar in size to the wheat 1Dx5 HMW glutenin subunit accumulates in the endosperm of transgenic maize from four independent transformation events. This protein reacts with a monoclonal antibody specific to the wheat 1Dx5 HMW glutenin subunit and was not detected in nontransgenic controls or in pollen, anthers, leaves, or embryos of plants grown from kernels expressing this protein in endosperm. We studied the inheritance of this gene through three sexual generations. Reciprocal crosses with nontransgenic plants and self-pollinations were performed, and the resulting kernels were analyzed for the presence of the 1Dx5 HMW glutenin subunit. These data, together with PCR analysis for the transgene, suggest the transgene is inefficiently transmitted through pollen in all four events. Therefore, the expressing (hemizygous) and non expressing (null) near isogenic kernels were produced on the same ear to study of the effect of the Glu-1Dx5 transgene expression on endogenous seed storage protein in endosperm. Seed weight of Glu-1Dx5 transgenic maize was not different between expressing and non-expressing transgenic plants or kernels. The 1Dx5 HMW glutenin subunit contents were 30.04 and 37.14 ng/mg in expressing endosperm of two events of transgenic maize when measured by ELISA. The 1Dx5 HMW glutenin subunit in endosperm of transgenic maize accounted for from 0.92% to 3.82% of total zeins by HPLC analysis. Kernels expressing the Glu-1Dx5 transgene had different levels of zeins and nitrogen than non-expressing sibling kernels.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Sangtong, Varaporn, "The 1Dx5 high molecular weight subunit gene from wheat in transgenic maize " (2001). Retrospective Theses and Dissertations. 673.