Induction of recombinant proteins in wheat seed and vegetative tissues

Cereals offer advantages for molecular farming because seeds have naturally evolved for protein storage. In seeds, the recombinant proteins can be targeted to storage organelles, such as Protein Bodies (PBs) derived from the endoplasmic reticulum (ER) and Protein Storage Vacuoles (PSVs) for stable accumulation and storage. Gamma-zein, the maize prolamin storage protein, and partial sequences thereof can be fused to proteins of interest to induce the formation of novel PB-like organelles not only in its native environment (maize endosperm) but also in other plant cells and in eukaryotic cells generally. We report the constitutive expression of recombinant fusion protein i.e. repeated and Pro-X domain of gamma-Zein and red fluorescent protein (DsRed) in transgenic wheat plants. The red fluorescent protein (DsRed) was used as tool for tracking subcellular localization of recombinant proteins and also as fluorescent selection marker to monitor transgene expression during wheat transformation. In the present study we explored endogenous and ectopically induced protein storage organelles as preferred intracellular destination for recombinant protein in wheat different tissues i.e. leaf, endosperm and embryo. Our finding has confirmed that the model fusion protein has induced the formation of an additional population of protein bodies containing the recombinant protein in seed and vegetative tissues. To better understand the trafficking of recombinant proteins in ectopic tissues, the subcellular localization of recombinant proteins in seed and green tissues were examined. We confirmed the feasibility of using γ-Zein for targeting recombinant protein into artificial protein bodies those were preferably retained in the protein storage vacuoles (PSV) in seed tissues. In vegetative tissues, the induced protein bodies were found highly mobile organelles, exhibiting different pattern of movement with no specific direction. The presence of the model fusion protein was validated in all three transgenic wheat plant tissues tested. Also, no degradation products were detected in transgenic wheat seeds after five months storage. The expression of recombinant fusion proteins had no harmful effect on the viability and development of transgenic wheat plants