Information on the inserted DNA sequence
The spring barley (Hordeum vulgare L.) cultivar Golden Promise was modified by Agrobacterium tumefaciens-mediated transformation of immature embryos using strain AGL1 carrying the binary vector pBRACT209 (AsGLOp::HE_GFP-OM-4A-3). The inserted T-DNA contains an antigen expression cassette encoding the recombinant rHE_GFP fusion protein and a selectable marker cassette encoding hygromycin B phosphotransferase. The transformation construct was designed for preferential accumulation of the recombinant antigen in barley grain endosperm.

(1) Antigen expression cassette

Expression of the recombinant rHE_GFP coding sequence is controlled by the endosperm-specific AsGLO promoter derived from the globulin 1 gene of oat (Avena sativa), with transcription terminated by the nopaline synthase terminator (NOSt) derived from Agrobacterium tumefaciens. The AsGLO promoter originates from the barley hordein storage-protein regulatory region and exhibits endosperm-preferential activity, resulting in expression predominantly within developing grain tissues. This tissue-specific promoter was selected to promote accumulation of recombinant vaccine antigens within the seed, facilitating antigen bioencapsulation and oral delivery through feed.

The rHE_GFP coding sequence is a chimeric sequence encoding a recombinant fusion protein comprising a fragment of the Infectious Salmon Anaemia Virus (ISAV) hemagglutinin-esterase (HE) antigen and Green Fluorescent Protein (GFP).  The HE coding sequence encodes the viral hemagglutinin-esterase surface glycoprotein. This protein functions in receptor recognition and constitutes one of the principal protective antigens used in experimental ISAV vaccine development. The HE coding sequence was translationally fused to the Green Fluorescent Protein (GFP) coding sequence derived from Aequorea victoria. GFP was incorporated into the fusion protein to enable visualization and monitoring of recombinant antigen expression and localization in plant tissues.

The rHE_GFP coding sequence includes additional elements not shown in the genetic construct diagram:  The N-terminal signal peptide (ZmCKX1) directs the recombinant protein into the secretory pathway, the C-terminal  signal (KDEL) promotes retention and accumulation of the recombinant protein in the endoplasmic reticulum. The recombinant construct additionally contains two 6×His tags for immunodetection and purification, separated by a flexible (GGGGS)₂ linker; an enterokinase cleavage site (DDDDK) between the antigen component and GFP; and a Myc epitope tag (EQKLISEEDL) for immunodetection (Micúchová et al., 2024). The chimeric coding sequence was optimized for barley codon usage.

(2) Selectable marker cassette

The T-DNA additionally contains a hygromycin B phosphotransferase (hpt) gene derived from Escherichia coli. The hpt gene encodes an aminoglycoside phosphotransferase enzyme that detoxifies hygromycin B and permits selection of transformed cells during tissue culture and plant regeneration. Expression of the selectable marker is controlled by the Cauliflower mosaic virus 35S promoter (35Sp) and the nopaline synthase terminator (NOSt) derived from Agrobacterium tumefaciens.