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::GP_LTB-S2-1-1). The inserted T-DNA contains an antigen expression cassette encoding the recombinant rGP_LTB 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 rGP_ LTB 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 rGP_ LTB coding sequence is a chimeric sequence encoding a recombinant fusion protein comprising a fragment of the Spring Viraemia of Carp Virus (SVCV) envelope glycoprotein (GP) and heat-labile enterotoxin B subunit (LTB) derived from Escherichia coli. The GP fragment corresponds to amino acid residues 243–337 of the SVCV envelope glycoprotein (95 amino acids). The SVCV glycoprotein is a viral envelope protein involved in receptor-mediated attachment and entry into host cells and is immunogenic, capable of eliciting host immune responses. LTB is a non-toxic mucosal adjuvant that binds GM1 ganglioside receptors and is frequently incorporated into recombinant oral vaccine formulations to enhance antigen uptake and stimulation of mucosal immune responses.

The rGP_LTB 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; 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.