Citation

We previously developed a molecularly uniform rice-based oral cholera vaccine (MucoRice-CTB) by using an overexpression system for modified cholera toxin B-subunit, CTB (N4Q) with RNAi to suppress production of the major rice endogenous storage proteins. To establish MucoRice-CTB for human use, here we developed hygromycin phosphotransferase selection marker-free MucoRice-CTB by using two different Agrobacterium tumefaciens, each carrying a distinct T-DNA for co-transformation. In the marker-free candidates from co-transformants by segregation in the seed progeny, we selected a line with high CTB expression, line 51A, which we advanced to the T6 generation by self-pollination to obtain a homozygous line without down-regulation of CTB expression. Southern blot analyses showed that three copies of the CTB gene, but not the backbone of the T-DNA binary vector, were inserted into the rice genome of MucoRice-CTB line 51A. By whole genome resequencing, we showed that the transgenes in this line were inserted into intergenic regions in chromosome 3 and chromosome 12. We determined that two full-length copies, each containing the CTB and RNAi expression cassettes, were inserted in a tandem reverted orientation into chromosome 3. An additional copy of the CTB over-expression cassette with a truncated RNAi cassette was inserted into chromosome 12. These findings provide useful information for the establishment of a seed bank of marker-free MucoRice-CTB for human use. Keywords Selection marker-freeGene locationMucoRice-CTBOral vaccineOryza sativaCholera