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Determination of genomic location and structure of the transgenes in marker-free rice-based cholera vaccine by using whole genome resequencing approach
- Mejima, Mio, Kashima, Koji, Kuroda, Masaharu, Takeyama, Natsumi, Kurokawa, Shiho, Fukuyama, Yoshiko, Kiyono, Hiroshi, Itoh, Kimiko, Mitsui, Toshiaki, Yuki, Yoshikazu
- Plant cell, tissue, and organ culture 2015 v.120 no.1 pp. 35-48
- Agrobacterium radiobacter, RNA interference, Southern blotting, cholera, cholera toxin, chromosomes, gene overexpression, homozygosity, humans, intergenic DNA, oral vaccination, progeny, rice, self-pollination, storage proteins, transfer DNA, transgenes, vaccines
- 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.