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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.