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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

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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.

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Cholera toxin


Cholera toxin B-subunit


Enzyme-linked immunosorbent assay


Mass spectrometry


Phosphate-buffered saline


Polymerase chain reaction


RNA interference


SDS-polyacrylamide gel electrophoresis


Transfer DNA


Thermal asymmetric interlaced polymerase chain reaction


Wild type


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We are grateful to Drs. Masaaki Oyama, Hiroko Kozuka-Hata, Satoshi Kaneto, Shintaro Sato, and Mr. Yuji Suzuki for useful discussions and technical support. This work was supported by Grants from the Programs of Special Coordination Funds for Promoting Science and Technology and a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y. Y., H. K.); the Ministry of Health, Labor and Welfare of Japan (Y. Y., H. K.); the New Energy and Industrial Technology Development Organization (NEDO) (H. K.); and the Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-step); and the Research and Development Program for New Bio-industry Initiatives of the Bio-oriented Technology Research Advancement Institution (Y. Y.).

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Correspondence to Yoshikazu Yuki.

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Mejima, M., Kashima, K., Kuroda, M. et al. Determination of genomic location and structure of the transgenes in marker-free rice-based cholera vaccine by using whole genome resequencing approach. Plant Cell Tiss Organ Cult 120, 35–48 (2015).

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  • Selection marker-free
  • Gene location
  • MucoRice-CTB
  • Oral vaccine
  • Oryza sativa
  • Cholera