Ai Sasou1, Yoshikazu Yuki2, Ayaka Honma1, Kotomi Sugiura1, Koji Kashima3, Hiroko Kozuka-Hata4, Masanori Nojima5, Masaaki Oyama4, Shiho Kurokawa1, Shinichi Maruyama3, Masaharu Kuroda6, Shinjiro Tanoue7, Narushi Takamatsu7, Kohtaro Fujihashi8, Eiji Goto9, Hiroshi Kiyono1,8,10,11. 1. Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 2. Division of Mucosal Immunology, IMSUT Distinguished Professor Unit, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. yukiy@ims.u-tokyo.ac.jp. 3. Asahi Kogyosha Co., Ltd., Tokyo, Japan. 4. Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 5. Center for Translational Research, IMSUT Hospital, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 6. Crop Development Division, NARO Agriculture Research Center, Niigata, Japan. 7. Astellas Pharma Inc., Tokyo, Japan. 8. Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 9. Faculty of Horticulture, Graduate School of Horticulture, Chiba University, Chiba, Japan. 10. Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan. 11. Chiba University-University of California San Diego Center for Mucosal Immunology, Allergy, and Vaccine, Division of Gastroenterology, Department of Medicine, University of California, San Diego, California, USA.
Abstract
BACKGROUND: We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker-free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. RESULTS: We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. CONCLUSIONS: All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.
BACKGROUND: We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker-free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. RESULTS: We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. CONCLUSIONS: All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.
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