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Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine
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 a...
Autores principales: | , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814724/ https://www.ncbi.nlm.nih.gov/pubmed/33468052 http://dx.doi.org/10.1186/s12864-020-07355-7 |
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author | Sasou, Ai Yuki, Yoshikazu Honma, Ayaka Sugiura, Kotomi Kashima, Koji Kozuka-Hata, Hiroko Nojima, Masanori Oyama, Masaaki Kurokawa, Shiho Maruyama, Shinichi Kuroda, Masaharu Tanoue, Shinjiro Takamatsu, Narushi Fujihashi, Kohtaro Goto, Eiji Kiyono, Hiroshi |
author_facet | Sasou, Ai Yuki, Yoshikazu Honma, Ayaka Sugiura, Kotomi Kashima, Koji Kozuka-Hata, Hiroko Nojima, Masanori Oyama, Masaaki Kurokawa, Shiho Maruyama, Shinichi Kuroda, Masaharu Tanoue, Shinjiro Takamatsu, Narushi Fujihashi, Kohtaro Goto, Eiji Kiyono, Hiroshi |
author_sort | Sasou, Ai |
collection | PubMed |
description | 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-020-07355-7. |
format | Online Article Text |
id | pubmed-7814724 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-78147242021-01-21 Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine Sasou, Ai Yuki, Yoshikazu Honma, Ayaka Sugiura, Kotomi Kashima, Koji Kozuka-Hata, Hiroko Nojima, Masanori Oyama, Masaaki Kurokawa, Shiho Maruyama, Shinichi Kuroda, Masaharu Tanoue, Shinjiro Takamatsu, Narushi Fujihashi, Kohtaro Goto, Eiji Kiyono, Hiroshi BMC Genomics Research Article 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-020-07355-7. BioMed Central 2021-01-19 /pmc/articles/PMC7814724/ /pubmed/33468052 http://dx.doi.org/10.1186/s12864-020-07355-7 Text en © The Author(s) 2021 Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Article Sasou, Ai Yuki, Yoshikazu Honma, Ayaka Sugiura, Kotomi Kashima, Koji Kozuka-Hata, Hiroko Nojima, Masanori Oyama, Masaaki Kurokawa, Shiho Maruyama, Shinichi Kuroda, Masaharu Tanoue, Shinjiro Takamatsu, Narushi Fujihashi, Kohtaro Goto, Eiji Kiyono, Hiroshi Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine |
title | Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine |
title_full | Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine |
title_fullStr | Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine |
title_full_unstemmed | Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine |
title_short | Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine |
title_sort | comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of mucorice-ctb 51a line, a rice-based oral cholera vaccine |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814724/ https://www.ncbi.nlm.nih.gov/pubmed/33468052 http://dx.doi.org/10.1186/s12864-020-07355-7 |
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