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CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 sys...

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Autores principales: Le, Huy, Nguyen, Nhung Hong, Ta, Dong Thị, Le, Thao Nhu Thi, Bui, Thao Phuong, Le, Ngoc Thu, Nguyen, Cuong Xuan, Rolletschek, Hardy, Stacey, Gary, Stacey, Minviluz G., Pham, Ngoc Bich, Do, Phat Tien, Chu, Ha Hoang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773711/
https://www.ncbi.nlm.nih.gov/pubmed/33391326
http://dx.doi.org/10.3389/fpls.2020.612942
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author Le, Huy
Nguyen, Nhung Hong
Ta, Dong Thị
Le, Thao Nhu Thi
Bui, Thao Phuong
Le, Ngoc Thu
Nguyen, Cuong Xuan
Rolletschek, Hardy
Stacey, Gary
Stacey, Minviluz G.
Pham, Ngoc Bich
Do, Phat Tien
Chu, Ha Hoang
author_facet Le, Huy
Nguyen, Nhung Hong
Ta, Dong Thị
Le, Thao Nhu Thi
Bui, Thao Phuong
Le, Ngoc Thu
Nguyen, Cuong Xuan
Rolletschek, Hardy
Stacey, Gary
Stacey, Minviluz G.
Pham, Ngoc Bich
Do, Phat Tien
Chu, Ha Hoang
author_sort Le, Huy
collection PubMed
description Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.
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spelling pubmed-77737112021-01-01 CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds Le, Huy Nguyen, Nhung Hong Ta, Dong Thị Le, Thao Nhu Thi Bui, Thao Phuong Le, Ngoc Thu Nguyen, Cuong Xuan Rolletschek, Hardy Stacey, Gary Stacey, Minviluz G. Pham, Ngoc Bich Do, Phat Tien Chu, Ha Hoang Front Plant Sci Plant Science Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing. Frontiers Media S.A. 2020-12-17 /pmc/articles/PMC7773711/ /pubmed/33391326 http://dx.doi.org/10.3389/fpls.2020.612942 Text en Copyright © 2020 Le, Nguyen, Ta, Le, Bui, Le, Nguyen, Rolletschek, Stacey, Stacey, Pham, Do and Chu. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Le, Huy
Nguyen, Nhung Hong
Ta, Dong Thị
Le, Thao Nhu Thi
Bui, Thao Phuong
Le, Ngoc Thu
Nguyen, Cuong Xuan
Rolletschek, Hardy
Stacey, Gary
Stacey, Minviluz G.
Pham, Ngoc Bich
Do, Phat Tien
Chu, Ha Hoang
CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds
title CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds
title_full CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds
title_fullStr CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds
title_full_unstemmed CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds
title_short CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds
title_sort crispr/cas9-mediated knockout of galactinol synthase-encoding genes reduces raffinose family oligosaccharide levels in soybean seeds
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773711/
https://www.ncbi.nlm.nih.gov/pubmed/33391326
http://dx.doi.org/10.3389/fpls.2020.612942
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