Creating directed mutation through CRISPR/Cas9 gene editing system to improve soybean seed quality
Soybean seeds are a source of food for humans, feed for animals and an ingredient in the food industry. However, the high content of the raffinose family (RFO) in soybean seeds causes indigestion and reduces the efficiency of nutrient absorption. Therefore, studies on improving soybean seed quality through reducing RFO content have been of interest in recent years.
After two years of implementation, the project "Research and application of CRISPR/Cas9 technology in creating gene mutation to reduce the accumulation of raffinose to improve soybean seed quality", code VAST02.04 / 19-20, directed by Assoc. Prof. Dr. Pham Bich Ngoc, the Institute of Biotechnology, has been accepted at the grassroots level and ranked excellent. The project created mutant soybean lines with much lower RFO content than the control plants, and high levels of easy-to-digest sugar.
In this study, the scientists used the CRISPR/Cas9 gene editing system to mutate the group of genes encoding Galactinol synthase to reduce the amount of raffinose indigestible sugar (RFOs) in soybean seeds. Vectors carrying CRISPR/Cas9 structure were designed and transferred into soybean plants via Agrobacterium tumefaciens bacteria. In the T0 generation, different mutant soybean lines were obtained. Prospective types continued to be sown to obtain the expected mutant lines of two genes GmGOLS1A and GmGOLS1B in the T1 and T2 generations.
Figure 1A: Determination of directed mutation through sequencing of genes of interest in the T2 generation. GmGOLS1A-WT, GmGOLS1B-WT: GmGOLS1B-WT gene sequence encoding Galactinol synthase of the soybean control plant; Figure 1B: T2 generation dissociation of mutant soybean lines D1.1; Analysis was performed on 2% agarose gel
The results of biochemical analysis on the seeds of the T2 generation with genetic stability to determine the sugar content of soybean seeds showed a clear change in the seed composition of the mutant soybean lines. Of which, the total content of RFO (Raffinose Family Oligosaccharides) indigestible sugar in single-gene mutant lines decreased by 32% compared to the control plants, and in mutant lines both genes decreased 34.1% compared to the control plants (Fig. 2 A).
In addition, although there was no difference in the amount of stachyose between the single mutation and the mutant concurrent two genes, compared with WT seed (Non-mutant control soybean) stachyose content in the mutant lines was down to 30%. In T3 seeds, the sucrose concentration in single-mutant lines decreased by 25.4% compared to WT, while sucrose levels in mutant lines of the two genes were not significantly different from WT (Figure 2C). However, when calculating the ratio of individual carbohydrates to total dissolved carbohydrates, an increase in the sucrose ratio was observed in both single-gene mutants and two-gene mutation. (Figure 2B).
Ingredients of sugar in the seeds of soybean lines. Figure 2A: Total carbohydrate content and total RFOs indigestible sugar measured by HPLC; Figure 2B: Ratio of stachyose and sucrose to total dissolved carbohydrates; Figure 2C: C: Carbohydrate concentrations in soybean seeds; (DT26: soybean mutant control - WT; D1.1.5-4, D1.1.7-1, D1.1.7-2, D1.1.14-3: Mutant soybean seeds T3)
Other components, including moisture, protein, fat, and starch in the seeds were also analyzed. Measurement results showed no significant difference in starch ratio between mutant lines and WT. However, the ratio of protein and fat in the single-gene mutant line was higher (40.06% and 21.9% respectively) than WT (38.30% and 19.8% respectively). Meanwhile, no change of these values was recorded for mutant lines of two genes at the same time. This result indicates that the two genes GmGOLS1A and GmGOLS1B are not only involved in RFO biosynthesis.
Figure 3A: Growth of DT26 (WT) and T2 mutant soybean lines (D1.1.5-4, D1.1.7-1, D1.1.7-2, D1.1.14-3) in net houses of the Institute of Biotechnology
Figure 3B: Evaluation of germination of mutant soybean lines compared with WT line
Figure 3C: Growth parameters, including number of parts, number of branches, and leaf size of soybean lines growing in the net house
In addition, the scientists also evaluated the growth and development of soybean mutant lines in net house conditions. Results showed that mutant lines had changes in RFO content in seeds that grew and developed normally, with no difference compared to the control plants (Figures 3A and 3C). In addition, the germination rate of the T3 soybean seeds was also evaluated. The comparison showed that the mutation did not affect the germination strength of the soybean seeds (Figure 3B). Thus, in terms of morphology and growth, the soybean mutant plants did not show any difference compared to the control plants under the same growing conditions.
The results of the project are the basis for further studies on sugar biosynthesis in soybean as well as some other crops. Research also contributes to confirm the role as well as the interaction between genes related to enzymes involved in sugar biosynthesis in seeds. This is the first success in gene editing on Vietnamese soybean varieties, opening up potential for application in improving soybean varieties in the country. In addition, the use of soybean varieties with low indigestible sugar will improve consumers’ health, and improve efficiency in production and breeding. The research results of the project have been published in the International Journal of Frontiers in Plant Science: https://www.frontiersin.org/articles/10.3389/fpls.2020.612942/full
Translated by Phuong Huyen
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