Overview
To develop soybeans with high folate content, a set of biosynthetic components was constructed and their feasibility was successfully confirmed. First, vector modules were built to overexpress key enzymes involved in folate biosynthesis specifically in soybean seeds. Second, these vector modules were introduced into soybeans using the Agrobacterium-mediated cotyledon node method, resulting in the generation of transgenic plants. Finally, it was verified that the introduced modules effectively led to a one-fold increase in the accumulation of active folate in soybean seeds.
Part 1. Selection of Key components for High-folate Soybean Bio-system
Four enzymes, GCH1 (GTP cyclohydrolase-1), ADCS (Aminodeoxychorismate Synthase), HPPK (Hydroxyphenylpyruvate Dioxygenase Kinase), and DHFR (Dihydrofolate Reductase), were selected as candidate genes to design elements for increasing folate content in soybeans. These enzymes act on different nodes of the folate biosynthesis pathway, respectively (de La Garza et al., 2007; Liang et al., 2019; Zamberlan, 2024). All of these enzymes are expressed at certain levels during soybean seed development.
In addition, four genes with high expression in the middle and late stages of seed maturation were screened, and their promoters were used to drive the expression of the candidate folate synthesis enzymes. These four genes are Oleosin-1, Oleosin-2, LEA5, and LEA7.
Part 2. Construction of Overexpression Vectors
We constructed the screened promoters and candidate genes into the pTF-Flag-35S expression vector, and obtained the following 4 vector constructions: GmOleosin-1p:GmGCH1, GmOleosin-2p:GmADCS, GmLEA5p:GmHPPK, GmLEA7:GmDHFR. The accuracy of the inserted sequences was verified by sequencing.
Part 3. Acquisition and Detection of Genetically Modified Soybeans
Soybean genetic transformation was carried out using the constructed expression vector, with HC6 selected as the recipient variety. This variety not only has high genetic transformation efficiency but is also a widely cultivated spring-planted soybean in South China. The half-cotyledon node method was employed for the transformation process, which includes explant preparation, infection and co-cultivation, induction and screening of clustered buds, stem elongation culture, and rooting culture.
A total of 149 transgenic seedlings were obtained from 4 transformation events, and PCR verification confirmed 115 of these as positive. Among the individual transformation events, 37 positive seedlings were obtained from GmOleosin-1p:GmGCH1 transformation, 29 from GmOleosin-2p:GmADCS transformation, 21 from GmLEA5p:GmHPPK transformation, and 28 from GmLEA7:GmDHFR transformation.
Part 4. Extraction of Folate
The process for extracting folate from soybean dry seeds was optimized based on the published extraction steps (Sun et al., 2022), and a folate extract with high purity was obtained.
Part 5. Establishment of Detection Method for Active Components of Folate
Five forms of folate standards, including folic acid (FA), tetrahydrogen folic acid (THF), 5-methyl tetrahydrofolate (5M-THF), 5,10-methylene-tetrahydrofolic acid (5,10M-THF), and 10-formyl folic acid (10F-THF), were selected and detected by HPLC-MS using an Agilent 1290UPLC-6470A triple quadruple mass spectrometer. All these five folate components were found to be accurately identifiable.
Part 6. Identification of Active Folate Components in Genetically Modified Soybeans
The five folate components mentioned above were detected in genetically modified positive seedlings, and it was found that different candidate genes exert varying effects on the final folate content. Overexpression of the DHFR enzyme doubled the 5M-THF content in soybean seeds, rising from 410 μg/100g seeds in the control group to 867 μg/100g seeds in the transgenic plants. This marked increase was confirmed across multiple transgenic plant lines. Other folate components showed slight increases, though these changes were not statistically significant.
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