Cycle1：Selection and Construction of Core Elements for High-Folate Soybean Biosynthesis

Design

First, many enzymes are involved in folate biosynthesis, and we need to figure out which enzymes in soybeans can be selected to effectively increase folate levels. Second, since our goal is to boost folate content in soybean seeds, we need to decide on the right promoter to drive the expression of folate synthetase specifically in seeds. Finally, we will integrate the chosen promoter and folate synthetase gene into an expression vector, which will then be used for soybean genetic transformation.

Based on literature research, GCH1 (GTP Cyclohydrolase-1) gene and ADCS (Aminodeoxychorismate Synthase) gene encode the rate-limiting enzymes in folate synthesis. These enzymes catalyze the conversion of GTP to dihydroneopterin triphosphate (DHNTP) and chorismate to aminodeoxychorismate (ADC), respectively, thereby supplying the necessary precursor substances for folate production. These two enzymes have been widely used in bioengineering other plants like Arabidopsis and rice (de La Garza et al., 2007; Liang et al., 2019; Zamberlan, 2024). For this reason, GCH1 and ADCS from soybeans were chosen as our primary candidate genes. Next, HPPK (Hydroxyphenylpyruvate Dioxygenase Kinase) and DHFR (Dihydrofolate Reductase) were also selected as candidate genes. These two enzymes are responsible for making dihydropteroate (a building block for dihydrofolate) and tetrahydrofolate, respectively (Waller et al., 2010). Since soybeans can naturally produce folate, these enzymes are all present at measurable levels in developing soybean seeds.

During soybean seed development, 4 genes that show high activity in the middle to late stages of seed ripening were screened. The promoters from these genes were then used to control the activity of the candidate enzymes involved in folate production. The four genes identified are Oleosin-1, Oleosin-2, LEA5, and LEA7, respectively.

Build and Test

The screened promoters and candidate genes were constructed into the pTF-Flag-35S expression vector, and the following 4 vector constructions were obtained: GmOleosin-1p:GmGCH1, GmOleosin-2p:GmADCS, GmLEA5p:GmHPPK, GmLEA7:GmDHFR.

Learn

Since soybeans can naturally produce folate and the key genes in their folate synthesis pathway show some level of activity during seed development, we didn't plan to use multi-gene stacking systems (which allow multiple genes to be expressed at once) when first designing the synthetic components. But when we work on improving the project later, we'll focus first on using these multi-gene systems. This should let us boost several enzyme reactions at the same time in a single step. Next, when building the vectors, we'll look into adding components that can remove the vector backbone from transgenic plants. This would make the plants safer, help them pass regulatory reviews more easily down the line, and speed up the process of getting them into production.

Cycle2：Acquisition and Detection of Genetically Modified Soybeans

Design

The constructed expression vector was used for soybean genetic transformation, with the high-efficiency genetic transformation soybean variety HC6 selected as the recipient. This variety is also a widely promoted spring-planted cultivar in South China. Soybean genetic transformation was carried out using the half-cotyledon node method, which involves the following steps: explant preparation, infection and co-cultivation, induction and screening of clustered buds, stem elongation culture, and rooting culture.

Build

We mastered and completed the entire soybean genetic transformation process, successfully obtaining transgenic soybean plants.

Test

A total of 149 transgenic seedlings were obtained from 4 transformation events, with 115 positive seedlings confirmed by PCR. For each transformation event, 37 positive seedlings were produced by GmOleosin-1p:GmGCH1 transformation, 29 by GmOleosin-2p:GmADCS transformation, 21 by GmLEA5p:GmHPPK transformation, and 28 by GmLEA7:GmDHFR transformation.

Learn

For the transgenic plants of the current generation obtained, the transgenic vector signal was only detected at the genomic DNA level. The high expression of the experimental target gene in soybean seeds by the overexpression vector at the transcriptional level was not detected. The expression level of the target gene will be detected in the next generation.

Cycle3：Extraction of Folate and Detection of Its Active Components

Design

This section involves two key tasks. First, a process for extracting folate from dry soybean seeds must be established to ensure the effective folate components in soybean seeds can be obtained. Second, the detection method for the active components of folate needs to be determined.

Build

Based on previously published folate extraction steps (Sun et al., 2022), we optimized the process for extracting folate from dry soybean seeds, resulting in folate extracts with high purity.

Five folate standards were selected, including folic acid (FA), tetrahydrofolic acid (THF), 5-methyl-tetrahydrofolate (5M-THF), 5,10-methylene-tetrahydrofolic acid (5,10M-THF), and 10-formyl-folic acid (10F-THF). These standards were analyzed using HPLC-MS with an Agilent 1290UPLC-6470A triple quadruple mass spectrometer. All five folate components were found to be accurately identifiable.

Test

Detection of the five folate components mentioned above in transgenic positive seedlings revealed 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 our transgenic plants. This marked increase was validated across multiple independent transgenic plants. Other folate components showed slight increases, though these changes were not statistically significant. Since 5M-THF represents the primary active form of folate, these findings indicate a significant advancement in folate biosynthesis research in soybeans.

Learn

We selected 5 forms of folate for detection, and more forms of folate should be chosen for comprehensive detection in the future. Meanwhile, some forms of folate are extremely easy to be inactivated. For example, FA is extremely easy to be degraded upon exposure to light. We will enhance the management of sample storage and transportation next.