Dihomo-Gamma-linolenic acid (DGLA) is a polyunsaturated fatty acid that plays important roles in human health. However, our human body can produce only a very limited amount of DGLA from linoleic acid (LA) through biochemical reactions catalyzed by enzymes. Scientific studies have shown that DGLA supplementation can significantly improve the efficacy of anti-cancer and anti-aging drugs. Soybean oil is rich in linoleic acid (LA) (up to 51%), but it does not contain DGLA because the crop lacks the enzymes for DGLA biosynthesis. Our overall goal is to develop transgenic soybean varieties that produce high DGLA content for pharmaceutical uses. With funding from ND Soybean Council for the past three years, we have developed transgenic soybean plants expressing the enzymes D6D and D6E required for DGLA biosynthesis. The transgenic plants were generated from soybean cultivar William 82 and Thorne with different gene constructs. Fatty acids analysis indicated some of the transgenic plants produced a high level of GLA (accounting for up to 30% in the extracted oil) and a good level of DGLA (accounting for up to 12% in the extracted oil). In this proposal, we will screen more transgenic plants we generated from the transformation experiments for DGLA content in soybean oil and select the plants with higher DGLA production. We will also test the function of DGLA-enriched soybean oil in age-related conditions. Completion of the project will enhance the value of soybean product and expand the markets of soybean since the DGLA-enriched soybean oil has great potential for anti-cancer and anti-aging therapies.

1. Evaluate more transgenic soybean plants for GLA, DGLA and other oil contents
2. Develop soybean varieties with GLA- and DGLA-enriched soybean oil
3. Test GLA- and DGLA-enriched soybean oil as a treatment for age-related conditions (by Dr. Christopher Wiley)

Transgenic soybean varieties that produce high DGLA content in oil will be developed for soybean production to meet pharmaceutical industry needs for anti-cancer and anti-aging treatments.

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The Executive Summary

Why the research is important to ND soybean farmers

DGLA has been demonstrated to be potentially useful for cancer therapy and anti-aging treatment in humans, but sources of DGLA are very limited and cannot be found in the major vegetable oil producing crops because they lack the two enzymes (D6D, ?6 desaturase and D6E, ?6 elongase) to catalyze the conversion of linoleic acids (LA) into DGLA. By introducing the genes for expressing D6D and D6E into soybean plants, we developed transgenic soybean plants that produce GLA and DGLA in soybean oil. However, the function of GLA- and DGLA-enriched soybean oil in anti-aging treatment remain to be tested. By developing new soybean varieties that produce DGLA-enriched soybean oil, the value of soybean product can be increased and the markets of soybean will be expanded because DGLA-enrich oil has a huge potential for use in cancer therapy and for consumption by normal humans to reduce cancer- and aging-related risks.

Research conducted

We increased seeds from homozygous individuals (T1) derived from the four gene constructs (pBAtC: BCA-D6D-KTi-D6E, pBAtC: BCA-D6D-BCA-D6E, pBAtC: BCA-D6D-FAD2-D6E and pBAtC:BCA-D6D-Gmubi3-D6E) and analyzed contents of fatty acids including GLA and DGLA in twenty soybean samples. We extracted GLA- and DGLA-containing soybean oil from three transgenic soybean lines homozygous for the transgene. These oil samples were used for experiments on mice. We applied DGLA ethyl ester (DGLA-EE) on old mice and evaluated its effect on liver fibrosis and inflammation. We also tested DGLA/GLA-enriched soybean oil in a mouse model of chemotherapy-induced premature aging for its anti-aging function.

Findings of the research

We generated different transgenic soybean lines using the four gene constructs from two soybean varieties (Willimans and Thorne). We found that the transgenic soybean plants with different gene constructs varied in DGLA production depending on the promoters used. Some transgenic soybean lines produced little or none DGLA, while some other lines produced up to 33% GLA and 10% DGLA in the soybean oil. When old mice were given DGLA ethyl ester (DGLA-EE), markers of liver fibrosis and inflammation were reduced relative to vehicle-treated control mice. This result further confirmed the potential effectiveness of DGLA as a therapeutic that targets aging. DGLA-enriched soybean oil was also evaluated in mice pre-treated with a chemotherapeutic drug (doxorubicin, aka Adriamycin, abbreviated DOXO), but few phenotypic changes were observed in treated animals regardless of DGLA status. Senescent cells were also increased follow DGLA-enriched soybean oil in DOXO-treated mice, indicating that DGLA-enriched soybean oil has the opposite effect of DGLA-EE on senescence in this model.

Benefits/Recommendations to North Dakota soybean farmers and industry

With few sources of free DGLA currently available, DGLA-enriched soybean oil could act as a supply for this compound, but it may require further refinement from the triglyceride to the free fatty acid before any therapeutic benefits would be produced.

Soybean oil accounts for over 40 percent of the intake of linoleic acids (LA) and alpha-Linolenic acid (ALA) in the United States. However, no DGLA is produced by the soybean crop because it lacks the enzymes for DGLA biosynthesis. By introducing the genes for expressing the two enzymes, ?6 desaturase and ?6 elongase, into soybean plants, we developed transgenic soybean plants that produce GLA and DGLA in soybean oil. The soybean-derived DGLA product has a huge potential market because of its function in cancer therapy and anti-aging. By planting soybean varieties producing DGLA-enriched soybean oil, the farmers will have a better economic return due to the extended and enhanced value of soybean oil in healthcare and pharmaceutical industry, especially in cancer therapy and anti-aging treatment.