Dihomo-gamma-linolenic acid (DGLA) is a polyunsaturated fatty acid. Recent studies in Dr. Qian lab (College of Health Professions, NDSU) discovered that DGLA has anti-cancer effects on various types of cancer cells because DGLA can be metabolized to a cancer inhibitory molecule by an enzyme produced by cancer cells and tumors. The research also showed that DGLA-supplementation can significantly improve the efficacy of new anti-cancer strategies/dugs discovered by Dr. Qian at NDSU. In mammals, only very limited DGLA (<0.02 %) can be converted from linoleic acid through biochemical reactions catalyzed by enzymes. Soybean oil is rich in linoleic acid (up to 51%), but does not contain DGLA because the crop lacks the enzymes for DGLA biosynthesis. Our ultimate goal is to develop transgenic soybean lines with high DGLA content for cancer therapy use. With funding from ND Soybean Council for the first year, we cloned genes expressing the two enzymes for production of DGLA, introduced them into soybean through Agrobacterium-mediated transformation, and generated seven transgenic plants from soybean cultivar William 82. In this proposal, we will evaluate the transgenic soybean plants for DGLA content and continue to improve expression of the genes for DGLA production in soybean plants by genetic engineering. Completion of the project will enhance the value of soybean product and expand the markets of soybean since the DGLA-enriched oil/soybean can be used not only for cancer patients in clinic to treat cancers with new drugs discovered by NDSU but also for normal human population to decrease cancer incident.

1. Enhance expression of the two genes (delta-6-desaturase and delta-6-elongase) required for DGLA biosynthesis in transgenic soybean plants.
2. Generate more transgenic plants with high DGLA-enriched oil in other soybean varieties.
3. Evaluate transgenic soybean plants for DGLA and other oil contents.

Transgenic soybean varieties with high DGLA content in oil will be developed for corn production to meet the needs of cancer therapy.

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Objectives of the research:

1. Enhance expression of the two genes for ?6 desaturase (D6D) and ?6 elongase (D6E) required for DGLA biosynthesis in transgenic soybean plants.
2. Generate more transgenic plants with high DGLA-enriched oil in other soybean varieties.
3. Evaluate transgenic soybean plants for DGLA and other oil contents.

Completed work:

• A new vector (pBAtC:Soybean-H1) was constructed for improving expression of the two genes for delta-6-desaturase (D6D) and delta-6-elongase (D6E) required for DGLA biosynthesis.
• Soybean cultivar Williams 82 was transformed by the Agrobacterium-mediated transformation system with the vector pBAtC:Soybean-H1 and 24 T0 transgenic soybean plants were produced. Among them, 21 plants were confirmed to have the genes for D6D and D6E and are being grown in the greenhouse to produce T1 seeds.
• Soybean cultivar Thorne was also transformed by the Agrobacterium-mediated transformation approach with the vector pBAtC:Soybean-H1 and 22 T0 transgenic soybean plants were generated. Among them, 17 plants were confirmed to carry the genes for D6D and D6E.
• Genetically stable transgenic Williams 82 plants producing a high content of GLA were developed and enough seeds were increased in greenhouse for potential field experiment.

Preliminary results:

• The genes expressing the two enzymes delta-6-desaturase (D6D) and delta-6-elongase (D6E) were synthesized and cloned into the corresponding sites of the binary vector pBAtC. Our previous study indicated that the promoter of beta-conglycinin alpha subunit gene from soybean was highly effective for driving the expression of the D6D gene. To enhance D6E expression in soybean seeds, the beta-conglycinin alpha subunit promotor from soybean was also used for driving the expression of the genes for D6E in the binary vector. We constructed a new vector (pBAtC:Soybean-H1), which carried the beta-conglycinin alpha subunit promoter for both gene for D6D and D6E separately.
• We introduced pBAtC:Soybean-H1 into the Agrobacterium strain EHA105 for soybean transformation.
• Using Agrobacterium-mediated transformation, we introduced the vector pBAtC:Soybean-H1 into two soybean cultivars Williams 82 and Thorne. A total of 24 T0 transgenic plants were obtained for Williams 82 and 21 of them were confirmed to contain the genes for D6D and D6E by PCR analysis. For Thorne, 22 T0 transgenic plants were obtained and 17 of them were confirmed to carry the genes for D6D and D6E based on PCR analysis.
• We also developed genetically stable transgenic soybean plants that showed high expression of D6D, which converts linoleic acid (LA) into gamma-Linolenic acid (GLA). Normal soybean oil does not contain GLA, but the genetically modified soybean plants produced GLA accounting for 30% of the soybean oil. GLA has been reported to have anti-inflammatory properties and may be used as supplemental to benefit some patients.

Work to be completed:

• Gene expression analysis will be conducted on the transgenic soybean plants.
• Homozygous transgenic plants will be identified by PCR analysis.
• DGLA and other oil contents in seeds of the transgenic soybean plants from the two soybean cultivars William 82 and Thorne will be analyzed to determine the amount of DGLA in the soybean oil.
• Genetically stable transgenic plants having a high level of DGLA production will be selected and registered as new special varieties for production of DGLA-enriched oil.
• The function of DGLA-enriched soybean oil in cancer therapy will be evaluated in the future.

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The Executive Summary
Research conducted
DGLA (dihomo-gamma-linolenic acid) is a 20-carbon polyunsaturated fatty acid, which plays an important role in human health, especially for halting cancer growth. However, major vegetable oils lack DGLA since the main oil-producing crops (soybean, corn, canola and so on) do not have the two genes encoding the enzymes [(delta-6-desaturase (D6D) and delta-6-elongase (D6E)] for DGLA biosynthesis. Our research goal is to develop transgenic soybean lines that express D6D and D6E for converting linoleic acid (LA) to DGLA and producing DGLA-enriched oil for cancer therapy use. We made a new gene construct with genes for expression of D6D and D6E and transgenic soybean plants were generated from two soybean cultivars William 82 and Thorne through Agrobacterium-mediated transformation. The fatty acids of transgenic soybean samples were analyzed and GLA (gamma-linolenic acid) and DGLA were detected in the transgenic soybean seeds. We will continue to improve the DGLA productivity in soybean plants.

Why the research is important to ND soybean farmers
Soybean oil accounts for over 40 percent of the intake of linoleic acids (LA) and alpha-Linolenic acid (ALA) in the United States because of its widespread use, but the crop fails to produce DGLA because it lacks the enzymes for DGLA biosynthesis. The research will develop new soybean varieties that produce DGLA-enriched soybean oil, which has a huge potential for use in cancer therapy and for consumption by normal humans to reduce cancer-related risks. The research will enhance the value of soybean product and expand the markets of soybean.

Final findings of the research
Transgenic soybean plants with the two genes required for DGLA biosynthesis have been developed. Some of the transgenic soybean plants produced up to 33.3% GLA and up to 10.25% DGLA in the soybean oil.
Benefits/Recommendations to North Dakota soybean farmers and industry

By growing new soybean varieties that produce 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.

Soybean oil accounts for over 40 percent of the intake of linoleic acids (LA) and alpha-Linolenic acid (ALA) in the United States because of its widespread use, but the crop fails to produce DGLA because it lacks the enzymes for DGLA biosynthesis. By introducing the genes for expressing the enzymes delta-6-desaturase and delta-6 elongase into soybean plants, we anticipate to develop transgenic soybean varieties that can produce DGLA-enriched soybean oil. The product has a huge potential market because of its function in cancer therapy. By planting the 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.