Updated March 26, 2020:
Key Activities
The major activities associated with this project include synthesis of RNAi silencing construct for the soy BADC genes, soybean transformation, and seed composition phenotyping. The first two activities were performed with external assistance. GeneArt synthesized the RNAi cassette that was subcloned into a binary vector containing a glycinin (seed-specific) promoter. The University of Missouri Transformation Facility led by Dr. Zhanyuan Zhang is currently performing the soy transformation. Seed composition analysis will be performed in the Thelen lab using both gravimetric and gas/liquid chromatography-mass spectrometry platforms in his group. MU Plant Growth Facilities will provide the greenhouse space for transgenic soy propagation on a fee-for-service basis.
Key Accomplishments
We have determined the ideal nucleotide sequences for the creation of a binary hairpin construct for RNAi silencing of both BADC isoform 1 and 2 in soybean. The sequence of this hairpin RNAi cassette was used for gene synthesis through the company GeneArt. The synthetic DNA was returned and cloned into the pMU103 binary vector. The pMU103 binary vector contains a seed-specific glycinin promoter to drive expression of this tandem RNAi cassette exclusively during seed maturation. We sequence confirmed this binary construct is authentic. We have been working with the Soybean Transformation Facility at Mizzou and University of Nebraska-Lincoln to make transgenic soybean. Both facilities have had our synthesized construct for silencing all BADC forms in soybean specifically during seed maturation and showed some progress by providing ten and one putative transformant, respectively. We have confirmed these by genomic PCR to be valid transgenic lines. We are now in the process of phenotyping these lines. This will be focus of the next round of funding.
KPIs (as listed in submitted proposal)
1) At least three independent transgenic soybean lines with 70-100% reduction in total BADC gene expression as compared to non-transgenic reference lines.
2) A significant and reproducible increase in soy seed oil content of at least 5% dry weight in at least three independent transgenic events as compared to non-transgenic reference lines.
3) No significant change in soy seed protein content in high oil transgenic lines compared to non-transgenic reference lines.
4) Identification of either government or industrial partners capable of transferring this trait into elite soy breeding lines through either selective breeding (gene mutation) or transgenic silencing.
Progress towards each KPI
1) Presently, we have eleven transgenic soybean lines. All have been confirmed by genomic PCR. Phenotyping has commenced.
2) No progress at present time.
3) No progress at present time
4) We currently have a corporate partner on the pending worldwide patent and exclusive licensing options therein. They are moving this trait into canola and camelina for tentative field trials in 2018.
Expected Outputs/Deliverables
1) A binary vector RNA interference construct capable of reducing expression of all isoforms of BADC genes in any soybean variety through RNA interference. This construct will contain gene sequences specific to BADC isoforms 1 and 2 in tandem as part of a coupled RNAi gene silencing strategy. We have used this gene silencing strategy in Arabidopsis as proof-of-principle.
2) Transgenic soybean lines with significantly (t-test, one-way ANOVA) higher levels of seed oil than nontransgenic reference lines.
Results on Deliverables
1) Completed. The RNAi cassette was designed and synthesized by GeneArt, and cloned into the pMUC binary vector. We are moving it into additional binary vectors for soy and camelina as well.
2) Ongoing. We have eleven confirmed transgenic soybean lines that produced seed, all independent events.
We did not anticipate soybean transformation would be so challenging with this project. This delayed the project substantially and we have been trying to catch up ever since. Additionally, we did not anticipate that genomic PCR would be so difficult with soybean. This took a while to verify this as all of our procedures employed Taq polymerase which is incompatible with the high GC content of soybean. This was discovered through iterative analysis.
As we still need to determine if transgenic soybean with reduced BADC expression produce higher oil seed, it is difficult to answer this question at the present time. We anticipate a marked increase in seed oil in soybean as repeatedly observed in Arabidopsis when one or two of the BADCs are silenced either by RNAi or T-DNA knockout. Soybean containing higher seed oil without a penalty on protein composition or seed harvest index would add commodity value that could be rewarded by seed processing companies.
We have not yet established the composition of BADC RNAi transgenic seed.
Soybean cultivars with decreased expression of the BADC gene, a negative regulator of fatty acid synthesis, were produced. These lines are not yet characterized for oil and protein content due to technical difficulties, so it is still unknown if this change has resulted in improved oil content in the soybean seed. However, since the approach has been successful increasing seel oil content in Arabidopsis and the technical difficulties encountered while testing showed clear, qualitative differences from control and low oil varieties, the results are interesting and demonstrate the oil production pathway being manipulated was well chosen.