Location: Crop Improvement and Genetics Research
Project Number: 2030-21000-020-07-R
Project Type: Reimbursable
Start Date: Oct 1, 2013
End Date: Sep 30, 2015
1) Test several novel exchange (EXCH) vectors containing the genome engineering components for Recombinase Mediated Cassette Exchange (RMCE) in transgenic soybean “founder” lines that contain target sequences for unidirectional site-specific recombinase enzymes Bxb1 and CinH. Bxb1 is capable of controlled integration while CinH is specific for excision of genomic DNA. The presence and orientation of their specific recognition sites is predicted to drive the system to completion in a two-step sequential process. 2) Use site-specific RMCE for sequential gene stacking in soybean founder lines to modify oil production and to stack improved seed meal traits. The first RMCE targeting will add to the genome an RNAi (gene suppression) construct to lower both Fad3 and Fad2-1 activities, thereby reducing linolenate and linoleate production. A second round of RMCE will be used to stack genes that are designed to lower oligosaccharide content, increase sucrose content, and/or increase protein accumulation in the seed. The genes used in the seed meal improvement experiments will be chosen based on the funding organization’s recommendations.
The suppression of both the Fad2-1 and Fad3 genes has previously been demonstrated to increase oleate concentration in soybeans. A set of pEXCH-1 vectors designed to suppress Fad2-1 and Fad3 via RNA interference (RNAi) and containing expression cassettes for CinH and Bxb1 recombinases will be assembled using standard molecular biology cloning techniques. The various expression cassettes will be tested for functionality through transient assays for either recombinase activity or RNAi expression. Agrobacterium-mediated transformation will be used to perform site-specific gene integration into previously constructed founder lines that contain target sites for Bxb1 recombinase. Molecular analyses will be performed to confirm targeted transgene integration of the Fad2-1 and Fad3 RNAi transgenes and selectable marker gene removal. The unidirectional activity of these recombinase enzymes will produce stable transgene loci. Thus, the GE soybean lines produced are expected to have predictable and stable levels of expression of the high oleate trait. These lines will be propagated and their seed oil composition characterized. This will complete the first round of Recombinase-mediated Cassette Exchange (RMCE). In parallel, a second set of EXCH vectors (pEXCH-2) designed to improve seed meal composition will be constructed. These will be tested for recombinase functionality using transient assays. They will be introduced via Agrobacterium transformation into one or more of the high oleic GE soybean lines. This will comprise the second round of RMCE. Molecular analyses will be performed to confirm targeted transgene integration of the pEXCH-2 transgenes and selectable marker gene removal.