Stacking Multiple Transgenes at a Selected Genomic Site via Repeated Recombinase-Mediated DNA Cassette Exchanges

Abstract

Recombinase-mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of transgenes can be stacked to the same site through multiple rounds of RMCE. A gene-silencing cassette, designed to simultaneously silence soybean (Glycine max) genes fatty acid ω-6 desaturase 2 (FAD2) and acyl-acyl carrier protein thioesterase 2 (FATB) to improve oleic acid content, was first inserted by RMCE at a precharacterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley (Hordeum vulgare) high-lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS), to improve the contents of the essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four overexpression cassettes to the previously integrated FAD2-FATB gene-silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes.

Influence of carbon to nitrogen ratios on soybean somatic embryo (cv. Jack) growth and composition

“…3A). The oil levels observed in these experiments are in good agreement with the oil content of somatic embryos reported by other researchers (Schmidt et al , 2005; Li et al , 2010; He et al , 2011). Although a linear regression line, derived from a plot of the oil and protein data, showing that a 1% reduction in oil content led to an apparent 1.82% increase in protein content, a value that was close to the 2% rule of thumb suggested for soybean by Clemente and Cahoon (2009), the correlation was very weak (Supplementary Fig.…”

Cas9-Guide RNA Directed Genome Editing in Soybean

“…If appropriate recombinase recognition sites, such as yeast-originated FRT1 and FRT87, are placed in a genome, the genomic site can be used as a landing site to accept genes through more predictable RMCE (Nanto et al, 2005;Chawla et al, 2006;Louwerse et al, 2007;Li et al, 2009Li et al, , 2010. A drawback of current RMCE technology is that the landing sites are created by random transformation and often not at preferred loci.…”

An Improved Variant of Soybean Type 1 Diacylglycerol Acyltransferase Increases the Oil Content and Decreases the Soluble Carbohydrate Content of Soybeans

“…Recombination-mediated cassette exchange (Li et al, 2009(Li et al, , 2010b) was used to create transgenic soybean events that expressed the GmDGAT1b-MOD variant under the control of the promoter of the soybean b-conglycinin a-prime subunit gene (Glyma.10G246300) known to confer strong seed-preferred gene expression (Beachy et al, 1985). A total of four events were created that harbor a single-copy transgene at the same location in the soybean genome (Supplemental Fig.…”

“…The FRT sites were included for subsequent gene targeting by recombinasemediated cassette exchange (RMCE; Li et al, 2009Li et al, , 2010b. Additionally, QC599A contains the nopaline synthase transcription terminator (NOS) after the HPT.…”

“…S2D; Li et al, 2009Li et al, , 2010b. Chlorsulfuron-resistant events were screened for single copy by qPCR analysis, and embryos were matured and dried as described.…”