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. 2010 Oct;154(2):622-31.
doi: 10.1104/pp.110.160093. Epub 2010 Aug 18.

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

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

Zhongsen Li et al. Plant Physiol. .
Free PMC article

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.

Figures

Figure 1.
Figure 1.
Alteration of fatty acid biosynthesis for high oleic acid and high oil production. Two genes, FATB and FAD2, are silenced, leading to the increase of 18:1 and decrease of saturated fatty acids 16:0 and 18:0. The DGAT gene encoding a key enzyme for fatty acid accumulation in oil bodies is overexpressed, leading to increased oil. ACP, Acyl carrier protein; KASII, β-ketoacyl-ACP synthase II; Δ9 DES, Δ-9 desaturase; FATA, acyl-acyl carrier protein thioesterase 1; FATB, acyl-acyl carrier protein thioesterase 2; FAD2, ω-6 desaturase; FAD3, ω-3 desaturase; DGAT, diacylglycerol acyltransferase, TAG, triacylglycerol; ER, endoplasmic reticulum. FATA or FATB in smaller font indicates a minor role for the step.
Figure 2.
Figure 2.
Schematics of DNA transgenes. A, The first target DNA, QC288A329A, contains the constitutive promoter SCP1 driving a mutant ALS gene. A FRT1 site (black triangle) is placed between the SCP1 promoter and the ALS gene, and a FRT87 site (white triangle) is placed at the 3′ end. B, Predicted first RMCE DNA, QC288A436A, from the transformation of QC288A329A target with the first donor, QC436. All the components between the FRT1 and FRT87 sites of QC288A329A are replaced by the components between the FRT1 and FRT87 sites of QC436. The third recombination site, FRT12 (black and white triangle), is introduced. C, Predicted second RMCE DNA, QC288A436A438A, from the retransformation of the first RMCE DNA, QC288A436A, now used as the second target, with the second donor QC438. The components between the FRT1 and FRT12 sites of QC288A436A are replaced by the components between the FRT1 and FRT12 sites of QC438. Relative positions of qPCR assays (vertical arrows), PCR primers, and MfeI-cleavage sites are marked. Black bars represent Southern hybridization probes specific to SCP1, HPT, FATB-2, and DGAT1. FLP QC292 and predicted excision DNA QC288ME, which lost all the components between the FRT1 and FRT87 sites, were described previously (Li et al., 2009).
Figure 3.
Figure 3.
Analysis of the first-round SSI events. PCR assays specific to the genomic borders of the B target site hosting different transgenes was done using combinations of the 5′ border, 3′ border, and transgene-specific primers (Fig. 2). A, Expected PCR fragments of the 5′ border (left), 3′ border (center), and excision QC288ME (right) of the first RMCE, QC288A436A, are 886, 1,116, and 986 bp. The expected 9,108-bp-long full-length QC288A436A is too large to be amplified (right). B, Expected PCR fragments of the 5′ border (left) and 3′ border (center) of target QC288A329A and the FRT12 region of RMCE QC288A436A (right) are 967, 1,180, and 693 bp. Wild-type DNA (wt) was included as a negative control. The four events were all chimeras containing the RMCE, target, and excision transgenes at the embryogenic callus stage. The FlashGel DNA markers are 4, 2, 1.25, 0.8, 0.5, 0.3, 0.2, and 0.1 kb (Lonza Rockland).
Figure 4.
Figure 4.
Transgene expression in the second-round SSI events. The expression of genes DHPS, BHL8, and CGS in mature somatic embryos of selected second-round SSI events was checked by western blotting. Only the RMCE event B531 expressed all three proteins DHPS (A), BHL8 (B), and CGS (C). Event B532 expressed only BHL8. Event B541 expressed both DHPS and BHL8 but not CGS. Event B511 expressed both DHPS and CGS but not BHL8. Event B512 expressed only DHPS. Positive controls containing respective genes are transgenic events from unrelated projects. The protein markers are in kD. wt, Wild type.
Figure 5.
Figure 5.
Analysis of the second round RMCE event. PCR assays specific to the genomic borders and internal regions of the second RMCE DNA, QC288A436A438A, were done on RMCE T0 plant B531-1 using various primer combinations (Fig. 2). The hemizygous (RMCE/excision) B531-1 ancestors hemizygous B53, homozygous B5 and B, and wild-type DNA (wt) were included as controls. A, The expected 886-bp 5′ border of both QC288A in B and QC288A436A in B53 (left) and the 561-bp 3′ border of QC288A in B (center) were amplified. The full-length 4,742-bp QC288A of B, 6,331-bp QC288A329A of B5, and 986-bp QC288ME (excision) of B53 and B531 were amplified (right). The expected full-length 9,108-bp QC288A436A of B53 and 21,925-bp QC288A436A438A of B531-1 were too large to be amplified. B, The expected 967-bp 5′ border of both QC288A329A in B5 and QC288A436A438A in B531 (left) and the 1,180-bp 3′ border of QC288A329A in B5 (center) were amplified. The same B5 band was also amplified from B53 that contained chimerically B5 DNA QC288A329A. The expected 1,116-bp 3′ border of both QC288A436A in B53 and QC288A436A438A in B531-1 were amplified (right). C, A 693-bp fragment unique to the FRT12 region of QC288A436A was amplified only in B53 (left). An 840-bp fragment unique to the FRT12 region (center) and a 711-bp fragment unique to the 5′ region of QC288A436A438A were amplified only in B531-1 (right). The FlashGel DNA markers are 4, 2, 1.25, 0.8, 0.5, 0.3, 0.2, and 0.1 kb. D, A 2,946-bp segment covering the DHPS, MYB2 terminator, and UBIQ10 promoter of QC288A436A438A was lost in T0 plant B531-1 although intact in embryogenic callus B531. The expected 5,753-bp Ph3-1/CRS-10 band (left) and 9,216-bp Dgat-1R/CRS-10 band (right) of intact QC288A436A438A were amplified from the embryogenic callus B531 and QC438 donor DNA but not from the T0 plant B531-1 leaf DNA, which produced approximately 3-kb smaller bands. Some nonspecific bands were amplified by the long-range PCR. The 1-kb DNA markers are 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, and 1 kb (Invitrogen).
Figure 6.
Figure 6.
Confirmation of gene stacking by Southern hybridization. Genomic DNA of B53 T1 plants, homozygous RMCE B53-1 and B53-2, hemizygous (RMCE/excision) B53-3 and B53-4, and excision B53-5 and B53-6, the T0 plant B531-1 (RMCE/excision), and homozygous B5 and B ancestor plants were digested with MfeI and hybridized sequentially with probes SCP1, HPT, FATB-5, and DGAT1. The expected sizes of Southern bands are specific to transgenes integrated at the B target site, where the transgenic QC288A lost 17-bp 5′ end and 49-bp 3′ end sequences. There is an MfeI site 2,131 bp upstream and 1,230 bp downstream, respectively, of the transgenes that contain MfeI sites (Fig. 2). A, The SCP1 probe hybridized to the expected 12,205-bp band of QC288A436A in B53-1 and B53-2, the 3,987-bp band of QC288ME in B53-5 and B53-6, and both bands in hemizygous plants B53-3 and B53-4. The same 3,987-bp QC288ME band and a 3,681-bp QC288A436A438A band were hybridized in B531-1. As expected, the same 3,681-bp band also specific to QC288A329A was detected in B5 and a 7,839-bp QC288A band was detected in B. B, The HPT probe hybridized to the same 12,205-bp QC288A436A band in B53-1, B53-2, B53-3, and B53-4 and to the same 7,839-bp QC288A band in B. C, The FATB-5 probe hybridized to two endogenous bands in all samples in addition to the same 12,205-bp QC288A436A band in B53-1, B53-2, B53-3, and B53-4. Instead of the expected 12,931-bp band of QC288A436A438A, a larger band, likely the 16,402-bp band expected from modified QC288A436A438A with its 2,946-bp DHPS-MYB2-UBIQ10 deleted, overlapped with the top wild-type band (wt) in B531-1. D, The DGAT1 probe detected in B531-1 the same 16,402-bp band of the modified QC288A436A438A instead of an expected 6,352-bp QC288A436A438A band. The DIGVII DNA markers are 8,576, 7,427, 6,106, 4,899, and 3,639 bp (Roche).

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