Stacking Multiple Transgenes at a Selected Genomic Site Via Repeated Recombinase-mediated DNA Cassette Exchanges

Overview

Journal Plant Physiol

Specialty Physiology

Date 2010 Aug 20

PMID 20720171

Citations 17

Authors

Zhongsen Li

Bryan P Moon

Aiqiu Xing

Zhan-Bin Liu

Richard P McCardell

Howard G Damude

S Carl Falco

Affiliations

Soon will be listed here.

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.

Citing Articles

Target lines for recombinase-mediated gene stacking in soybean.

Jiang L, Li R, Han Z, Zhao X, Cao D, Ow D Theor Appl Genet. 2022; 135(4):1163-1175.

PMID: 35325256 DOI: 10.1007/s00122-021-04015-6.

Targeting Nitrogen Metabolism and Transport Processes to Improve Plant Nitrogen Use Efficiency.

The S, Snyder R, Tegeder M Front Plant Sci. 2021; 11:628366.

PMID: 33732269 PMC: 7957077. DOI: 10.3389/fpls.2020.628366.

Efficient Gene Stacking in Rice Using the GAANTRY System.

Hathwaik L, Horstman J, Thomson J, Thilmony R Rice (N Y). 2021; 14(1):17.

PMID: 33547973 PMC: 7867672. DOI: 10.1186/s12284-021-00460-5.

Recombinase-mediated integration of a multigene cassette in rice leads to stable expression and inheritance of the stacked locus.

Pathak B, Srivastava V Plant Direct. 2020; 4(7):e00236.

PMID: 32760877 PMC: 7391932. DOI: 10.1002/pld3.236.

Uniform Expression and Relatively Small Position Effects Characterize Sister Transformants in Maize and Soybean.

Betts S, Basu S, Bolar J, Booth R, Chang S, Cigan A Front Plant Sci. 2019; 10:1209.

PMID: 31708936 PMC: 6821721. DOI: 10.3389/fpls.2019.01209.

References

Galili G, Amir R, Hoefgen R, Hesse H . Improving the levels of essential amino acids and sulfur metabolites in plants. Biol Chem. 2005; 386(9):817-31. DOI: 10.1515/BC.2005.097. View

Heppard E, Kinney A, Stecca K, Miao G . Developmental and growth temperature regulation of two different microsomal omega-6 desaturase genes in soybeans. Plant Physiol. 1996; 110(1):311-9. PMC: 157722. DOI: 10.1104/pp.110.1.311. View

Zhu X, Galili G . Lysine metabolism is concurrently regulated by synthesis and catabolism in both reproductive and vegetative tissues. Plant Physiol. 2004; 135(1):129-36. PMC: 429340. DOI: 10.1104/pp.103.037168. View

Cohen S, Houben A, Segal D . Extrachromosomal circular DNA derived from tandemly repeated genomic sequences in plants. Plant J. 2007; 53(6):1027-34. DOI: 10.1111/j.1365-313X.2007.03394.x. View

Townsend J, Wright D, Winfrey R, Fu F, Maeder M, Joung J . High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature. 2009; 459(7245):442-5. PMC: 2743854. DOI: 10.1038/nature07845. View

Damude H, Kinney A . Engineering oilseed plants for a sustainable, land-based source of long chain polyunsaturated fatty acids. Lipids. 2007; 42(3):179-85. DOI: 10.1007/s11745-007-3049-1. View

HESSE H, Kreft O, Maimann S, Zeh M, Willmitzer L, Hofgen R . Approaches towards understanding methionine biosynthesis in higher plants. Amino Acids. 2001; 20(3):281-9. DOI: 10.1007/s007260170044. View

Xing A, Moon B, Mills K, Falco S, Li Z . Revealing frequent alternative polyadenylation and widespread low-level transcription read-through of novel plant transcription terminators. Plant Biotechnol J. 2010; 8(7):772-82. DOI: 10.1111/j.1467-7652.2010.00504.x. View

Kinney A . Metabolic engineering in plants for human health and nutrition. Curr Opin Biotechnol. 2006; 17(2):130-8. DOI: 10.1016/j.copbio.2006.02.006. View

10.

Cahoon E, Shockey J, Dietrich C, Gidda S, Mullen R, Dyer J . Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux. Curr Opin Plant Biol. 2007; 10(3):236-44. DOI: 10.1016/j.pbi.2007.04.005. View

11.

Zhu X, Galili G . Increased lysine synthesis coupled with a knockout of its catabolism synergistically boosts lysine content and also transregulates the metabolism of other amino acids in Arabidopsis seeds. Plant Cell. 2003; 15(4):845-53. PMC: 152333. DOI: 10.1105/tpc.009647. View

12.

Damude H, Kinney A . Enhancing plant seed oils for human nutrition. Plant Physiol. 2008; 147(3):962-8. PMC: 2442541. DOI: 10.1104/pp.108.121681. View

13.

Shukla V, Doyon Y, Miller J, DeKelver R, Moehle E, Worden S . Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature. 2009; 459(7245):437-41. DOI: 10.1038/nature07992. View

14.

Cai C, Doyon Y, Ainley W, Miller J, DeKelver R, Moehle E . Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol. 2008; 69(6):699-709. DOI: 10.1007/s11103-008-9449-7. View

15.

Nanto K, Yamada-Watanabe K, Ebinuma H . Agrobacterium-mediated RMCE approach for gene replacement. Plant Biotechnol J. 2006; 3(2):203-14. DOI: 10.1111/j.1467-7652.2005.00118.x. View

16.

Roesler K, Rao A . A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein. Protein Sci. 2000; 9(9):1642-50. PMC: 2144711. DOI: 10.1110/ps.9.9.1642. View

17.

Ow D . Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol. 2002; 48(1-2):183-200. View

18.

Louwerse J, van Lier M, van der Steen D, de Vlaam C, Hooykaas P, Vergunst A . Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens. Plant Physiol. 2007; 145(4):1282-93. PMC: 2151714. DOI: 10.1104/pp.107.108092. View

19.

Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson M . Heritable targeted mutagenesis in maize using a designed endonuclease. Plant J. 2009; 61(1):176-87. DOI: 10.1111/j.1365-313X.2009.04041.x. View

20.

Chiba Y, Ishikawa M, Kijima F, Tyson R, Kim J, Yamamoto A . Evidence for autoregulation of cystathionine gamma-synthase mRNA stability in Arabidopsis. Science. 1999; 286(5443):1371-4. DOI: 10.1126/science.286.5443.1371. View