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

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, designe...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 2010-10, Vol.154 (2), p.622-631
Main Authors: Li, Zhongsen, Moon, Bryan P, Xing, Aiqiu, Liu, Zhan-Bin, McCardell, Richard P, Damude, Howard G, Falco, S. Carl
Format: Article
Language:English
Subjects:
Amino acids
Biological and medical sciences
Biosynthesis
Breakthrough Technologies
Callus
Corynebacterium glutamicum
Deoxyribonucleic acid
DNA
DNA probes
Fatty acids
Fatty Acids - biosynthesis
Fundamental and applied biological sciences. Psychology
Genomics
Glycine max
Glycine max - genetics
Hordeum vulgare
Mutagenesis, Insertional - methods
Plant physiology and development
Plants
Plants, Genetically Modified - genetics
Polymerase chain reaction
Somatic embryos
Soybeans
Transformation, Genetic
Transgenes
Yarrowia lipolytica
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Summary: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.
ISSN:0032-0889
1532-2548
1532-2548
DOI:10.1104/pp.110.160093