Gene Targeting by Homologous Recombination as a Biotechnological Tool for Rice Functional Genomics

The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting (GT), has broad implications for basic and applied research. Rice (Oryza sativa), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, an...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 2007-06, Vol.144 (2), p.846-856
Main Authors: Terada, Rie, Johzuka-Hisatomi, Yasuyo, Saitoh, Miho, Asao, Hisayo, Iida, Shigeru
Format: Article
Language:English
Subjects:
Alcohol Dehydrogenase - genetics
Biological and medical sciences
Breakthrough Technologies
Bryopsida - genetics
Callus
Fundamental and applied biological sciences. Psychology
Gene targeting
Gene Targeting - methods
Genes
Genes. Genome
Genetic vectors
Genomes
Genomics - methods
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Mosses
Oryza - genetics
Oryza sativa
Plants
Plants, Genetically Modified
Polymerase chain reaction
Recombination, Genetic
Rice
Transgenes
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Summary:The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting (GT), has broad implications for basic and applied research. Rice (Oryza sativa), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, and the single-copy gene Waxy on chromosome 6 has been modified with a frequency of 1% per surviving callus by GT using a strong positive-negative selection. Because the strategy is independent of gene-specific selection or screening, it is in principle applicable to any gene. However, a gene in the multigene family or a gene carrying repetitive sequences may preclude efficient homologous recombination-promoted GT due to the occurrence of ectopic recombination. Here, we describe an improved GT procedure whereby we obtained nine independent transformed calli having the alcohol dehydrogenase2 (Adh2) gene modified with a frequency of approximately 2% per surviving callus and subsequently isolated eight fertile transgenic plants without the concomitant occurrence of undesirable ectopic events, even though the rice genome carries four Adh genes, including a newly characterized Adh3 gene, and a copy of highly repetitive retroelements is present adjacent to the Adh2 gene. The results indicate that GT using a strong positive-negative selection can be widely applicable to functional genomics in rice and presumably in other higher plants.
ISSN:0032-0889
1532-2548
1532-2548
DOI:10.1104/pp.107.095992