期刊
PLANT AND CELL PHYSIOLOGY
卷 55, 期 3, 页码 658-665出版社
OXFORD UNIV PRESS
DOI: 10.1093/pcp/pct183
关键词
Agrobacterium-mediated transformation; Gene targeting; Negative selection; Rice
资金
- Bio-Oriented Technology Research Advancement Institution (BRAIN) of Japan [PROBRAIN (Program for Promotion of Basic Research Activities for Innovative Biosciences)
- Ministry of Agriculture, Forestry and Fisheries of Japan [Genomics for Agricultural Innovation] [GMC-0001]
- Ministry of Education, Culture, Sports, Science and Technology of Japan [the Strategic Promotion Program for Basic Nuclear Research] [210105]
- Japan Society for the Promotion of Science [KAKENHI] [25450002]
- Grants-in-Aid for Scientific Research [25450002] Funding Source: KAKEN
Gene targeting (GT) is a powerful tool manipulating a gene of interest in a given genome specifically and precisely. To achieve efficient GT in higher plants, both positive and negative selection markers are required. In particular, a strong negative selection system is needed for enrichment of cells to eliminate those cells in which random integration of the introduced DNA has occurred in GT experiments. Currently, non-conditional negative selection marker genes are used for GT experiments in rice plants, and no conditional negative selection system is available. In this study, we describe the development of an efficient conditional negative selection system in rice plants using Escherichia coli cytosine deaminase (codA). We found that a mutant codA gene, codA(D314A), acts more efficiently than the wild-type codA for negative selection in rice plants. The codA(D314A) marker was further used as a negative selection marker for GT experiments in rice. Our conditional negative selection system effectively eliminated the cells in which random integration event(s) occurred; the enrichment factor was approximately 100-fold. This enrichment factor was similar to that found when Corynebacterium diphtheriae toxin fragment A was used. Our results suggest the codA(D314A) marker gene as a promising negative selection marker for GT of rice.
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