2013 Fiscal Year Final Research Report
Screening of factors affecting gene targeting efficiency.
| Project/Area Number |
24780010
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Breeding science
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| Research Institution | National Institute of Agrobiological Sciences |
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Principal Investigator |
ENDO Masaki 独立行政法人農業生物資源研究所, ゲノム機能改変研究ユニット, 研究員 (40546371)
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| Project Period (FY) |
2012-04-01 – 2014-03-31
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| Keywords | 形質転換 / ジーンターゲッティング / 植物 / アグロバクテリウム / DNA二重鎖切断 |
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| Research Abstract |
Arabidopsis tester lines possessing an I-SceI recognition site and an inducible I-SceI expression system were established for monitoring protein attachment at DNA double strand break (DSB) site and for monitoring gene targeting events. By using this experimental material, it was revealed that gene targeting efficiency was increased by a factor of 5-10 by DSB induction during transformation of gene targeting vector.
It is known that T-DNAs preferentially integrate at DNA DSB sites. Previous studies show that VirD2, which is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer is important for efficient T-DNA integration in to the plant genome. However there is no direct evidence to show the role of VirD2 in T-DNA integration step. For detecting in vivo VirD2 attachment at DSB sites, I established chromatin immunoprecipitation assay using protoplast of this Arabidopsis tester line with transfection of VirD2 expression cassette and DSB induction.
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