| Project/Area Number |
11660011
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Breeding science
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| Research Institution | National Institute for Basic Biology |
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Principal Investigator |
TERADA Rie Okazaki National Research Institute, National Institute for Basic Biology, Research Associate, 基礎生物学研究所, 助手 (30137799)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHISHIGE Inagaki Okazaki National Research Institute, National Institute for Basic Biology, Research Associate, 基礎生物学研究所, 助手 (50280764)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | targeting / rice / Agrobacterium transformation / diphtheria toxin protein / negative selection marker / DT-A chain gene / Waxy / DFR / ターゲッティング |
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| Research Abstract |
In order to study molecular mechanism of epigenetic phenomena caused by gene transformation, gene targeting system will serve as a powerful instrument for manipulation of genes without changing the loci of the genes in question. The objective of research is to develop a rice targeting system towards a study of epigenetic gene expression of rice Waxy and DFR (dihydroflavonol-4-reductase) genes. Since gene targeting is induced through homologous recombination, targeting vector includes homologous sequences of Waxy or DFR genes with its flanking sequences in addition to a positive (hygromycin phosphotransferase (hph)) and negative selection markers. The Waxy and DFR genes and its flanking sequences were cloned from Japonica rice ver. Shimokita and ver. Murasakiine respectively. By using these clones targeting vectors for Waxy and DFR loci were constructed. Positive and negative selection is generally used in mouse gene targeting to choose out rarely caused homologous recombination among a
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huge number of illegitimate recombinations. We have chosen the diphtheria toxin (DT) A chain gene as a negative selection marker. In order to treat a huge number of transformation events we have established a highly effective transformation system via Agrobacterium. Compared to general method about 10 times higher frequency of trans formation was obtained by our improved method, in which parameters about Agrobacterium inoculation were optimized. By using the high frequency rice transformation method the lethal effect for rice calli of the negative selection marker of DT gene was analyzed. More than 99% of mortality of rice calli transformed with the DT gene was observed. Based on these highly effective transformation system via Agrobacterium and the positive and negative selection, the Waxy targeting vector was applied for gene targeting. Average 50〜60 calli were survived among 500〜2000 transformation events induced in one time of experiment. In order to find out gene targeting event PCR analysis of these survived calli is on going. These results strongly suggest that the combination of the developed methods of transformation and of positive negative selection described above, have much possibility to be a powerful system for gene targeting of rice plant. Less
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