The Establishment of the Gene Transfer System of Higher Plants Using an Auxotrophic Mutant.
| Project/Area Number |
01480001
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
遺伝学
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| Research Institution | The University of Tokyo |
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Principal Investigator |
KOMEDA Yoshibumi The University of Tokyo, Molecular Genetics Research Laboratory, associate professor, 遺伝子実験施設, 助教授 (10124215)
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| Co-Investigator(Kenkyū-buntansha) |
UEGUCHI Miyako Osaka Prefectural Institute of Public Health, research fellow, 研究員 (80201622)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥5,000,000 (Direct Cost: ¥5,000,000)
Fiscal Year 1990: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1989: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Arabidopsis thaliana / thiamine requiring / Escherichia coli thiaB gene / Agrobacterium / gene transfer / ビタミンB_1要求性 / 大腸菌<thiB>___ー遺伝子 / thiB遺伝子 / thiC遺伝子 / CAT |
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| Research Abstract |
The nucleotide sequence was determined of the Escherichaia coli K-12 thiB gene coding for thiamine synthetic enzyme, thiamine-phosphate pyrophosphorylase. The analysis permitted us to assign the site of transcriptional control and to deduce the amino acid sequence of the enzyme. Thus, the bacterial controlling region was deleted and replaced with the Cauliflower Mosaic Virus 35S RNA promoter, which has been shown to function as a plant promoter. The resulting plasmid carrying the fusion (35S promoter and thiB coding region) was introduced into Agrobacterium tumefasciens, which was used for the infection of explants of thi1 Arabidopsis thalina. At first, the gene of the hygromycin phosphotransferase in the plasmid was used for selection and trasformants carrying hygromycin resistant character were selected. In plates with the addition of thiamine, hygromycin-resistant calli were grown and shoots were regenerated. Then, transgenic plants were established carrying hygromycin-resitant trait. These plants of transgenic generation (T1 generation) were self-pollinated and we got seeds of next generation (T2 generation). When T2 seeds were sown on plants without the addition of thiamine, about 75% plants grew happily and 25% showed necrosis and died. Accordingly, the fused gene carrying CaMV 35S promoter and thiB sequence recovered the defect of thi1 mutati on of Arabidopsis thaliana. The growth of these transgenic plants were shown to be a little poorer than that of thi1 mutants growt with the addition of thiamine, showing the difference of E. coli enzyme. This thiB gene is now manipulated to become a selectable marker of transformations of higher plants.
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Report
(3 results)
Research Products
(2 results)
