| Project/Area Number |
06660005
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Breeding science
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YOSHIDA Kaoru t The Univ.of Tokyo, Assistant Foculty of Agriculture Assistant Prof., 農学部, 助手 (70183994)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Differential display / Cloning / RT-PCR / Regeneration / Somatic embryo / Adventitious shoot / Rice / Protein / Differential screening / 分子生物学 |
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| Research Abstract |
To detect gene expressions during regenration, we applied two novel methods to rice culture system, in which we could separate morphogenic pathways, embryogenesis and organogenesis.
1.Simplified differential display
Reverse-transcribed cDNAs from mRNA populations isolated from organogenetic calli, embryogenic calli, and unorganized calli were used as templates for PCR with a short arbitrary RAPD primer. Eight differentially expressed PCR fragments were cloned and used as probes for Northern blots. All clones could detect specific transcripts corresponding to the specificity observed in PCR products. The results revealed that PCR patterns of cDNAs are reliable to judge their specificity and that the method is also effective to detect and clone differential expressed genes even if their expressions are in the low abundant class. Nucleotide sequences of both ends of cDNAs were detemined. They were used to search the Genbank database for sequence homology. Two clones out of eight were identified as Saccharomyces cerevisiae p68 gene (RNA helicase gene) and Drosophila melanogaster bithoraxoid region, respectively.
2.Immunosubraction
To enrich embryogenesis-specific proteins, we used the affinity chromatography. The proteins that were expressed in unorganized calli were immunosubtracted from total proteins of embryogenic calli using the antiserum against total proteins of unorganized calli. Comparison of 2-D PAGE profile of immunosubtracted proteins with that of total proteins before immunosubtraction allowed us to detect 7 proteins specific to somatic embryogenesis. The result indicated that the method of immunosubtraction is an effective method to concentrate embryogenesis-specific proteins at low abundance.
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