| Project/Area Number |
61580133
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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 |
物質生物化学
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| Research Institution | Tokyo University of Agriculture and Technology |
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Principal Investigator |
OHNO Takeshi Tokyo University of Agriculture an Technology, Assoc. Prof., 農学部, 助教授 (00011726)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1987: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1986: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | winged bean / Kunitz-type chymotrypsin inhibitor / cDNA cloning / plant gene / シグナルペプチド / プロテアーゼインヒビター / cDNA / クローニング / λgt 10 |
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| Research Abstract |
The Kunitz-type chymotrypsin inhibitor, WCI-3, is the most predominant inhibitor in winged bean (Psophocarpus tetragonolobus) seeds. The WCI-3 appears in immature seeds around 35 days after flowering and increases until around 50 days. We analyzed the mode of expression, isolated the WCI-3 cDNA clone and determined the sequence.
(1)Analysis of translatably active mRNA: Total poly(A)^+RNA was isolated from 30-, 35- and 40-day-old immature seeds and translated in a rabbit reticulocyte system. The specific product immunoprecipitated with anti WCI-3 antiserum was detected in the products of 35- and 40-day-old samples, but not in that of 30-day-old one. The apparent molecular weight of the specific product was about 2,000 daltons larger than that of the purified WCI-3. This suggests that WCI-3 will be bynthesized as a precursor molecule and mature after processing.
(2)Isolation of WCI-3 cDNA clone and its nucleotide sequence: The WCI-3 cDNA clones were isolated from a <lambda>gt 11 cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA was obtained which encoded 207-amino acid residues. The deduced sequence of 183 amino acids from 25th to the C-terminus coincided completely with the amino acids sequence of the purified WCI-3. The extra N-terminal 24-amino acid segment is rich in hydrophobic amino acids and deduced to be a signal peptide.
(3)Analysis of WCI-3 mRNA by Northern hybridization: Northern hybridization analysis using the cloned cDNA probe showed that the WCI-3 mRNA was approximately 900 nucleotide long and accumulated in 35- and 40-day-old immature seeds but not in 30-day-old ones. These results showed that the expression of chymotrypsin inhibitor WCI-3 was regulated by the accumulation of the mRNA in developping seeds.
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