| Project/Area Number |
12839006
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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 |
生物資源の変換と展開
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
ADACHI Motayasu Graduate School of Agriculture, Research Associate, 農学研究科, 助手 (60293958)
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| Co-Investigator(Kenkyū-buntansha) |
MARUYAMA Nobuyuki Graduate School of Agriculture, Research Associate, 農学研究科, 助手 (90303908)
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| Project Period (FY) |
2000 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | cruciferin / glycinin / crystal structure / physicochemical function / transgenic plant |
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| Research Abstract |
The first objective of this study was determination of the crystal structure of cruciferin from rapeseed by x-ray crystallography. Since native cruciferin forms heterogenic hexamer composed of at least three kinds of subunit, it is actually difficult to make its crystal for x-ray crystallography. Thus, we constructed the E. coli expression system to prepare recombinant procruciferin. The cDNA was obtained after RT-PCR reaction using mRNA purified from its developing seeds. As a result of investigation for various expression conditions, the recombinant procruciferin occupied 15% of total E. coli protein as a soluble protein. Using this expression system, wild type procruciferin and some mutants were prepared to make crystals and characterize their physicochemical properties. The amino acid residues forming intra salt bridge and C287 residue were mutated. In addition, variable regions of the procruciferin were modified. Another objective was the production of engineered rape seed plant that expresses soybean glycinin in its seed. Because the glycinin has better functional and nutritional properties, we can expect the improvement by adding the better properties to the rape seed. The cotyledon of the rape seed was transformed by agrobacterium method, and the expression of glycinin in the seed was observed by western blotting. Eventually, flowering F1 generation was obtained.
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