| Project/Area Number |
60560087
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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 |
応用生物化学・栄養化学
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| Research Institution | Nagoya University |
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Principal Investigator |
NAKAMURA Kenzo Nagoya University, School of Agriculture, Associate Professor, 農学部, 助教授 (80164292)
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| Co-Investigator(Kenkyū-buntansha) |
ASAHI Tadashi Nagoya University, School of Agriculture, Professor, 農学部, 教授 (10023392)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1986: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1985: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Storage proteins / Vacuole / Precursor processing / Intracellular protein transport / cDNA cloning / Gene structure / 植物への遺伝子導入 |
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| Research Abstract |
Cell fractionation studies indicated that the storage proteins of the sweet potato tuberous root and the potato tuber are deposited in vacuoles. Precursors for these proteins were synthesized by membrane-bound polysomes, and the precursor for the sweet potato storage protein sporamin was 4 kd larger than the mature form. By immunological screening of cDNA expression libraries constructed by a novel method employing an E. coli expression vector-primed cDNA synthesis, full-length cDNA clones for sporamin and three different storage proteins of the potato tuber were identified. An improved E. coli expression vector for the construction and identification of full-length cDNAs has also been developed. Nucleotide sequencing of these cDNA clones indicated that precursors for potato storage proteins contain typical signal peptide sequences at their N-termini. The N-terminal extra-sequence of sporamin precursor, however, contained 16 amino acid residues enriched with basic amino acids following the signal peptide segment. In vitro processing studies suggested that the sporamin precursor undergoes two-step processings where the signal peptide is removed co-translationally followed by the post-translational removal of the segment enriched with basic amino acids. The structure and the processing mechanisms of the sporamin precursor resembles to those of the precursors for yeast vacuolar proteins and animal cell lysosomal proteins. When sporamin precursor was expressed in yeast cells using an yeast expression vector, at least part of them were transported into vacuole. We have also succeeded introducing and expressing a chimeric gene in which sporamin cDNA is joined to a strong plant virus promoter in tobacco. The sporamin precursor expressed in tobacco callus cells seems to be processed into mature size.
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