Structure of plant protease inhibitor gene and regulation of the expression.

Research Project

Project/Area Number	61580133
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	物質生物化学
Research Institution	Tokyo University of Agriculture and Technology
Principal Investigator	OHNO Takeshi Tokyo University of Agriculture an Technology, Assoc. Prof., 農学部, 助教授 (00011726)
Project Period (FY)	1986 – 1987
Project Status	Completed (Fiscal Year 1987)
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)
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
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.