| Project/Area Number |
01560096
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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 | Kobe University |
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Principal Investigator |
YAMAGATA Hiroshi Kobe Univ. Faculty of Agriculture, Associate Professor, 農学部, 助教授 (00159203)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Melon / Serine proteinase / Fruit enzyme / Protein synthesis / cDNA cloning |
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| Research Abstract |
Melon serine proteinase (MSP) is a major protein in the fruits of melon. The time and the site of MSP synthesis is restricted to the developing young fruits. The goal of this project is to clear the molecular mechanisms of the gene expression and the protein synthesis of the MSP.
The tissue blotting assay and the incorporation of the labeled amino acid into the protein in the tissue block suggested that the MSP is synthesized in the seed sac or its peripheral tissue.
Using guanidinium isothiocyanate and ultra centrifugation, a mRNA which had a high template activity of protein synthesis in vitro can be isolated. The differences were observed among the in vitro products of mRNA from the several parts in the fruits. A cDNA was synthesized from the mRNA isolated from the center parts of the fruits and ligated to the lambda gt 11 arm and then in vitro packaged phage was introduced into E. coli Y1090R-. This constructed cDNA library was screened using anti-MSP rabbit antibody and 6 positive clones could be isolated. The length of the 5 cDNAs of these clones was 700 to 1,900 bp. The longest clone, lambda MSP 28 had a cDNA whose length is thought to be near full-length of the MSP mRNA. These 5 cDNA clone were subcloned into phagemid pBluescript and their sequencing are now being carryied out.
On the other hand, genome DNA was extracted from young melon leaves using CTAB, and was partially hydrolyzed by Sau 3A. Resulting the DNA of 10 to 20 kb was ligated to the EMBL 3 arm and the genome libraly was prepared. In the following research, the structure of the isolated cDNA and the MSP gene will be analyzed and clear the cis elements responsible for the fruit specific expression of the MSP gene.
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