2004 Fiscal Year Final Research Report Summary
Stricture and characteristics of chitinases from mature grapes
| Project/Area Number |
14360018
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
園芸・造園学
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| Research Institution | University of Yamanashi |
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Principal Investigator |
TAKAYANAGI Tsutomu University of Yamanashi, Department of Research Interdisciplinary Graduate school of medicine and Engineering, Professor, 大学院・医学工学総合研究部, 助教授 (00252007)
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| Co-Investigator(Kenkyū-buntansha) |
OKUDA Tohru University of Yamanashi, Department of Research Interdisciplinary Graduate school of medicine and Engineering, Associate Professor, 大学院・医学工学総合研究部, 助教授 (10252008)
YOKOTSUKA Koki University of Yamanashi, Department of Research Interdisciplinary Graduate school of medicine and Engineering, Professor, 大学院・医学工学総合研究部, 教授 (60020450)
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| Project Period (FY) |
2002 – 2004
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| Keywords | Chitinase / PR-protein / Grape / Vitis / Disease resistance |
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| Research Abstract |
Plant chitinases are thought to be closely related to the plants' defense system against phytopathogens. The structure and characteristics of chitinases separated from Muscat Bailey A (Bailey x Muscat Hamburg) grape skins were investigated. The chitinase fraction was obtained from the extract of the grape skins by affinity chromatography with chitin beads (Chitopearl BL-01) and then separated by cation-exchange chromatography (SP-Sepharose HP). The cation-exchange chromatography yielded two protein peaks (CHI-1 and CHI-2) that exhibited chitinase activity. The molecular weights of CHI-1 and CHI-2 were estimated to be 29 kDa and 27 kDa, respectively, by SDS-PAGE. The optimum pHs for CHI-1 and CHI-2 activity were 3.5-4.5 and 4.5-5.0, respectively. The enzymes were stable over the pH range of 3.0 to 7.0. The optimum temperature for CHI-1 activity (70℃) was higher than that for CHI-2 activity (50℃). CHI-1 was further separated by chromatofocusing into four chitinase isozymes, CHI-1a, -1b, -1c, and -1d. The N-terminal amino acid sequences of the chitinases (CHI-1a, -1b, -1c, -1d and CHI-2) were highly homologous with that of the class IV chitinase of Vitis vinifera. CHI-1 and CHI-2 showed an inhibitory effect on the growth of Botrytis cinerea (gray mold) and Glomerella cingulata (ripe rot). Chitinase activity in the grape skins of Muscat Bailey A was higher than those in the three Vitis vinifera cultivars (Cabernet Sauvignon, Koshu and Chardonnay). The grape skins of Muscat Bailey A, Cabernet Sauvignon and Koshu contained CHI-1 and CHI-2, whereas those of Chardonnay contained only CHI-1. These results suggested that the quantity and number of chitinase isozymes in grape skins are related to the disease resistance level of the grapevines.
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