2002 Fiscal Year Final Research Report Summary
Control of gene expression by gene engineering technique and induction mechanism of the enzyme activity
| Project/Area Number |
12680145
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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 |
食生活
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| Research Institution | Nagoya Women's University |
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Principal Investigator |
OBA Kazuko Nagoya Women's University, Department of Food Science and Nutrition, Professor, 家政学部, 教授 (80023480)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUOKA Makoto Nagoya University, Bio science center, Professor, 生物分子応答研究センター, 教授 (00270992)
FUJIE Ayumi Nagoya Women's University, Department of Food Science and Nutrition, Research associate, 家政学部, 助手
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| Project Period (FY) |
2000 – 2002
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| Keywords | L-ascorbic acid / antisense inhibition / L-galactonolactone dehydrogenase / potato / sweet potato / tobacco BY-2(Nicotiana tobacum CV. Bright Yellow 2) / transgenic plant / wounding |
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| Research Abstract |
1. Control of gene expression of L-galactono-1, 4-lactone dehydrogenase in tobacco.
In higher plants, the terminal step of L-ascorbic acid (AsA) biosynthesis is catalyzed by the enzyme L-galactono-1, 4-lactone dehydrogenase (EC 1.3.2.3, GLDH). We generated AsA-deficient transgenic tobacco BY-2 cell lines by antisense expression of the GLDH cDNA that was amplified from BY-2 cells using PCR. Tow transgenic cell-lines, AS1-1 and AS2-2, having a marked expression of antisense RNA were analyzed. Antisense suppression of GLDH mRNA led to a significant decline in the GLDH activity. The AsA levels in the transgenic cell lines were found to be 30% lower than the wild-type BY-2 cells. In synchronous cultures, division of AS1-1 and AS2-2 cells was restrained with a concomitant decrease in mitotic index that was probably due to a decline in AsA levels. The rate of cell growth was also found to be less than that of the wild-type cells. The calli of AS1-1 and AS2-2 appeared to be sticky and soft. Microscopic analysis revealed that AS1-1 and AS2-2 cells were abnormally slender. Thus, we observed that decline in the AsA levels has an adverse effect on the division, growth and structure of a plant cell.
2. Induction mechanism of GLDH in sweet potato root and potato tuber after wounding.
cDNA of GLDH from sweet potato was over expressed in Escherichia coli. The specific polyclonal antibody against the GLDH protein was prepared by immunization of a rabbit. The antibody recognized GLDH protein from potato tuber and vigna mungo as well as the sweet potato protein. GLDH activity in sweet potato root and potato tuber increased in one-day after wounding and followed by the increase in the level of vitamin C in the tissue. Increased patterns of both GLDH activity and GLDH protein measured by western blotting were similar, suggesting that GLDH protein was synthesized de novo in response to wounding, and resulted in the increase in GLDH activity.
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