| Project/Area Number |
15580109
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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 |
Food science
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
HONJOH Ken-ichi Kyushu University, Faculty of Agriculture, Research Associate, 大学院・農学研究院, 助手 (00264101)
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| Co-Investigator(Kenkyū-buntansha) |
IIO Masayoshi Kyushu University, Faculty of Agriculture, Professor, 大学院・農学研究院, 教授 (40038279)
MIYAMOTO Takahisa Kyushu University, Faculty of Agriculture, Associate Professor, 大学院・農学研究院, 助教授 (70190816)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | freezing tolerance / trehalose / trehalase / trehalose 6-phosphate synthase / trehalose 6-phosphate phosphatase / transgenic plants |
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| Research Abstract |
We tried to isolate a cDNA clone corresponding to a gene encoding trehalase from Nicotiana tabacum and express the coding region of the clone in E.coli. As the expressed protein formed an inclusion body, it did not show its enzymatic activity. However, it was used as an antigen for construction of polyclonal antibodies. Expression of trehalase in wild type tobacco plants was investigated by using the antibiodies. The result showed that the level of expresson of the enzyme was very low. Twenty-two transgenic tobacco plants, carrying the gene encoding trehalase in antisense direction, were also constructed. However, the accumulation of trehalose was not confirmed in the transgenic plants.
Next, we tried to isolate cDNA clones corresponding to genes encoding trehalose 6-phosphate synthase (tps) and trehalose 6-phosphate phosphatase (tpp) from N.tabacum For obtaining the PCR products, primers were designed based on the conserved regions of two enzymes from other higher plants. The sizes of amplified PCR fragments corresponding to tps and tpp were about 650 bp and 450 bp, respectively. The nucleotide sequences of the amplified fragments were confirmed. By using the fragments as probes, a cDNA library was screened. After screening of the library for tpp gene, one positive clone was obtained. The cDNA clone was 1854-bp size and coded for 384 amino acids. The result of homology search showed that the deduced amino acid sequence of the cDNA clone was quite similar to those of TPPs form other higher plants. On the other hand, no cDNA clone corresponding to tps gene was obtained. By combination of 5'RACE and 3'RACE methods, nucleotide sequence of the partial fragment of cDNA for tps was determined. The size was 2177 bp and the fragment codes for 676 amino acids.
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