Gene Cloning and Expression of Recombination Gene of Buckwheat Protein FE25
| Project/Area Number |
06680024
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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 |
家政学
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| Research Institution | Ehime University |
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Principal Investigator |
TAKUMI Kenji Ehime University, Faculty of Education, Professor, 教育学部, 教授 (90035428)
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| Co-Investigator(Kenkyū-buntansha) |
KOGA Tetsuro Tokushima University, School of Medicine, Department of Nutrition, Tutor, 医学部, 助手 (20093859)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1994: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | buchwheat storage protein / FE25 Globulin / Eco RI and Sal-1 restriction enzymes / pCU8 plasmid / Sucrose density gradient / poly (A) -RNA / cDNA encoding FE25 globulin / cGNA library / 25kDaサブユニット / Sau3AI制限酵素 / λEMBL3 / 15〜20Kbp DNA新片 |
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| Research Abstract |
This study has been attempted for the purpose of development an improved geno-type of buckwheat grain, by which it could be expected that some intrinsic faults of the grain, including rough-textured nature, or allergenic property would be removed or weaken. Thus to construct and expression of cDNA encoding buckwheat globulin (25FE), which is the major storage protein of the grain, is a main goal of the study.
In 1994, total seed protein RNA was prepared from the endosperm of 10-day after flower and then poly (A) -enriched RNA was isolated from the RNA by two cycles of adsorption and elution from oligo (U) -agarose column. Starting with 25 mu g of poly (A) -RNA,first- and second-strand cDNA reactions were performed by using avian myeloblastosis virus (AMV) reverse transcriptase.
In 1995, the constructed cDNA was digested with both Eco R1 and Sal-1, and cDNAs larger than 500 bp were purified on a Sepharose 4B minicolumn and inserted into pCU8 for expression. The colonies were reprica plated onto nitrocellulose filters and the library was screened by anti-buckwheat globulin FE25 antibody following with ^<125>I-labeled goat anti-rabbit IgG.Only 4 colonies were detected that reacted specifically with the antisera. At present we are investigating tha colonies for the identification of cDNA encoding FE25 globulin.
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Report
(3 results)
Research Products
(11 results)
