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Gene Cloning and Expression of Recombination Gene of Buckwheat Protein FE25

荞麦蛋白FE25重组基因的克隆及表达

基本信息

批准号：
06680024
负责人：
TAKUMI Kenji
金额：
$ 1.41万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680024/
关键词：
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

项目摘要

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.

本研究的目的是开发一种改良的荞麦籽粒基因型，通过这种基因型，可以预期消除或减弱籽粒的一些内在缺陷，包括粗糙的质地性质或过敏性。1994年，我们从花后10天的荞麦胚乳中提取总蛋白RNA，然后通过两次吸附和oligo（U）-琼脂糖柱洗脱，从RNA中提取富含poly（A）的RNA。从25 μ g poly（A）-RNA开始，用禽成髓细胞瘤病毒（AMV）逆转录酶进行第一和第二链cDNA反应，1995年，用Eco R1和Sal-1消化构建的cDNA，在Sepharose 4 B微型柱上纯化大于500 bp的cDNA，并插入pCU 8中进行表达。用抗荞麦球蛋白FE 25抗体和~（13）<125>I标记的羊抗兔IgG对文库进行筛选，只有4个克隆能与抗血清发生特异性反应。目前，我们正在研究这些菌落，以鉴定编码FE 25球蛋白的cDNA。