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ANALYSIS OF ALLERGENS AND USEFUL GENES IN BUCKWHEAT SEED STORAGE PROTEINS

荞麦籽贮藏蛋白中过敏原和有用基因的分析

基本信息

批准号：
15580010
负责人：
FUJINO Kaien
金额：
$ 2.43万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580010/
关键词：
buckwheat rutin allegen seed storage protein tatary buckwheat glucosidase rutinase アレルギー

项目摘要

Buckwheat (Fagopyrum esculentum M.) gene EL0847 that showed an increase in transcript during the early stages of seed development was isolated from immature seeds harvested 14 days after pollination by differential screening. The deduced amino acid sequence of EL0847 shows several repeats of alanine, glutamate and valine which suggests a structural role for the encoded protein, and reveals similarity to the deduced amino acid sequence of Hev b5, which is believed to be a major allergen from natural rubber latex. In an E.coli. expression system, the translation product of a cDNA encoding the EL0847 was not recognized by sera from allergic patients.Based on the N-terminal amino acid sequence of the rutinase which showed flavonol 3-glucosidase activity, a degenerate primer were synthesized and 3' RACE PCR amplification was carried out. The deduced amino acid sequence of rutinase gene shows similarity to the beta-glucosidase in plants. Antiserum was raised against the translation product of a cDNA encoding the rutinase in an E.coli. expression system and immunoblotting of total protein from tatary buckwheat seeds (Fagopyrum tataricum G.) revealed that two proteins reacted with the antiserum. The immunoblotting and genomic Southern analysis data indicated the rutinase gene were encoded by gene family. In an E.coli, and a S.pombe expression system, the translation product of a cDNA encoding the rutinase showed little flavonol 3-glucosidase activity.

从授粉后14天收获的荞麦（Fagopyrum esculentum M.）未成熟种子中分离出在种子发育早期转录增加的EL0847基因。EL0847的氨基酸序列显示了几个重复的丙氨酸、谷氨酸和缬氨酸，这表明编码蛋白的结构作用，并揭示了与Hev b5的氨基酸序列相似，Hev b5被认为是天然胶乳的主要过敏原。在大肠杆菌中。结果表明，编码EL0847的cDNA翻译产物不能被过敏患者血清识别。根据显示黄酮醇3-葡萄糖苷酶活性的芦丁酶n端氨基酸序列，合成简并引物，进行3′RACE PCR扩增。推导出的芦丁酶基因的氨基酸序列与植物中的β -葡萄糖苷酶相似。对大肠杆菌中编码芦丁酶的cDNA的翻译产物进行了抗血清的培养。对苦荞种子（Fagopyrum tataricum G.）总蛋白的表达系统和免疫印迹分析表明，两种蛋白与抗血清发生反应。免疫印迹和基因组Southern分析结果表明，该基因属基因家族编码。在大肠杆菌和S.pombe表达系统中，编码芦丁酶的cDNA翻译产物显示出很少的黄酮醇3-葡萄糖苷酶活性。