ANALYSIS OF ALLERGENS AND USEFUL GENES IN BUCKWHEAT SEED STORAGE PROTEINS

荞麦(Fagopyrum esculentum M.)通过差异筛选，从授粉14d后收获的未成熟种子中分离到了在种子发育早期转录增加的基因EL0847。EL0847的推导氨基酸序列显示了丙氨酸、谷氨酸和缬氨酸的几个重复序列，这表明该编码蛋白具有结构作用，并与HeV b5的推导氨基酸序列相似，后者被认为是天然胶乳的主要过敏原。在大肠杆菌中。针对过敏患者血清不能识别EL0847编码基因的翻译产物，根据具有黄酮醇3-葡萄糖苷酶活性的芦丁酶N端氨基酸序列，设计简并引物，进行3‘RACE PCR扩增。推测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。在大肠杆菌中制备了针对编码芦丁酶的cDNA翻译产物的抗血清。苦荞种子总蛋白的表达体系及免疫印迹研究发现有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，芦丁酶基因是由基因家族编码的。在大肠杆菌和S.pombe表达系统中，编码芦丁酶的cDNA的翻译产物显示很少的黄酮醇3-葡萄糖苷酶活性。

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.