苦荞麦主要过敏蛋白N端基因片段的克隆及序列分析

为了获得苦荞麦主要过敏蛋白全长基因并深入开展苦荞过敏蛋白的研究,本实验在先前获得的苦荞麦主要过敏蛋白C端(TBa)基因序列的基础上,设计不同的引物,以开花20d左右的苦荞麦果实为材料提取总RNA,并以此RNA为模板,通过RT-PCR和5’-RACE等方法,扩增,克隆获得苦荞麦主要过敏蛋白N端(命名为TBb)cDNA序列。序列分析结果表明,该序列由1005bp组成,开放阅读框为960bp,可编码一个由320个氨基酸残基组成的功能蛋白。同源性分析显示,此核苷酸序列与甜荞麦过敏性贮藏蛋白及豆球类蛋白的同源性达到90%。由此核苷酸序列推导出的氨基酸序列与甜荞麦13S贮藏蛋白有84%的同源性,与甜荞麦主要过敏蛋白有76%的同源性。苦荞麦主要过敏蛋白N端基因的获得为该蛋白的重组表达及其生物学活性等研究建立了前期基础。

Cloning and Sequence Analysis of N-terminal Gene of Major Allergenic Protein in Tartary Buckwheat

The N-terminal gene of major allergenic protein in tartary buckwheat (named TBb) was amplified from the total RNA of tartary buckwheat using RT-PCR and 5'-rapid amplication of cDNA ends (5'-RACE) methods. The TBb gene sequence consisted of 1005 nucleotides and a 960bp open reading frame (ORF) which encoded a protein of 320 amino acids residues. Results of homology analysis revealed that the TBb cDNA sequence shared 90% homology with the major allergenic storage protein and legumin-like protein of common buckwheat. The deduced amino acid sequence shared 84% and 76% homology with the legumin-like 13S storage protein and the major allergenic storage protein of common buckwheat respectively. The obtain- ment of the N-terminal gene of major allergenic protein in tartary buckwheat should lay an important foundation for further study of the tartary buckwheat allergen.