荞麦籽粒中抗真菌蛋白的分离纯化及抗真菌活性研究

Purification and Antibacterial Activity of Antifungal Protein from Buckwheat Seed

为了深入研究荞麦中抗真菌类蛋白质,选择荞麦品种‘晋荞2号’籽粒为试验材料,先经过筛、脱脂,然后利用缓冲液浸提、硫酸铵盐析提取籽粒中总蛋白,水浴锅中80℃下热处理去除杂蛋白,经过Resourse S阳离子交换层析和Superdex Peptide HR10/300分子筛层析两步分离纯化,建立了一套荞麦抗真菌蛋白分离纯化的工艺。SDS-聚丙烯酰胺凝胶电泳测定该抗真菌蛋白的相对分子质量,牛津杯法测定抑菌活性,倒置显微镜观察真菌形态变化。结果表明:试验获得的荞麦籽粒抗真菌蛋白在蛋白胶图上表现出单一条带,纯度较高,相对分子质量为8.2 kDa。该抗真菌蛋白对绿色木霉的生长有明显的抑制作用,并且呈现一定的剂量依赖性。形态学观察发现,菌丝生长受到抑制,表现出顶端部膨大、泡状物出现、原生质凝缩及片段化等特征。

In order to further study the antifungal proteins in buckwheat,the seed of'Jinqiao 2'of buckwheat varieties was selected as the experimental material.After screening and degreasing,the total protein in the seeds was extracted by buffer solution extraction and ammonium sulfate salting out.The miscellaneous protein was removed by heat treatment at 80℃in a water bath pot.After cationicex changing chromatography by Resource S and molecular sieve chromatography by Superdex Peptide HR10/300,a set of separating and purifying technology of buckwheat antifungal protein were set up.The relative molecular mass of the antifungalprotein was determined by SDS-polyacrylamide gel electrophoresis,the antifungal activity was determined by Oxford cup method,and the morphological changes of fungi were observed by inverted microscope.The results showed that the antifungal protein of buckwheat seeds showed a single band on the protein gel map and possessing high purity,and the relative molecular mass was 8.2 kDa.The antifungal protein significantly inhibited the growth of Trichoderma viride and showed a certain dosedependence.Morphological observation showed that the growth of mycelium was inhibited,showing the characteristics of top enlargement,vesicles,protoplasm condensation and the morphological fragmentation.