您好, 访客   登录/注册

荞麦籽粒中抗真菌蛋白的分离纯化及抗真菌活性研究

来源:用户上传      作者:李静舒 贺东亮

  摘 要: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。
  关键词:荞麦;抗真菌蛋白;离子交换层析;分子筛层析;抗真菌活性
  中图分类号:S517;Q516 文献标识码:A DOI 编码:10.3969/j.issn.1006-6500.2022.06.002
  Purification and Antibacterial Activity of Antifungal Protein from Buckwheat Seed
  LI Jingshu1,HE Dongliang2
  (1.Shanxi Open University,Taiyuan,Shanxi 030027, China; 2.Taiyuan Institute of Technology, Taiyuan,Shanxi 030008, China)
  Abstract: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.
  Key words: buckwheat;antifungal protein;cationic exchange chromatography; molecular sieve chromatography; antifungal activity
  高等植物在其N子贮藏、植株形成等生长阶段,会受到各种病原微生物污染的影响。但是经过长期的进化,高等植物也形成了一套自己的防御体系,来维持植物抗病性和病原微生物致病性的动态平衡。最常见的方式是在植物体内表达出具有抵抗病原微生物的物质,例如抗菌蛋白(antimicrobial protein,AMPs),它是某些植物体为了抵抗外界病原体的侵袭,而产生的一种或一类具有抗菌活性的非特异性蛋白质的总称[1-2]。植物源的抗菌蛋白具有其独特的优越性,它们的抗菌活性较强,抗菌谱范围广,可以直接作为杀菌制剂喷涂在植株上或混入植物生长的土壤中,因而受到研究人员的广泛关注。有报道称,在多种植物中发现并分离出抗菌蛋白,它们对细菌、真菌和某些病毒的生长活性具有明显的抑制作用。袁素素等[3]从望江南种子中提取出SU5抗菌蛋白,该蛋白对玉米小癍病菌等12种植物病原真菌具有抑制活性。张耀等[4]从芝麻菜种子中分离纯化出ZSU2抗菌蛋白,且该蛋白具有极强的热稳定性和酸碱稳定性。另外,植物源抗菌蛋白还可以作为基因工程改造的材料,经过修饰、加工,将抗病基因导入植株中,进一步提高植物的抗病能力。肖松华等[5]将天麻抗真菌蛋白基因插入到一种棉花品系中,获得三种抗落叶型黄萎病的棉花株系。

nlc202207261500



转载注明来源:https://www.xzbu.com/8/view-15436684.htm

相关文章

服务推荐