拟南芥中草酸结合蛋白的分离鉴定及功能分析
发布时间：2018-08-23 13:45
【摘要】：核盘菌(Sclerotinia sclerotiorum)是一种分布广泛且极具破坏性的植物病原菌,能够引起油菜、莴苣等多种作物的菌核病。草酸被认为是核盘菌中重要的致病因子之一,但其具体作用靶标尚不明确,研究草酸的作用模式以及探索草酸在植物中的结合蛋白有助于帮助解析草酸在植物中的作用靶标,并为后续防治核盘菌提供新的线索。基于此,本课题进行了以下研究:1、测定了不同草酸浓度、不同pH值的草酸根溶液对不产草酸核盘菌菌株Ep-1PB菌株致病力恢复情况的影响,以及在不同pH值下,草酸根在本氏烟原生质体的积累量。结果表明:外源添加草酸可部分恢复Ep-1PB菌株的致病力,效果显著高于柠檬酸。在一定浓度范围内,外源添加的草酸浓度和Ep-1PB菌株致病力呈正相关。此外,添加的草酸溶液pH越低,Ep-1PB菌株致病能力越强。在酸性条件下,草酸进入本氏烟原生质体的量显著高于中性和碱性条件。2、构建了一种分离拟南芥中草酸结合蛋白的方法,包括高效制备结合有草酸的凝胶珠以及分离利用该凝胶珠分离拟南芥中草酸结合蛋白。结果表明:获得结合有草酸的凝胶珠的制备反应体系的最佳pH为5.6-6.0,且在反应1h后添加草酸调节pH为6.0,dd H2O为最佳溶剂。3、从拟南芥中分离并鉴定出79种草酸结合蛋白。对这些蛋白质进行GO分析,结果表明这些蛋白参与了抗氧化压力、由RNA引起的基因沉默、细胞对光强度的应答等多种生物学进程。GO富集分析表明最大的草酸结合蛋白类别主要为抗氧化压力类,包括抗超氧化物、应对超氧化物代谢、移除超氧根离子等生物学进程。通过KEGG分析,发现这些草酸结合蛋白参与了氨基酸合成、碳代谢、三羧酸循环、光合作用的碳固定等通路。KEGG富集分析显示差异最显著的通路为苯丙素的合成和淀粉及蔗糖代谢这两种。本研究的创新之处包括:(1)明确了H~+可促进草酸根离子进入植物细胞从而帮助核盘菌致病(2)构建了一种分离植物草酸结合的蛋白的方法。(3)首次对植物体内的草酸结合蛋白进行了分离鉴定,并对相关蛋白进行了生物信息学相关分析,为进一步研究草酸在植物中的作用靶标提供了新的思路。
[Abstract]:Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is a widely distributed and destructive plant pathogen which can cause sclerotinia in many crops such as rape lettuce and so on. Oxalic acid is considered to be one of the important pathogenic factors in Sclerotinia sclerotiorum, but the specific target of oxalic acid is not clear. It also provides a new clue for the control of Sclerotinia sclerotiorum. Based on this, the following research was carried out to determine the effect of oxalate solution with different oxalic acid concentration and pH on the recovery of pathogenicity of non-oxalic acid-producing Sclerotinia sclerotiorum strain Ep-1PB, and the effects of different pH values on the pathogenicity of the strain. The accumulation of oxalate in the protoplasts of Bentner's tobacco. The results showed that exogenous oxalic acid could partially restore the pathogenicity of Ep-1PB strain, and the effect was significantly higher than that of citric acid. There was a positive correlation between oxalic acid concentration and pathogenicity of Ep-1PB strain in a certain concentration range. In addition, the lower pH of oxalic acid solution added, the stronger pathogenicity of Ep-1PB strain. Under acidic conditions, oxalic acid entered the protoplasts of Bensi tobacco significantly higher than that of neutral and alkaline conditions. A method for isolation of oxalic acid binding proteins from Arabidopsis thaliana was constructed. It includes preparation of oxalic acid bound gel beads and separation of oxalic acid binding proteins from Arabidopsis thaliana. The results showed that the optimum pH of the preparation system of gel beads with oxalic acid was 5.6-6.0, and 79 oxalic acid binding proteins were isolated and identified from Arabidopsis thaliana by adding oxalic acid to adjust pH 6.0 dd H2O for 1 hour. Go analysis of these proteins showed that these proteins were involved in antioxidant stress, gene silencing induced by RNA, Many biological processes, such as cell response to light intensity. Go enrichment analysis showed that the largest oxalic acid binding protein type was mainly antioxidant pressure, including resistance to superoxide, superoxide metabolism, removal of superoxide ion and other biological processes. KEGG analysis showed that these oxalic acid binding proteins were involved in amino acid synthesis, carbon metabolism and tricarboxylic acid cycle. KEGG enrichment analysis showed that the two most significant pathways for photosynthesis were phenylpropanin synthesis and starch and sucrose metabolism. The innovations of this study are as follows: (1) it is clear that H ~ can promote oxalate ions into plant cells and thus help Sclerotinia sclerotiorum cause disease. (2) A method of isolating oxalic acid-binding proteins from plants was constructed. (3) oxalic acid knot in plants was first identified. The synthase was isolated and identified. The bioinformatics correlation analysis of the related proteins provides a new idea for the further study of oxalic acid target in plants.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.44
本文编号：2199250
[Abstract]:Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is a widely distributed and destructive plant pathogen which can cause sclerotinia in many crops such as rape lettuce and so on. Oxalic acid is considered to be one of the important pathogenic factors in Sclerotinia sclerotiorum, but the specific target of oxalic acid is not clear. It also provides a new clue for the control of Sclerotinia sclerotiorum. Based on this, the following research was carried out to determine the effect of oxalate solution with different oxalic acid concentration and pH on the recovery of pathogenicity of non-oxalic acid-producing Sclerotinia sclerotiorum strain Ep-1PB, and the effects of different pH values on the pathogenicity of the strain. The accumulation of oxalate in the protoplasts of Bentner's tobacco. The results showed that exogenous oxalic acid could partially restore the pathogenicity of Ep-1PB strain, and the effect was significantly higher than that of citric acid. There was a positive correlation between oxalic acid concentration and pathogenicity of Ep-1PB strain in a certain concentration range. In addition, the lower pH of oxalic acid solution added, the stronger pathogenicity of Ep-1PB strain. Under acidic conditions, oxalic acid entered the protoplasts of Bensi tobacco significantly higher than that of neutral and alkaline conditions. A method for isolation of oxalic acid binding proteins from Arabidopsis thaliana was constructed. It includes preparation of oxalic acid bound gel beads and separation of oxalic acid binding proteins from Arabidopsis thaliana. The results showed that the optimum pH of the preparation system of gel beads with oxalic acid was 5.6-6.0, and 79 oxalic acid binding proteins were isolated and identified from Arabidopsis thaliana by adding oxalic acid to adjust pH 6.0 dd H2O for 1 hour. Go analysis of these proteins showed that these proteins were involved in antioxidant stress, gene silencing induced by RNA, Many biological processes, such as cell response to light intensity. Go enrichment analysis showed that the largest oxalic acid binding protein type was mainly antioxidant pressure, including resistance to superoxide, superoxide metabolism, removal of superoxide ion and other biological processes. KEGG analysis showed that these oxalic acid binding proteins were involved in amino acid synthesis, carbon metabolism and tricarboxylic acid cycle. KEGG enrichment analysis showed that the two most significant pathways for photosynthesis were phenylpropanin synthesis and starch and sucrose metabolism. The innovations of this study are as follows: (1) it is clear that H ~ can promote oxalate ions into plant cells and thus help Sclerotinia sclerotiorum cause disease. (2) A method of isolating oxalic acid-binding proteins from plants was constructed. (3) oxalic acid knot in plants was first identified. The synthase was isolated and identified. The bioinformatics correlation analysis of the related proteins provides a new idea for the further study of oxalic acid target in plants.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.44
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