【摘要】：核盤菌(Sclerotinia sclerotiorum)是一種分布廣泛且極具破壞性的植物病原菌,能夠引起油菜、萵苣等多種作物的菌核病。草酸被認為是核盤菌中重要的致病因子之一,但其具體作用靶標尚不明確,研究草酸的作用模式以及探索草酸在植物中的結合蛋白有助于幫助解析草酸在植物中的作用靶標,并為后續(xù)防治核盤菌提供新的線索�；诖�,本課題進行了以下研究:1、測定了不同草酸濃度、不同pH值的草酸根溶液對不產(chǎn)草酸核盤菌菌株Ep-1PB菌株致病力恢復情況的影響,以及在不同pH值下,草酸根在本氏煙原生質(zhì)體的積累量。結果表明:外源添加草酸可部分恢復Ep-1PB菌株的致病力,效果顯著高于檸檬酸。在一定濃度范圍內(nèi),外源添加的草酸濃度和Ep-1PB菌株致病力呈正相關。此外,添加的草酸溶液pH越低,Ep-1PB菌株致病能力越強。在酸性條件下,草酸進入本氏煙原生質(zhì)體的量顯著高于中性和堿性條件。2、構建了一種分離擬南芥中草酸結合蛋白的方法,包括高效制備結合有草酸的凝膠珠以及分離利用該凝膠珠分離擬南芥中草酸結合蛋白。結果表明:獲得結合有草酸的凝膠珠的制備反應體系的最佳pH為5.6-6.0,且在反應1h后添加草酸調(diào)節(jié)pH為6.0,dd H2O為最佳溶劑。3、從擬南芥中分離并鑒定出79種草酸結合蛋白。對這些蛋白質(zhì)進行GO分析,結果表明這些蛋白參與了抗氧化壓力、由RNA引起的基因沉默、細胞對光強度的應答等多種生物學進程。GO富集分析表明最大的草酸結合蛋白類別主要為抗氧化壓力類,包括抗超氧化物、應對超氧化物代謝、移除超氧根離子等生物學進程。通過KEGG分析,發(fā)現(xiàn)這些草酸結合蛋白參與了氨基酸合成、碳代謝、三羧酸循環(huán)、光合作用的碳固定等通路。KEGG富集分析顯示差異最顯著的通路為苯丙素的合成和淀粉及蔗糖代謝這兩種。本研究的創(chuàng)新之處包括:(1)明確了H~+可促進草酸根離子進入植物細胞從而幫助核盤菌致病(2)構建了一種分離植物草酸結合的蛋白的方法。(3)首次對植物體內(nèi)的草酸結合蛋白進行了分離鑒定,并對相關蛋白進行了生物信息學相關分析,為進一步研究草酸在植物中的作用靶標提供了新的思路。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S432.44

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本文編號：2199250