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受叶锈菌侵染的小麦叶片细胞间隙液中抗病相关蛋白的鉴定

标题: 受叶锈菌侵染的小麦叶片细胞间隙液中抗病相关蛋白的鉴定
英文标题: Identification of Pathogenesis Related Protein in Intercellular Washing Fluid of Rust-infected Wheat Leaves
作者: 吕术超
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: 小麦,叶锈菌,细胞间隙液,防卫反应,双向电泳,β-1,3-葡聚糖酶
英文关键词: wheat(Triticum aestvum L),leaf rust fungus(Puccinia triticina),Intercellular washing fluids (IWF),Defense response,Two-dimensional electrophoresis,β-1,3-glucanase
论文级别: 硕士
学位: 学位论文
导师: 侯春燕%王冬梅
专业: 植物学
提交时间: 2009
摘要: 本试验以小麦(Triticum aestivum)品种洛夫林10、5389和叶锈菌(Puccinia triticina) 生理小种260 为研究对象,分别提取亲和组合(5389*260)接菌后五天以及不亲和组合 (洛10*260)接菌后三天的小麦叶片细胞间隙液(IWF),将其注射到小麦幼苗叶片中, 测定被注射叶片的PAL、PO 活性及过敏性反应(HR)面积,并观察对随后接种的叶锈 菌菌丝扩展的影响以及IWF 对叶锈菌孢子萌发的影响.结果表明,无论来自亲和组合 或不亲和组合的小麦细胞间隙液均能诱导小麦PAL,PO 活性升高和产生HR,而且来 自不亲和组合的细胞间隙液所诱导的防卫反应活性要明显高于亲和组合.IWF 对随后接 种的表现亲和互作的叶锈菌菌丝扩展也有一定抑制作用,并且也能抑制孢子的萌发,初 步证明了IWF中存在抗叶锈菌相关的物质. 为了进一步分离获得小麦细胞间隙液中与诱导防卫反应及抗叶锈菌相关的物质,以 不亲和组合的细胞间隙液为研究对象,以小麦健叶细胞间隙液为对照,利用双向电泳技 术进行鉴定.首先建立了一套适用于小麦叶片细胞间隙液蛋白质分析的双向电泳体系. 本试验确定了用50%TCA 法沉淀细胞间隙液的蛋白质,适宜的蛋白上样量为800μg,主 动水化12h,通过不同pH范围胶条的选择确定了细胞间隙液蛋白主要分布在pI4-7之 间,一向到二向过渡时平衡时间为15min,最终获得了重复性好,分辨率高的蛋白质双 向电泳图谱.经观察发现,不亲和组合的小麦叶片细胞间隙液被诱导产生大量新的蛋白 质.对其中显著的两个蛋白点进行质谱分析和数据库检索,发现分别与β-1,3-葡聚糖内 切酶和β-1,3-葡聚糖酶前体具有较高的同源性,推测β-1,3-葡聚糖酶可能在诱发小麦对叶 锈菌的抗性反应中发挥作用. 为进一步验证β-1,3-葡聚糖酶是否在小麦抗叶锈菌侵染过程中发挥作用,首先测定 了接菌后不同时间点不同亲和性组合细胞间隙液中β-1,3-葡聚糖酶的活性,结果表明不 亲和组合的β-1,3-葡聚糖酶活性始终高于亲和组合,并在4h和72h 出现两个高峰,说明 β-1,3-葡聚糖酶在小麦早期抵抗叶锈菌的侵染过程中可能发挥重要作用.随后,将叶锈 菌孢子培养到含有一定浓度的外源β-1,3-葡聚糖酶的水培液中,发现孢子萌发并没有受 到抑制,仍然长出芽管,说明其在体外并不能抑制孢子的萌发.再向小麦叶片中注射外 源β-1,3-葡聚糖酶,并接菌,观察对菌丝扩展和HR的影响,试验结果表明外源β-1,3-葡 聚糖酶在亲和组合中可以抑制菌丝的生长,在不亲和组合中可以将出现HR的时间提 前,且HR 面积减小.以上试验初步证明β-1,3-葡聚糖酶参与到了小麦抵抗叶锈菌侵染 的过程. 综上所述,本试验首先确定了不亲和组合细胞间隙液诱导防卫反应活性较大,并有 抗病相关物质存在.随后对其进行双向电泳分析,鉴定得到了β-1,3 葡聚糖酶这一病程 相关蛋白.并且通过生理生化试验证明β-1,3 葡聚糖酶在小麦抵抗叶锈菌侵染过程中发 挥重要作用,为进一步探讨小麦抗叶锈菌的生理机制奠定了基础.
英文摘要: In this experiment, wheat cultivar Lovrin10,5389 and leaf rust fungus races 260 were defined as study objects. The intercellular washing fluids (IWF) extracted from wheat leaves of combinations(5389*260) after 5 days after inoculation (dai) and incombinations(L10*260) after 3 dai were injected into wheat leaves to determine the activities of PAL, PO and the area of hypersensitive reaction (HR). Then the effects of IWF on hyphal extension and spore germination were observed. The results indicated that both of the IWF from the compatible and incompatible combinations could induce activities of PAL, PO and HR area on leaves of wheat increasing. Furthermore, the defense response induced by IWF from incompatible combination was obviously higher than that of compatible combination. Meanwhile, they could inhibit the hyphal extension and spore germination in the incompatible combination either. This result took a proof of the existence of resistance substances in IWF.In order to isolate pathogenic related protein, the IWF from rust-infected leaves of incompatible combination were used as study objects, and the IWF of healthy wheat leaves served as control, both of them were identified by two-dimensional gel electrophoresis. A suitable test system of two-dimensional electrophoresis for proteomic study on wheat IWF was established in this experiment, which referred a series of optimized effectors as followed: (1) the 50%TCA method was used to precipitate protein of IWF, (2) 800μg of protein was the best loading amount, (3) active hydration was carried out for 12h, (4) the IPG of pI 4-7 was suitable to IWF of wheat, and finally, (5) the best balance time from the one direction to two direction was 15min. This result revealed that high reproducible and resolution two-dimensional gel electrophoresis maps of protein were obtained according to the key effectors mentioned above. A large number of new proteins which were induced by rust fungus in incompatible group were observed. Two significant protein dots in the maps were analyzed by mass spectrometry and then compared with the database in NCBI. The searched information indicated these two proteins had high homology with endo-β-1,3-glucanase andβ-1,3-glucanase precursor. Considered the given result, we supposed thatβ-1,3-glucanase may play a role in wheat resistance to leaf rust fungus. To verify the role ofβ-1,3-glucanase in the process of wheat resistance, firstly,β-1, 3-glucanase activity in IWF from two combinations at different time points after inoculation were detected.The results showed that the activity ofβ-1,3-glucanase from incompatible combination was always higher than that from compatible combination, in addition to that, two peaks were observed at 4hai and 72hai. It indicated thatβ-1,3-glucanase also played a role in wheat resistance to leaf rust fungus. Subsequently, the urediniospores were cultivated in the solution which contained different concentrations of exogenousβ-1,3-glucanase. We found that the germination of spores was not inhibited because of germ tube, which indicated thatβ-1,3-glucanase did not inhibit spore germination in vitro. Thirdly, exogenousβ-1, 3-glucanase were injected into wheat leaves, which inoculated with leaf rust fungus, to measurement its effect on the hyphal extension and the impact of HR. Our results showed thatβ-1,3-glucanase can inhibit the extension of hyphal area in compatible group; and the initiation of HR was getting earlier accompanied with HR area reducing in incompatible group. The experiment results proved thatβ-1, 3-glucanase may be involved in the resistance of wheat to leaf rust fungus infection.In conclusion, the inducing activity to wheat defense response of IWF from incompatible group was obviously higher than compatible group, and there was resistance substances in IWF. Then theβ-1,3-glucanase in IWF was identified by two-dimensional gel electrophoresis analysis, the result indicated thatβ-1,3-glucanase may take part in the wheat resistance to leaf rust through our experiments. The results might lead a way for further studies on the mechanisms of wheat-leaf rust resistance.