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小麦与叶锈菌互作过程中β-1,3-葡聚糖酶的表达特征分析

标题: 小麦与叶锈菌互作过程中β-1,3-葡聚糖酶的表达特征分析
英文标题: Research on the Expression Analysis of β-1,3-glucanases in the Process of Wheat and Leaf Rust Interaction
作者: 吴洪波
出版时间: 2012-01-01
所在大学: 河北农业大学
关键词: 小麦,叶锈菌,防卫反应,β-1,3-葡聚糖酶,半定量PCR,Western Blotting检测,酶活性测定,免疫金标记
英文关键词: wheat,Puccinia triticina,β-1,3-glucanase,defensive reaction,RT-PCR,Western Blotting,activity determination,transmission electron microscopic immunolocalization
论文级别: 硕士
学位: 学位论文
导师: 王冬梅
专业: 植物学
提交时间: 2012
摘要: 本试验室长期以来从事小麦抗叶锈菌侵染生理机制的研究工作,前期工作已经证明,在小麦与叶锈菌(Puccinia triticina)的不亲和互作中β-1,3-葡聚糖酶(β-1,3-glucanase,BG)表现上调表达,推测其在小麦抵抗叶锈菌的抗病反应中可能起到了重要作用.本试验以小麦(Triticum aestivum L.)品种洛夫林10和叶锈菌生理小种165与260为研究对象,洛夫林10与小种165构成亲和组合,与260构成不亲和组合,对不同亲和性组合中β-1,3-葡聚糖酶的表达特征进行了系统研究.获得主要结果如下:1、利用RT-PCR技术分析了不同组合中小麦被叶锈菌侵染后不同时间点β-1,3-葡聚糖酶的表达特征.结果表明,在不亲和组合中小麦叶片在接菌后4hβ-1,3-葡聚糖酶基因的表达量较接种后0h有明显增加,呈上调表达,一直至接种后120 h在叶片中的表达水平呈持续增加趋势;而在亲和组合中β-1,3-葡聚糖酶基因在接种后4h较0 h表现出轻微上调表达,之后也呈增加趋势,但其表达量要低于不亲和组合,在接种后48h其表达量开始下调,到120h恢复至0h水平.这一结果表明,β-1,3-葡聚糖酶基因的表达受叶锈菌侵染诱导,其转录水平在亲和组合和不亲和组合之间的表达差异意味着该基因可能与小麦抗叶锈菌侵染有关.2、通过Western blotting技术分析了不同组合中小麦被叶锈菌侵染后不同时间点β-1,3-葡聚糖酶的表达特征.结果表明,接种后4h,β-1,3-葡聚糖酶在不同组合中均开始表现上调表达,且呈递增趋势,但在不亲和组合中各时间点的表达量均高于亲和组合,且高表达的时间持续较长,而亲和组合在接种后期出现下降趋势.3、分析了不同组合中小麦被叶锈菌侵染后不同时间点β-1,3-葡聚糖酶活性的变化特征.发现酶活性的变化趋势同酶表达量的变化趋势大体相同,初步表明β-1,3-葡聚糖酶在小麦抗叶锈菌侵染过程中可能发挥重要作用.4、利用免疫金标记技术在原位水平对叶锈菌侵染小麦诱导产生的β-1,3-葡聚糖酶的时空表达特征进行了系统研究.试验结果表明:β-1,3-葡聚糖酶主要定位在寄主的细胞壁以及细胞间隙中,在不亲和组合中的表达高于亲和组合,且主要分布在菌丝周围,这种趋势在后期更加明显.这一结果暗示着β-1,3-葡聚糖酶可能直接作用于菌丝细胞壁,对菌丝的生长产生了抑制作用.综上所述,我们以小麦品种洛夫林10和叶锈菌生理小种260与165分别组成不亲和和亲和组合,通过半定量PCR、Western Blotting检测、酶活性测定和免疫金标记技术分别在转录水平、翻译水平、酶活性和原位水平对叶锈菌侵染诱导产生的β-1,3-葡聚糖酶进行了系统研究,证明β-1,3-葡聚糖酶对诱发小麦对叶锈菌的抗性反应起到重要作用,为进一步探讨小麦抗叶锈菌侵染的生理机制奠定了基础,对于全面阐释小麦抗叶锈菌侵染的防卫反应机理具有重要意义.
英文摘要: Our laboratory has long been engaged in the research on the physiological mechanisms of wheat Resistance to Wheat Leaf Rust Infection. Preliminary works have demonstrated that, in the incompatible wheat-leaf rust (Puccinia triticina) combinations,β-1,3-glucanase (β-1,3-glucanase, BG) has an important role in the resistance response induced by wheat leaf rust .In this experiment, wheat cultivar Lovrin10 and leaf rust fungus races 260 and 165 were defined as study object. Loverin 10 and 165 constitute the compatible combination , Loverin 10 and 260 constitute the incompatible combination. Systematic study ofβ-1,3-glucanase gene expression characteristics in both conbinations were carried out. Total RNA was extracted in both conbinations at 0h, 4h, 12h, 24h, 48h, 72h, 120h after wheat leaf rust infection andβ-1,3-glucanase gene expression characteristics were get by semi-quantitatively PCR after RT-PCR.The results showed that in both conbinations leaf rust infection could induceβ-1,3-glucanase gene expression, and the gene expression level in incompatible combination is higher than that in compatible combination. At 4h after inoculation,β-1,3-glucanase gene expression in the two combinations has showed a significant difference ,which may be related to host early resistance response .At 12h, 24h and 48h,β-1,3-glucanase gene expression has raised in both combinations, but the increase ofβ-1,3-glucanase gene in incompatible combination was significantly higher than compatible combination.At 72h and 120h,β-1,3-glucanase gene expression in incompatible combination continued to rise, however in compatible combinationβ-1,3-glucanase gene expression showed a downward trend.From the genetic level point of view, it is speculated thatβ-1,3-glucanase play an important role in the resistance response to wheat leaf infetion.To further demonstrate the reasons of the existence of these differences,it is studied from the protein level and physiological levels ofβ-1,3-glucanase which play a role in wheat resistance response induced by leaf rust fungus. Total protein was extracted in both combinations at above-mentioned time, andβ-1,3-glucanase were detected by western-blotting.The results showed thatβ-1,3-glucanase protein was detected at 4h after inoculation and gradually increased in both combinations. And the expression level in incompatible combination is higher than in compatible combination. In compatible combination enzyme expression levels began to reduce after 48 h, but in incompatible combinations the expression level maintained a high level. Then the enzyme activity in the bacteria leaves was detected and the activity trend is the same as the trend of the enzyme expression levels .All the results indicate thatβ-1,3-glucanase plays an important role in the wheat resistance response to leaf rust infection.Thenβ-1,3-glucanase spatial and temporal expression characteristics were studied in situ levels after leaf rust infection by immunolocalization.The results showed that:β-1,3-glucanase was mainly localized in the host and the host cell wall and cell gap solution and protein level in incompatibal combination is higher than in compatible combination, and this trend is more obvious in the later time. Resluts above prove thatβ-1,3-glucanase participates in the process of wheat resistance response to leaf rust infection and directly inhibitates the leaf rust infection.In summary, wheat varieties Loverin 10 and leaf rust physiological races 260 and 165 were chosen to form compatible and incompatible combinations. By semi-quantitative PCR, Western Blotting detection, enzyme activity assay and immuno-gold labeling technique at the transcriptional level, translation level, enzyme activity and in situ levels to studyβ-1,3-glucanase after leaf rust infection.The results have proved thatβ-1,3-glucanase plays an important role in the resistance responses induced by wheat leaf rust .The results lay a foundation for further study of physiological mechanisms of wheat resistance to leaf rust, and has an important role in our in-depth understanding of wheat defense mechanism to leaf rust.