小麦抗白粉病近等基因系的差异蛋白质组学研究

Differential Proteomic Analysis of Wheat Powdery Mildew Resistance Near Isogenic Line

作者: 专业:作物遗传育种 导师:张卫东 年度:2010 学位:硕士  院校: 山东农业大学

Keywords

wheat, powder mildew, physiological indicators, proteomics, pathogenesis-related proteins

        小麦白粉病是由白粉病菌(Blumeria graminis f. sp. tritici, Bgt)侵染引起的小麦重要真菌病害之一,它流行性强,危害严重,该病害发生流行常常造成小麦严重减产,甚至导致绝收。小白冬麦是北疆古老的冬麦地方品种,主要分布于新疆乌鲁木齐、景化及玛纳斯等地,其抗白粉病能力表现突出,为我国特有的地方抗病品种。小白冬麦所含的抗白粉病基因xbd定位于7B染色体,并与Pm5位点紧密连锁,至今保持着一定的抗白粉病能力和较低的毒性频率,对E01、E03、E05、E09、E15、E16、E18、E20、E26、E30和E31共11个小麦白粉菌菌株表现抗病。本研究选用两个近等基因系作为研究材料,易感白粉病的小麦品种百农3217,和含有抗白粉病基因xbd的百农3217的近等基因系(xbd/百农3217(BC7F8)、抗白粉病,中国农业科学院作物所惠赠)。从生理生化与蛋白质组学角度研究两个小麦材料对白粉病反应的差异,揭示(1)0-6天内,白粉菌侵染两个近等基因系材料,对过氧化氢含量及其相关代谢酶活性的影响。(2)三叶期幼苗接种白粉菌4天后,两个近等基因系材料的蛋白质组学差异。力图了解病原入侵寄主后所引起的生理变化与蛋白表达变化情况,揭示寄主小麦与病菌之间的互作机制,并获得有价值的抗白粉病特异蛋白。获得的主要结果如下:1.白粉菌侵染小麦0-6天,抗病品系相对于感病品系,H2O2含量显著升高,H2O2含量对小麦抗白粉病有很重要的作用。小麦对白粉病的抗病性可能与植物体内活性氧的积累有关,H2O2含量的变化是由一些抗氧化酶系统共同作用的结果。2. H2O2相关代谢酶活性,在感病品系中变化较大,显著高于抗病品系。在接种白粉病菌5d后,感病品系小麦体内SOD、GR、GPX、APX、GST酶活性显著升高,并显著高于抗病小麦品系,而抗病小麦品系体内SOD、GR、GPX、APX、GST酶活性并没有显著的变化。感病品系与病菌互作后,体内各种生理指标都发生了很大变化,说明病菌对感病品系侵染后,引起了一种应激反应,是对病害胁迫的一种适应调节,且近等基因系抗病品系对白粉病菌表现免疫,病菌并没有引起抗病品系体内各种酶类的很大变化。3.优化了小麦幼苗叶片蛋白的提取、分离和染色技术,获得了分离清晰、蛋白点多的电泳胶图。蛋白提取加入10%PVP研磨叶片,适量增加裂解液中CHAPS和硫脲的含量,并增加了超声处理步骤;等电聚焦水化液中加入4%DTT,增加除去离子步骤,电压至500V,延长时间至5h,等电聚焦电压维持8000V,时间至13h;SDS凝胶电泳调整电泳缓冲液中SDS含量至0.1%和琼脂糖固定液的浓度至0.5%。这样,利用pH 3-10,24cm胶条,经蛋白双向电泳,硝酸银染色和PDQuest软件分析,检测到2500个蛋白质点。4.经过双向电泳分离,在感病和抗病材料中检测出具有显著性差异的蛋白点230个,质谱鉴定、数据库搜索获得19个具有明确功能的蛋白质点。这些蛋白包括:抗性相关蛋白、光合和电子传递相关蛋白、代谢相关蛋白、其它功能蛋白等。抗性相关蛋白包括系统获得性抗性体系的病程相关蛋白,如β-1,3-葡聚糖苷酶(点2)、β-葡萄糖苷酶(点6)、茉莉酸诱导蛋白(点7),和SAR体系中诱导防卫蛋白如氧甲基转移酶(点1)、TIR-NBS-LRR类抗病相关蛋白(点8);光合和电子传递相关蛋白有光系统II蛋白(点15)、Rubisco活化酶(点11、12、13、16)、细胞色素b6(点4)、氧协同进化复合体前体(点14);代谢相关蛋白如苹果酸脱氢酶(点19)、脂肪酶I(点21)、谷氨酸合成酶(点10);及其它功能蛋白,如核糖体蛋白L11(点3),蛋白合成有关,腺苷高半胱氨酸酶(点20),核苷酸转运有关,钾离子通道蛋白属膜转运蛋白(点9),β-微管蛋白(点17)属于结构蛋白。
    Wheat powder mildew, caused by the fungus Blumeria graminis f.sp.Tritici (Bgt), is one of the most important wheat diseases worldwide. It can lead to nearly 40 percent of yield loss in some fields in epidemic years. Xiao Baidong is north of ancient local varieties of winter in Xinjiang, distributed mainly in Xinjiang Urumqi and Jinghua etc, and its powdery mildew resistance ability is outstanding, and is the special local resistant varieties. Xiao Baidong contained a resistant genes named xbd, and located in chromosome 7B with Pm5 closely, retained powdery mildew resistance capacity and low toxicity frequency up to now, is resistant to many wheat powder mildew bacteria (E01, E03, E05, E09, E15, E16, E18, E20, E26, E30, E31).This study chooses two near isogenic line as research materials, Bainong3217(susceptibility to powdery mildew) and xbd/Bainong 3217 (BC7F8)(resistant to powdery mildew). We studied wheat powdery mildew resistance on physiological and biochemical and proteomic method aiming at finding the differences between the two near isogenic line, and revealed: (1) After inoculated by Blumeria graminis, the wheat leaves were detected on concentrations of hydrogen peroxideanalysis (H2O2) and biological activities of relevant kinds of metabolic enzymes during 0-6 days. (2) Differential proteomic of the two near isogenic line, after seedling inoculation with four days later. Aimed at finding the interaction mechanisms between the two near isogenic line, and gaining valuable specific resistant proteins.The main results as follows:1. H2O2 content in the susceptible and resistant lines were significantly decreased and increased after inoculation with powdery mildew, respectively. H2O2 might play an important role in the wheat responses, several metabolic mechanisms might also involve in the responses, and H2O2 content could be influenced by these various enzymes.2. Metabolic enzymes activities related H2O2 in the leaves of susceptible line increased significantly during post-inoculation, and were higher than those in the resistant lines. After inoculated with bacteria in 5d, GPX, GR, SOD, APX, GST enzyme activity significantly increased in susceptible line, and this enzyme activity no significant changes in the resistant lines. Physiological indexes have changed significantly during the pathogen infection period, explain to interested disease strains infect bacteria is a kind of disease stress to adjust after a response to stress.3. Optimized the 2D technology including protein extraction, separation and staining techniques in leaves of wheat seedling, obtained a map which has clear separation and numbers of proteins. Add 10% PVP when grinding leaves, increase CHAPS and Thiourea content moderatly, and increased ultrasonic processing steps during protein extraction; Add 4% DTT in IEF hydration solution and add one step to remove the ions, enhancing the voltages to 500V and enlonging the time to 5h, isoelectric focusing voltage is 8000V, the time to 13h; Adjust SDS concentration to 0.1% and agarose concentration of fixative to 0.5% in SDS gel electrophoresis buffer solution. Thus, we detected 2500 protein spots by using pH 3-10, 24cm strip and 2D, silver staining and PDQuest software analysis.4. After 2D separation, we detected significantly different protein spots 230 between the susceptible and resistant lines, and obtained 19 proteins with clear features through MS and database searches. These proteins include: resistance-related protein, photosynthetic and electron transport proteins, metabolism-related protein, and other functional proteins. Resistance related proteins includ pathogenesis-related protein in systemic acquired resistance(SAR), such as beta-1,3-glucosidase(spot 2), beta-glucosidase(spot 6), 60 kDa jasmonate-induced protein(spot 7), and induction of defense proteins in SAR, such as O-methyltransferase (spot 1), TIR-NBS-LRR class disease resistance protein(spot 8); photosynthetic and electron transport proteins: optical system II protein(spot 15), ribulose-1,5-bisphosphate carboxylase(spot 11, 12, 13, 16), cytochrome b6(spot 4), oxygen-evolving complex precursor(spot 14); Metabolism-related protein: malate dehydrogenase(spot 19), lipase I(spot 21), plastidic glutamine synthetase(spot 10); And other functional proteins, such as ribosomal protein L11(spot 3), protein synthesis related, Adenosyl-homocysteinase(spot 20), nucleotide transporter related, potassium channel protein(spot 9) belongs to membrane transportproteins, beta tubulin (spot 17) belongs to structural protein.
        

小麦抗白粉病近等基因系的差异蛋白质组学研究

摘要8-10
ABSTRACT10-12
1 前言13-43
    1.1 小麦白粉病的研究进展13-24
        1.1.1 小麦白粉病的特征与发生原因14-16
        1.1.2 小麦白粉病的防治手段16-17
        1.1.3 小麦抗白粉病种质资源研究进展17-20
        1.1.4 小麦白粉病生理机制的研究进展20-24
    1.2 蛋白质组学及其研究进展24-33
        1.2.1 样品制备25-26
        1.2.2 双向凝胶电泳26-28
        1.2.3 液相色谱分离技术28-30
        1.2.4 质谱技术在蛋白质组学的应用30-33
        1.2.5 生物信息学33
    1.3 小麦的蛋白质组学研究进展33-36
        1.3.1 小麦逆境相关蛋白质组学研究34
        1.3.2 小麦抗病相关蛋白质组学研究34-35
        1.3.3 小麦遗传学差异研究35
        1.3.4 小麦发育相关蛋白质组学研究35-36
    1.4 蛋白质组学在作物病理学上的运用36-40
        1.4.1 小麦抗病蛋白质组研究36-38
        1.4.2 水稻抗病蛋白质组研究38-39
        1.4.3 玉米抗病蛋白质组研究39-40
        1.4.4 展望40
    1.5 论文设计:研究目的意义及技术路线40-43
        1.5.1 研究目的和意义40-42
        1.5.2 技术路线42-43
2 实验材料与方法43-55
    2.1 材料43-47
        2.1.1 研究材料43
        2.1.2 生化试剂43
        2.1.3 主要设备仪器43-44
        2.1.4 试剂配制44-47
    2.2 方法47-55
        2.2.1 白粉菌的接种47
        2.2.2 叶片生理指标的测定47
        2.2.3 蛋白提取47-48
        2.2.4 蛋白浓度测定Bradford 法蛋白质定量操作48-49
        2.2.5 双向电泳49-52
        2.2.6 凝胶染色52-54
        2.2.7 凝胶图谱分析54
        2.2.8 质谱54-55
        2.2.9 数据库搜索和蛋白鉴定55
3 结果与分析55-71
    3.1 小麦近等基因系与白粉病菌互作的生理生化研究55-61
        3.1.1 近等基因系叶片抗病表现55-56
        3.1.2 感病、抗病品系与白粉病菌互作后H2O2 含量变化56-57
        3.1.3 感病、抗病品系与白粉病菌互作后各种酶的活性变化57-61
    3.2 小麦幼苗叶片双向电泳技术体系的优化61-66
        3.2.1 小麦幼苗叶片提取蛋白方法的优化61-62
        3.2.2 小麦幼苗叶片蛋白双向电泳技术的优化62
        3.2.3 小麦幼苗叶片染色技术的优化62-63
        3.2.4 双向电泳实验失误分析63-66
    3.3 小麦抗白粉病近等基因系的差异蛋白质组学研究66-71
        3.3.1 双向电泳图谱分析66-67
        3.3.2 质谱及鉴定结果67-71
4 讨论71-77
    4.1 小麦近等基因系与白粉病菌互作的生理生化研究71-72
    4.2 小麦幼苗叶片双向电泳技术体系的优化72-73
    4.3 小麦抗白粉病近等基因系的差异蛋白质组学研究73-76
        4.3.1 抗病及防御相关蛋白73-75
        4.3.2 光合与电子传递相关蛋白75
        4.3.3 代谢相关蛋白75
        4.3.4 其它蛋白75-76
    4.4 进一步研究设想76-77
5 结论77-79
参考文献79-91
致谢91-92
攻读学位期间发表论文表情况92
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