【作者】 韩巧霞；

【导师】 郭天财； 康国章；

【作者基本信息】 河南农业大学 ， 作物栽培学与耕作学， 2013， 博士

【摘要】 选用普通小麦品种豫麦34，在植株处于药隔形成初期时，使其遭受模拟–5°C的冻胁迫伤害，进而利用基因芯片和蛋白质双向电泳技术，研究了冻胁迫1d和3d后叶片基因表达图谱和蛋白质组的变化，以深入探讨小麦春季冻胁迫响应的分子机制。主要研究结果如下：1处于幼穗发育药隔形成初期的5个普通小麦品种，在遭受5°C冻胁迫期间，不同品种植株叶片的相对电导率无显著差异。表明同一幼穗发育阶段小麦品种，对春季冻胁迫的敏感性无显著差异。因此，选用其中的豫麦34品种进行下一阶段试验。表型与显微观察结果表明，在抽穗前，叶片遭受的冻害程度明显高于叶鞘包裹的幼穗。2利用基因芯片方法，研究了豫麦34小麦植株遭受–5°C冻胁迫1d和3d后基因表达图谱的变化，结果表明在冻胁迫处理后1d或3d，或同时在2个时间点，600个基因的转录水平发生了2倍以上变化。进一步分析后，发现转录水平变化至少8倍以上的基因有99个，在这些基因中，有许多冷胁迫相关基因和信号分子，它们编码了重结晶抑制蛋白、冷相关蛋白、CBF转录因子、钙依赖的蛋白激酶，推测它们参与了小麦春季冻胁迫响应过程。将本研究结果与以前的小麦冻锻炼（freeze hardening）和大麦春季冻胁迫(–3°C)的表达谱进行了比较。发现在这3个研究中，许多基因，包括编码WCOR413、LEA、glycine-rich RNA-binding protein、铁蛋白（a ferritin）、水通道蛋白（an aquaporin2）和病原体诱导蛋白（a pathogen-inducedprotein）的基因，呈现相似的表达模式。表明这些基因可能在高等植物冻胁迫响应中发挥着重要作用。3利用双向电泳（2-DE）和基质辅助激光解吸/电离飞行时间质谱法（MALDI-TOF MS）辨析了遭受冻胁迫1d和3d后小麦叶片中的响应蛋白。结果表明有115个蛋白点的丰度在冻胁迫后发生了1.5倍以上的变化，并成功鉴定出75个蛋白点，其中52个上调，18个下调。所鉴定出的相关蛋白主要参与了信号转导（4个）、应激/响应/解毒（17个）、蛋白质代谢（13个）、光合作用（11个）、氨基酸代谢（5个）、碳水化合物代谢和能量途径（4个）。这些结果表明，发生丰度显著改变的这些蛋白可能主要参与了小麦植株响应春季冻胁迫响应过程。4将上述表达图谱结果与蛋白质组结果进行比较后发现，绝大多数转录水平发生显著改变的基因与蛋白丰度发生显著改变的差异蛋白，并不一致。仅有4个蛋白（基因）在表达图谱与蛋白组结果中同时出现，它们包括2个S-like RNases、1个Cold-responsive LEA/RAB-relatedCOR protein和1个Cu/Zn superoxide dismutase，这些蛋白均参与了植物的防御反应。更多还原

【Abstract】 In the present study, a common wheat cultivar Yumai34was selected tobe suffered from–5°C freeze stress. And then, in order to explor the molecularmechanism on spring freeze-stress in wheat plants, the gene expression profile andproteomics after1and3days of freeze-stress in leaves of wheat plants at antherconnective tissue formation phase of spike development were studied by cDNAmicroarray and two-dimensional electrophoresis (2-DE). The main results were asfollow.1The rates of relative electrolyte leakage of Five common wheat cultivars sufferedfrom5°C freeze-stress at anther connective tissue formation phase of spikedevelopment had no significant difference. It indicated that the susceptibility to springfreeze-stress had no significant difference in different wheat cultivars at the samespike developmental stage. The observed result on phenotype and microexaminationon young spikes demonstrated that before the spikes emerge from leaf sheath, leavescould be more easily injured by freeze-stress than young spikes within the leaf sheath.2A global transcriptional profile was created using the Affymetrix Wheat GeneChipmicroarray for one wheat cultivar (Yumai34) under5°C freeze stress after1and3days. After1and3days of freeze stress,600genes that were previously annotated asshowing changes in expression of at least than two-fold were measured at one or bothof the time points. After further analysis, we found99genes whose expression levelschanged at least eight-fold after1or3days of freeze stress. These genes encoded anice recrystallization protein, cold-related proteins, CBF transcription factors,calcium-dependent protein kinases, Na+/H+antiporters, aquaporins, and manymetabolic enzymes. The results of this study were compared with those of a previousstudy on the sub-freeze hardening response in wheat and spring freeze stress in barley.Many genes, including those encoding WCOR413, LEA, glycine-rich RNA-bindingprotein, ferritin, aquaporin2, and a pathogen-induced protein, showed similar expression levels in these studies. It indicated that these genes play important roles infreeze-stress response in higher plants.3Following three-day exposure to–5oC simulated spring freeze stress, wheat plantsat anther connective tissue formation phase of spike development were analysedfreeze-stress responsive proteins in leaves at1and3days after following freeze-stressexposure, using two-dimensional electrophoresis and matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry. Our results indicate that out of75protein spots successfully identified under freeze-stress conditions52spots wereupregulated and18were downregulated. These spring freeze-stress responsiveproteins were involved in signal transduction (4spots),stress/defence/detoxification(17spots), protein metabolism (i.e. translation,processing, and degradation)(13spots), photosynthesis(11spots), amino acidmetabolism(5spots), carbohydrate metabolism(3spots), and energy pathways(1spots), and may therefore be functionally relevant for many biological processes. Theenhanced accumulation of signal transduction proteins such as a C2H2zinc fingerprotein, stress/defence/detoxification proteins including LEA-related COR protein,disease resistance proteins, Cu/Zn superoxide dismutase, and two ascorbateperoxidases may play crucial roles in the mechanisms of response to spring freezestress in wheat plants.4Most of genes changed at transcriptional level and proteins changed at proteinabundance were inconsistently afrer comparation on the results between transcriptomeand proteome. Only4proteins/gens emerged in transcriptome and proteome at thesame time. Four proteins/gens included two S-like RNases, a Cold-responsiveLEA/RAB-related COR protein and a Cu/Zn superoxide dismutase. Theseproteins/genes all involved in defense reaction in wheat plants.更多还原