【作者】 马信；

【导师】 孔令让；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 小麦赤霉病(Fusarium Head Blight, FHB)是小麦的一种主要病害，主要的致病菌是禾谷镰刀菌(Fusarium graminearum，F.g)，赤霉病的流行严重影响小麦产量和品质。在侵染小麦时禾谷镰刀菌分泌脱氧雪腐镰刀菌烯醇(Deoxynivalenol, DON)毒素，可以促进赤霉菌在小麦穗部的扩展。DON可被一些特异的UDP-葡糖基转移酶(UDP-glycosyltransferases, UGT)糖基化，降低其毒性。本研究依据一对感、抗赤霉病近等基因系Apogee和Apogee73S2在赤霉菌侵染前后的表达谱数据，挖掘一些与赤霉病抗性相关的基因，通过生物信息学分析、表达分析以及功能分析等方法对克隆的2个UGT基因和1个1,3-1,4-β-葡聚糖水解酶(endo-1,3-1,4-β-glucanase，EGL)开展了初步的功能分析，主要研究结果如下：1.同源克隆获得TaUGT4，RACE克隆获得仅在抗病的Apogee73S2中存在的TaUGT5。TaUGT4编码461个氨基酸残基，位于2D染色体上，编码蛋白分布在细胞质中。TaUGT5编码476个氨基酸残基。两基因编码的蛋白在C-端均有PSPG box功能域。2.在禾谷镰刀菌、DON毒素处理下两个UGT基因表达均显著上调，说明两个基因参与了小麦对赤霉菌的应答响应。相比水杨酸(SA)处理，甲基茉莉酸(MeJA)更能有效地诱导两个基因的上调表达，说明JA可能在调节两个UGT基因的表达中起着重要作用。3. TaUGT5转化拟南芥，提高了转基因拟南芥对DON毒素的抗性，表明TaUGT5对DON具有一定解毒能力。4. MeJA和SA的处理均使感赤霉病小麦穗部的发病率下降，而且MeJA提高赤霉病抗性更加明显，表明JA在小麦抗赤霉病中起着重要作用。5. TaEGL1基因在抗赤霉病小麦苏麦3号和Apogee73S2中的基础表达水平高于感赤霉病小麦科农199和Apogee。用复性的原核表达蛋白TaEGL1处理小麦叶片，可导致叶片失绿，表现出细胞受损的症状，说明TaEGL1可能通过破坏细胞壁参与小麦的赤霉病发生。6. SA在感赤霉病小麦第一叶展开前处理可显著提高叶片对赤霉菌的抗性，而MeJA前处理抗赤霉病小麦则促使叶片感染赤霉菌，表明SA和JA作用机制的不同。通过以上研究，克隆获得两个新的UGT基因和一个EGL基因，并对三个基因的序列结构和表达特性开展了研究，通过拟南芥遗传转化、复性蛋白的功能分析以及激素的调节表达初步分析了三个基因的功能，为小麦抗赤霉病机制的研究以及抗赤霉病育种提供了参考依据。更多还原

【Abstract】 Fusarium head blight, caused primarily by Fusarium graminearum, is a destructive disease ofwheat throughout the world. FHB epidemics not only cause significant yield losses, but alsoreduce grain quality primarily. Deoxynivalenol (DON) produced by F. graminearum whichenhance the pathogen to spread could be converted into inactivating formation D3G by UDP-glycosyltransferases (UGTs). FHB resistance-related genes were selected according to thetranscript profile generated from a FHB-susceptible cultivar Apogee and its one near-isogenicline Apogee73S2with resistance to FHB. Two UGT genes and one EGL gene were cloned inthis study and their functions were analyzed. The main results are as follows:1. TaUGT4and TaUGT5were obtained by in silico cloning and RACE, respectively.TaUGT4, which was located on chromosome2D, encoded461amino acids protein.Subcellular localization of TaUGT4indicated that TaUGT4protein was localized incytoplasm. Both of TaUGT4and TaUGT5have PSPG-box motif at C-terminus.2. Both of TaUGT4and TaUGT5were obviously induced by F. g inoculation or DONtreatment which suggested that the two UGT genes involved in response to F. g inoculation.The expressions of the two UGT genes were also significantly induced by MeJA treatment,implying that JA signaling pathway might play important roles against F. g inoculation.3. The TaUGT5transgenic Arabidopsis showed enhanced tolerance against DON whichsuggested that TaUGT5had the ability to deactivate DON.4. The ability of resistance to FHB was enhanced in FHB-susceptible cultivar bytreatment with SA or MeJA, and the effect was more significant in wheat spikes treated byMeJA. This result also suggested that JA signaling pathway might play important roles inFHB resistance.5. The expression of TaEGL1was higher in FHB-resistant cultivar than that in FHB-susceptible cultivar. Leaf cells could be damaged by renatured TaEGL1produced byprokaryotic expression, which suggested that TaEGL1might participated in F. g inoculation.6. The leaves of FHB-susceptible cultivar exhibited high resistance to F. g by treating theseeding with SA before the first leaf stretching. On the contrary, The leaves of FHB-resistantcultivar showed more susceptible to F. g by treating the seeding with MeJA before the firstleaf stretching.In conclusion, we cloned two novel TaUGT genes and one TaEGL gene from wheat, andanalysed their sequence characteristics and expression patterns. Their functions were analyzedby genetic transformation using Arabidopsis and exogenous hormone regulation. The above works established a foundation for using these novel genes in wheat transformation andgenetic improvement.更多还原