【作者】 高巍；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 棉花是世界上最重要的经济作物之一。但是,棉花纤维的产量和质量受到棉花黄萎病的严重制约。棉花黄萎病是一种由大丽轮枝菌引起的土传性真菌维管束病害。当黄萎病暴发时,棉花的纤维品质和产量都会受到非常严重的影响,甚至会达到30%以上的减产。因此棉花黄萎病又被称为‘棉花的癌症’。然而目前在世界范围内都没有发现比较好的陆地棉抗黄萎病种质资源。遗传资源的缺乏,对发掘优良的抗病基因形成了挑战。另一方面,棉花黄萎病菌生理小种多,小种变异性强,加大了对棉花抗黄萎病菌的抗病机制以及抗病育种研究的难度。海岛棉相对于陆地棉品种往往表现出较好的耐病甚至抗病性,因此在海岛棉的抗病品种中,可能存在某些抗病基因或抗性机制。我们选择海岛棉品种海‘7124’作为研究材料,通过比较蛋白质组学的方法,来发掘棉花中的抗病相关基因,并研究其可能存在的抗病机制。通过双向电泳分析以及质谱鉴定,一共得到了188个在海’7124’根系中受黄萎病菌侵染后与水处理的平行对照相比表达量出现差异的蛋白。在这些差异表达的蛋白中,下调表达的蛋白数量达到47%,接近差异表达蛋白总数量的一半。通过生物学功能注释分类,这些差异蛋白一共可以分为17类。其中参与细胞过程、代谢过程和应激反应的蛋白数量最多。这些蛋白共同参与了棉花对黄萎病菌入侵的响应。说明棉花在受病原菌侵染后,正常情况下的细胞过程和生理代谢都发生了变化。棉酚是棉花特有的一类次生代谢物。在我们的蛋白表达谱中发现两个与棉酚代谢相关的酶GbCAD1和GbdHG-6-OMT。GbCAD1基因编码杜松稀合成酶,是棉酚合成的限速酶。通过病毒诱导的基因沉默(VIGS)技术干涉GbCAD1后,发现GbCAD1干涉植株棉酚含量显著降低。接种强致病力落叶型黄萎病菌’V991’后发现,干涉植株对黄萎病的抗性明显降低。说明棉酚可能影响棉花对黄萎病菌的抗性。在棉花根系中,检测黄萎病侵染后其它一些棉酚代谢相关基因的表达发现,棉酚合成在棉花受黄萎病侵染后是被激活的。而作为棉酚活性的负调控因子,编码脱氧半棉酚-6-氧甲基转移酶的GbdHG-6-OMT基因在病原菌侵染后下调表达。通过组织切片、组织化学染色以及棉酚含量的测定,进一步确认了棉花在黄萎病菌侵染后,植株体内的棉酚含量是大量积累的。这些结果表明棉酚的积累可能参与抵抗黄萎病菌的侵染。在研究棉酚代谢上游的调控因子及调控方式时,通过检测抗病相关植物激素水杨酸(SA)和茉莉酸甲酯(MeJA)处理后,棉酚代谢相关基因的表达,发现JA处理后,棉花植株体内棉酚代谢相关基因GbFPS、GbCADl和WRKY1表达上调。这说明棉酚代谢可能受茉莉酸信号的调控。BR介导的信号路径除了在植物的生长发育中发挥着重要的功能外,也参与了植物抗病。我们分离到两个在V991侵染后蛋白水平下调的14-3-3蛋白Gb14-3-3c和Gb14-3-3d。14-3-3蛋白是BR信号路径的负调控因子,通过调控BZR1和BZR2的蛋白定位,来影响BZR1和BZR2的活性,进而影响BR信号的传递以及下游基因的表达。而RT-PCR检测发现,在棉花受黄萎病侵染后,与BR信号路径正相关的关键基因GbBRI1和GbBZR1的表达量上调。说明黄萎病菌侵染后棉花BR信号路径被激活。外施BL或通过VIGS干涉Gb14-3-3c和Gb14-3-3d都能增强棉花对黄萎病菌的抗性。说明BR信号路径可能对棉花抗黄萎病起到正向调控的作用。检测BL处理后海’7124’根系中SA和JA相关基因的表达,发现JA信号路径相关基因的表达量上调。这说明在棉花中BR所介导的信号路径可能是处于JA信号路径的上游,或BR与JA信号路径有重叠或协同作用。SA和JA是植物抗病反应中两种非常重要的小分子激素。位于SA和JA信号传导结点的调节因子在这两种激素介导的抗病信号路径交叉网络中起着关键的调控作用。在我们的研究中分离到在转录水平和蛋白水平都受黄萎病菌诱导上调的硬脂酰去饱和酶GbSSI2。通过VIGS干涉掉GbSSI2后,干涉植株的茎和叶片组织都出现坏死斑,严重时会造成植株死亡。经分析发现,GbSSI2干涉植株体内的活性氧大量积累；同时SA含量升高,SA路径相关基因表达上调；而JA含量降低,JA相关基因的表达受到抑制。对干涉植株叶片进行离体接种实验,发现干涉GbSSI2降低了棉花对黄萎病菌的抗性。说明GbSSI2可能参与负调控SA信号路径,正调控JA路径和棉花抗黄萎病。此外,RT-PCR分析发现棉花受黄萎病侵染后,JA信号路径相关基因的表达上调。进一步说明了JA信号路径在棉花抗黄萎病中可能存在的正向调节作用。从棉花与黄萎病菌互作的转录表达谱中,分离得到一个受黄萎病菌侵染后差异表达的Homeodomain转录因子HDTFl。通过序列比对分析发现,其属于HD家族PINTOX类成员。表达模式分析发现,HDTF1在棉花的叶片组织中优势表达；受黄萎病菌及灰霉病菌侵染后显著下调表达；同时其表达还受SA的强烈诱导,但MeJA处理后,HDTF1的表达会受到抑制。利用VIGS方法把棉花中HDTF1基因干涉后,植株对黄萎病菌以及灰霉病菌的抗性明显增强。研究结果表明,HDTF1可能作为一个负调控因子,调节棉花的抗病性。随后的研究发现HDTF1干涉植株体内JA含量增多,JA合成以及下游响应相关基因表达被激活。而干涉植株体内SA含量以及SA路径相关基因表达相对于野生型植株都没有显著差异。说明HDTF1介导的抗性,可能不依赖于SA信号路径,或者是通过负调节JA信号路径来影响棉花的抗性。HDTF1在棉花抗黄萎病菌中的具体调控机制还有待进一步深入的研究。更多还原

【Abstract】 Cotton is one of the most important commercial crops all over the world. However, cotton production is influenced by Verticillium wilt every year. Verticillium wilt is caused by Verticillium dahliae, which is a soil-borne fungi caused vascular disease. The quality and yield of cotton will be subjected to serious reduction, even more than30%reduction of production when the outbreak of Verticillium wilt. So Verticillium wilt is called the cancer of cotton. It is a challenge to explore resistance genes, because of lacking cotton germplasm with high resistance to V. dahliae. On the other hand, it is difficult to uncover the molecular mechanism of cotton resistance to V. dahliae, because the race of V. dahliae mutates rapidly in nature. Sea island cotton exhibit more resistance to V. dahliae compared with upland cotton, therefore there may be some resistance genes or resistance mechanisms in sea island cotton. We used sea island cotton ’7124’ with high resistance to V. dahliae as the research materials, and utilized comparative proteomics to discover resistance mechanisms during the interaction of cotton and V. dahliae.By two-dimensional electrophoresis and mass spectrometry analysis, totally188differentially expressed proteins were identified from ’7124’ roots after infection by V. dahliae. In these differentially expressed proteins,47%of them was down regulated. According to gene ontology analysis, these differentially expressed proteins can be divided into a total of17categories. The number of proteins which involved in cell processes, protein metabolic processes or stress responses is more than others. These proteins may participate in the response of cotton to V. dahliae. After cotton infection by V. dahliae, cellular metabolism and physiological processes were influenced, and it may be the response to pathogen invasion.Gossypol is one of cotton-specific secondary metabolites. GbCAD1and GbdHG-6-OMT, two enzymes involved in gossypol metabolism, were found to differentially expressed in our protein profile. GbCADl, a cadinene synthase gene, is a key regulator in gossypol synthesis. After silencing GbCADl by VIGS, the gossypol content of GbCAD1-silenced plants was reduced compared with mock. Meanwhile, the resistance to V. dahliae was significantly reduced after GbCAD1was silenced. So gossypol may affect the cotton resistance to V. dahliae. Some other gossypol metabolism-related genes were found to up regulated in cotton roots after infection by V. dahliae, suggesting that gossypol metabolism was activated in cotton after infection. As a negative regulator of the activity of gossypol, GbdHG-6-OMT encodes desoxyhemigossypol-6-O-methyltransferase, and was down regulated after pathogen infection. By histochemical analysis, gossypol was accumulated in cotton after infection by V. dahliae. It is suggested that the accumulation of gossypol in cotton may enhance the resistance to V. dahliae. Gossypol metabolism-related genes (GbFPS, GbCAD1,WRKY1) were found to up regulated in cotton plants with methyl jasmonate treatment, indicating that the metabolism of gossypol may be regulated by jasmonate signal.In addition to playing an important role in plant growth and development, BR-mediated signal pathway is also reported to involve in plant immunity. Four Gb14-3-3proteins, the homologous genes of which in Arabidopsis were reported to interact with the BIN2-phosphorylated targets in the BZR1protein, were all down-regulated significantly in the protein expression profile. According to RT-PCR analysis, the positive regulated genes (GbBRI1and GbBZR1) in BR signal pathway were up regulated in cotton after infection with V. dahliae, indicating BR signal pathway was activated in infected cotton. Application of exogenous BL or silencing of Gb14-3-3by VIGS can enhance cotton disease resistance to V. dahliae, suggesting that BR signal pathway may contribute to cotton disease resistance. After treated cotton with BL, genes related to JA signal pathway were up regulated using qRT analysis, indicating that the BR signal pathway might be upstream JA signal pathway in cotton, and JA signal pathway might be have crosstalk with BR signal pathway.SA and JA are two very important small molecule hormones in plant disease resistance responses. Some genes located at the junction of the SA and JA signal pathway play important roles in regulating crosstalk of these two hormones. In our study, GbSSI2was isolated from protein profile, which was up regulated in the transcript and protein levels in cotton after infection with V. dahliae. After silencing GbSSI2by VIGS, GbSSI2-silenced plants appeared necrosis on stems and leaves; severe necrosis even caused plant death. After ROS detection, the H2O2was accumulated in the GbSSI2-silenced plants; the SA content was accumulated and SA signal related genes were up regulated; while JA content was decreased and JA signal-related genes were suppressed in the silenced cotton plants. After the leaves of silenced plants infected by V. dahliae, the GbSSI2-silenced leaves were more susceptive to V. dahliae. All the mentions above indicate that GbSSI2may influence the cotton resistance by altering SA-and JA-mediated defense signaling. By RT-PCR analysis, JA signal related genes were up regulated in cotton after infection with V. dahliae, indicating JA signal pathway might contribute to cotton disease resistance to V. dahliae.The molecular mechanism that underlies Verticillium dahliae resistance in cotton is poorly understood at present. Here we characterize HDTF1, which is cloned from transcription expression profile of cotton response to V. dahliae. HDTF1was predicted to encode a nuclear homeodomain transcription factor and HDTF1expression was down-regulated in cotton upon Verticillium dahliae and Botrytis cinerea inoculation. To elucidate the possible involvement of HDTF1in cotton-pathogen interactions, we employed virus-induced gene silencing to generate HDTF1-silenced cotton. Silencing HDTF1significantly enhanced cotton resistance to the fungal pathogens V. dahliae and B. cinerea. In addition, HDTF1silencing caused the accumulation of the phytohormone JA in addition to the activation of a JA-related signal pathway. We could not detect altered SA levels or differential expression in the SA-related genes of HDTFl-silenced plants. Our studies suggest that silencing of HDTF1transcripts in cotton resulted in improved resistance to V. dahliae and B. cinerea, and HDTF1was involved in regulating the JA signaling pathway.更多还原