【作者】 邱德运；

【导师】 王石平；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 在植物的抗病反应中，信号分子水杨酸和茉莉酸介导着两个截然不同的抗病信号途径，而且通常认为这两个途径存在一定程度的拮抗。大量的研究表明，WRKY转录因子作为植物所特有的一类转录因子家族在上述植物抗病反应中扮演着重要的角色。白叶枯病和稻瘟病是影响水稻生产的两个重要病害，尽管目前在水稻基因组中已经鉴定了109-112个WRKY基因，但是关于此家族成员在水稻抗病反应中的作用仍然不是很清楚，特别是关于WRKY因子在抗病信号传导途径中的地位。本研究通过运用各种功能基因组学的研究手段，研究了一个受病原诱导的水稻WRKY转录因子OsWRKY13在水稻抗病反应中的主要功能。利用农杆菌介导的遗传转化在感病品种牡丹江8中超量表达OsWRKY13，共分离检测结果表明，无论是苗期还是成株期都增强了转基因植株对白叶枯病和稻瘟病的抗性；相反的是，反义抑制的明恢63转化植株也表现了对白叶枯病感病性的提高。采用定量RT-PCR的方法进一步检测了上述转基因植株中病程相关蛋白和其它与水杨酸及茉莉酸代谢相关的基因的表达情况。在接种前后的至少在一个检测时间点OsWRKY13超量表达植株中比对照牡丹江8聚集了更多的PR1α基因的转录本，而且反义表达植株中互补了这种表达模式；同时，PR10和JIOsPR10基因的表达量在OsWRKY13超量表达植株中比对照牡丹江8的明显要低；而对11个与水杨酸、茉莉酸或者环氧脂类信号传导途径相关基因的表达分析显示，OsWRKY13的表达量提高至少在一个时间点显著地诱导了基因CHS，PAD4和ICS1的表达，但是部分抑制基因OsWRKY13的表达量对这3个基因的表达没有影响；同时，超量表达OsWRKY13则显著抑制了基因AOS2和POX的表达，而且抑制OsWRKY13显著提高了这两个基因的表达。利用酵母单杂交实验证明了OsWRKY13对PR1α，AOS1，AOS2和LOX的启动子具有很强转录激活能力，而对ICS1，NH1和PAD4则没有；同时利用超量表达植株的核蛋白和重组蛋白OsWRKY13-GST进行的体外DNA结合和蛋白质磷酸化实验进一步表明，OsWRKY13能特异的结合到W-盒或者类W-盒上，而且这种结合受到病原物侵染的调控影响，推测这种影响部分来自对蛋白质磷酸化状态产生的改变。结合超量表达植株中水杨酸含量提高和茉莉酸含量下降的结果，我们认为OsWRKY13通过激活水杨酸信号途径和抑制茉莉酸信号传导途径，参与水稻的抗病反应。为了进一步分析OsWRKY13所调控的下游基因以及其调控方式，本研究对超量表达植株芯片分析中所获得的差异表达基因进行了深入的分析。首先，差异表达基因的Geneontology(GO)分类显示OsWRKY13正调控类黄酮代谢，负调控萜类和脂类代谢过程，而上述过程正好分别与水杨酸和茉莉酸的代谢途径相关；其次，进一步统计分析了差异表达基因启动子区保守顺式元件，并通过采用穷举法的方法扫描了差异表达基因启动子区域的保守模体，结果发现在上升表达的基因中显著富集了W-盒(GTTGACC)，而在下降的基因中富集了MYB转录因子的结合位点，同时GCC-盒在下降基因的启动子区域中呈明显的减少趋势，并且这种顺式调控元件的变化正好与差异表达的转录因子相吻合。因此，推测OsWRKY13很可能协同MYB和AP2／ERF转录因子调控下游基因，影响类黄酮代谢和脂类代谢过程。此外，本研究还分析了OsWRKY13与其它水稻WRKY家族成员的关系以及与拟南芥AtWRKY70共调控的基因。结果表明，OsWRKY13超量表达后负调控许多水稻WRKY基因，特别是与ABA信号相关的；同时在拟南芥AtWRKY70与OsWRKY13参与的抗病反应中，衰老和抗氧爆破过程存在一定的保守性。综合上述研究结果，可以看出OsWRKY13通过复杂的调控方式，影响着水杨酸和茉莉酸合成以及类黄酮和脂类代谢过程，最后参与水稻对白叶枯病和稻瘟病的抗病反应。更多还原

【Abstract】 During the plant defense responses, the components involved in the cross-talkbetween salicylic acid (SA)- and jasmonic acid (JA)-dependent defense pathways againstpathogens and insects have been partially revealed in dicot plants. Many evidences haveshowed that the WRKY transcription factors as unique to plants are also involved inmodulation of SA- or JA-responsive gene expression. Bacterial blight and fungal blast aretwo of the most devastating diseases of rice worldwide. Although 109-112 WRKY geneshave been identified in the rice genome, the functions of most in rice disease resistanceare unknown, especially for their role in SA- and JA-dependent signaling transductionpathways. Here we show that OsWRKY13 plays a pivotal role in rice disease resistancethrough different methods in functional genomic research.Overexpression of OsWRKY13 in rice susceptible cultivar Mudanjiang 8 canenhance transgenic plants resistance to bacterial blight and fungal blast, two of the mostdevastating diseases of rice worldwide, at both the seedling and adult stages. Conversely,transgenic plants of OsWRKY13-antisense suppression appeared to be more sensitive tobacterial blight (Xoo strain PXO61). Furthermore, different pathogenesis-related genes,SA synthesis-related genes and JA synthesis-related genes were examined in transgenicplants by quantitative RT-PCR. The results showed that OsWRKY13-overexpressingplants accumulated significantly more PR1αtranscripts than wild-type plants in at leastone examine time-point, and OsWRKY13-suppressed plant complemented that expressionpattern. Moreover, the analysis of eleven genes involved SA synthesis, JA synthesis oroxylipin metabolism indicated that accumulation of OsWRKY13 transcripts significantlyinduced the expression of CHS, PAD4, and ICS1 in at least one time point examined, incontrast, partially suppressing OsWRKY13 expression showed no influence on theexpression of the three genes compared with wild-type plant. Overexpressing OsWRKY13significantly repressed the expression of AOS2 and POX, while suppressing OsWRKY13expression significantly increased the expression of the two genes compared withwild-type plants. Further analysis of yeast one hybrid suggested that OsWRKY13possessed specific and strongly DNA-binding ability to the promoters of PR1α, AOS1,AOS2 and LOX, not to the promoters of ICS1, NH1 and PAD4. And the gel mobility shiftassay showed that nuclear proteins from OsWRKY13-overexpressing plants andOsWRKY13-GST fusion protein had stronger binding signal to the probe, harboring theW-like box, than that from wild type or control. Notably, that DNA-binding ability wasinfluenced by challenged bacterial pathogen certified by a in vitro phosphorylationanalysis, which suggesting the phosphorylation or de-phosphorylation status ofOsWRKY13 play a crucial role in its regulation function. Considering the increasing ofendogenous SA concentration and decreasing of JA concentration inOsWRKY13-overexpressing plants, we concluded that OsWRKY13 participate in ricedefense response as an activator of the SA-dependent pathway and a repressor of JA-dependent pathways.To determine more downstream OsWRKY13-regulated genes and its regulationmechanism in OsWRKY13-overexpressing plants, we performed microarray analysisusing the Affymetrix oligonucletide rice chip array. Firstly, the analysis of geneontology(GO) classification in differential expressed genes showed that OsWRKY13 activelyregulated the flavonoids metabolism and negatively regulated terpene or lipid metabolism,those of which always related to the SA or JA signaling pathways, respectively. Secondly,the statistic analysis of conserved cis-regulatory element and discovering of putativeconserved motif in the promoters of OsWRKY13 downstream genes through performingan enumerative method showed that the promoters of most OsWRKY13 up-regulatedgenes over-represented a W-box element (GTTGAC), and Myb3 binding site enriched indown-regulated genes whereas the GCC-box depleted in down-regulated genes. Thoseresults are consistent with the previous results of differential expressed transcriptionfactors. Thus, OsWRKY13 regulated the metabolism of flavonoids and lipid by regulatedthe downstream genes through interaction with other MYB and AP2/ERF transcriptionfactors.Otherwise, we also explored the relationship between OsWRKY13 and other riceWRKY family members as well as Arabidopsis AtWRKY70. Interestingly, OsWRKY13negatively regulation the expression of most of other rice WRKY family members,especially for those related to ABA signaling pathway. Comparing analysis indicatedthere was a putative conservation of aging and anti-oxidation burst mechanism inAtWRKY70- and OsWRKY13-mediating defense responses.In summary, our results showed that OsWRKY13 participated in rice resistance tobacterial blight and fungal blast by regulating SA- and JA- related signaling pathways orflavonoids and lipid metabolism, through different regulation models.更多还原