【作者】 曹应龙；

【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】 白叶枯病是水稻中重要的病害之一，农业生产中对于白叶枯病的防治主要利用抗病基因，水稻中鉴定了大量的抗白叶枯病基因，抗病基因的克隆将为农业生产提供抗源，同时对抗病基因功能的研究以及应用这些抗病基因有着重要的指导意义。水稻抗白叶枯病基因Xa26和Xa4都定位于水稻11染色体长臂的末端，Xa4是在中国水稻抗白叶枯病育种中应用较广的基因，Xa26是在三系杂交水稻恢复系明恢63中鉴定的广谱抗病基因。采用图位克隆的策略分离克隆这两个基因具有重大的理论和应用价值。利用农杆菌介导的转化方法对Xa4和Xa26的候选基因进行稳定的遗传转化，对转化植株进行接种鉴定，确定基因。首先将Xa26的两个候选基因MRKa，MRKb，利用其自身启动子转化到粳稻感病品种牡丹江8中，接种发现所有携带MRKb的转基因植株都获得了对白叶枯病的抗性，对其T1代植株接种表明所有抗性和转基因的表型共分离，说明MRKb即为Xa26基因。牡丹江8背景的转基因植株抗性相对于供体品种明恢63抗性显著提高，为了进一步研究粳稻背景对Xa26抗性的影响，将Xa26导入到另外两个感病的粳稻品种中花11和02428中，结果发现这两个背景下Xa26抗性也有明显增强，并且由成株期抗性变为全生育期抗性。以上结果表明粳稻背景有利于Xa26发挥功能。为了更深入地了解Xa26的功能，利用玉米泛素启动子在中花11和牡丹江8背景中超量表达Xa26，接种结果表明超量表达可以进一步提高抗性，并可以在一定程度上拓宽抗谱，而对于水稻生长发育并无明显影响，预示Xa26有着广阔的应用前景。同时我们还利用一个病原诱导的启动子在相同背景下表达Xa26，转基因植株的抗性明显减弱。这表明Xa26介导的抗性受到遗传背景和表达强度的影响。利用定量PCR的方法分析不同背景下不同启动子驱动的转基因植株中Xa26的表达量，发现自身启动子调控Xa26的所有转基因植株表达量都明显高于两个籼稻品种明恢63和IRBB3，这说明Xa26在粳稻背景中的表达量明显高于籼稻背景，表达量的升高可能是Xa26抗性增强最直接的原因。为了进一步证明Xa26表达量与抗性的相关性，又在明恢63背景下超量表达Xa26，转基因植株的抗性明显增强。这些结果表明Xa26发挥作用具有明显的剂量效应。Xa26在不同背景下在接种后的表达模式不同，在牡丹江背景转基因植株中Xa26的表达有明显的上升，而明恢63和IRBB3中却有一个显著的下降后再上升的趋势，但中花11背景的植株没有明显的变化。同时分析了Xa26在不同生育期的表达量的变化，发现Xa26在苗期表达量较低，而随着生长发育的进行表达量逐步升高，在转基因植株中也有相同的发现，这个结果表明不同时期的表达量差异决定了不同时期的抗性差异。我们还发现Xa26的表达量的提高可以加快抗病反应下游基因OsWRKY13和NH1对白叶枯病菌的应答。这些结果说明了Xa26基因的表达受到生长发育的影响，并且Xa26表达量的提高可以影响下游基因的表达。采用超量表达的方法研究了Xa26家族成员MRKa和MRKc的功能，在中花11中超量表达MRKa，转基因植株表现为中等抗性；而MRKc的转基因植株却没有获得抗性，表明在抗病基因家族中一些成员会有抗性的残留。构建了一个MRKa和Xa26的嵌合基因：Xa26LRR-TM-MRKaK，可以介导对白叶枯病的部分抗性，这说明通过人为的方法可以获得新的抗病基因。同时对MRKa，Xa26，MRKc的表达模式研究发现三个基因的表达模式类似，并且基因表达部位都集中在维管组织周围，说明抗病基因家族的表达模式在进化上有一定的保守性。并且白叶枯病是一个维管病害，抗病基因在维管周围的大量表达有利于其更好的发挥作用。利用RNAi技术抑制明恢63中Xa26的表达后植株抗性下降，而转基因植株的生长发育并没有受到明显影响，表明抗病可能是Xa26基因的主要功能。此外在Xa4的研究中，将候选基因B4-RKa，TQ-RKa，TQ-RKb，TQ-RKd转化到几个水稻品种中都没有获得抗性，表明这几个基因都不是Xa4，进一步分析发现还有一个基因可能是Xa4的候选基因：B4-WAK，这个基因在叶鞘中表达较高，接种后有微弱的上升。测序发现IRBB4和带有Xa4的另外两个品种特青及93-11中WAK基因序列一致，超量表达B4-WAK发现转基因植株对白叶枯病有一定的抗性。B4-WAK是否为Xa4基因还有待进一步确定。更多还原

【Abstract】 Rice bacterial blight is a devasting disease trigged by Xanthomonas oryza sative pv oryza, causing tremendous yield loss each year. Therefore, disease control is of great importance. A very effective way among such control is the utilization of host resistance genes. Till now, many genes were identified in rice, some of which were cloned. It will help us prompting our knowledge of molecular basis of disease resistance; facilitate genetic improving of rice disease resistance.Xa26 and Xa4 are two rice major resistance genes against bacterial blight. They are located on the terminal long arm of Chromosome 11. Xα4 was once widely used in rice breeding. Xα26 derived from Minghui 63 with wide-spectrum resistance. Based on that, cloning of those two genes makes sense.Candidate genes transformation method was used to identify genes. Previous study showed that MRKαand MRKb might be candidates of Xα26. Thereby those two genes with complete ORFs and promoters were introduced into susceptible japonica cultivar Mudanjiang 8 by agrobacteria-mediated transformation. After inoculation with Xoo, all TO transgeneic plants carrying MRKb exhibited resistance against bacterial blight. Further T1 analysis showed that such phenotype were cosegaregated with the ingressed MRKb, powerfully proving MRKb was Xα26. Intriguingly, transgenic plants diplayed a higher level of resistance after inoculation than its donor line Minghui 63. The same results were observed when Xα26 were introdued into other two susceptible japonica cultivar, Zhonghua 11 and 02428. This might suggest a vital rloe genetic background played in the resistance reaction. Further oversepressing Xα26 under ubiquitin promoter in Zhonghua 11 and Mudanjiang 8 endowed the transgenic plants with higher level of resistance, also exhibiting wide resistance-spectrum. Howerer, reducing its expression level using a pathogen induced promoter in the same genetic background caused a decresed resistance level in transgenic plants.The above results were confirmed by real-time quantitative PCR: expression level of Xα26 under origin pomotor in japonica bacgroud is markedly higher than that in the indica background. This might interpret why transgenic plants of japonica background immuned from more bacterial than those of indica background, probablely duing to greater amounts of Xα26 mRNA. When overexpressing Xα26 in its donor cultivar Minghui 63, higher resistance level was speculated in transgenic plants. Concluding from above results implies a potential mechanism Xα26 might act in the resistance reaction: the effect of Xα26 in disease resistance was directly correlated with its mRNA level, functioning with a dosage effect way.Another appealing result rose up when checking Xα26 expression profile in two genetic backgrounds after inoculation with Xoo. Xα26 transcribed remarkedly rise after being infected by incompatible bacterial strain in Mudanjiang 8. Such trend was not so evident when Xα26 expressed in another elite japonica cultivar-Zhonghua 11. However, analysis of gene expression in indica cultivar (Minghui 63 & IRBB3) showed a typical on and off pattern. Expression analysis revealed that OsWRKY13 and NH1, downstream members of resistance signal pathway, were positively regulated once there was an increased expression of Xα26.As Xα26 belongs to a gene family, therefore we checked whether other members of this RLK family were able to confer resistance. It was achieved by overexpressing MRKαand MRKc. Transgenic plants of MRKαexhibited moderate resistance to pathogen, while those of MRKc were susceptible. It seems that members of the resistance gene family were yet with residual effect against pathogen. We introduced a chimeric gene, Xα3/Xα26LT-MRKαK which could partialy enhance resistance to Xoo. MRKαand MRKc showed similar expression pattern as Xα26, which expressed only in the vascular systems of different tissues. The expressional characteristic of MRKαand MRKc perfectly fits the function of genes conferring resistance to Xoo, a vascular pathogen.RNAi method used to study the function of Xα26, all transgenic plants showed the enhanced susceptible phenotype after inoculated with Xoo, and showed no influence to the growth of plants indicated that Xα26 may had the unique function: disease resistance.Xα4 is currently under cloning. Several candidate genes such as B4-RKα, TQ-RKα, TQ-RKb, TQ-RKd were already excluded. Another possible candidate gene was believed to be Xα4, as transgenic plants overexpressing this gene showed moderate resistance. This gene weakly expressed in plants, with a slight rise exposing to pathogen. Comparing its sequence among IRBB4, Teqing, 93-11 indicated there was no diversity among these three cultivars which contain Xα4. Further analysis should be done to confirm its role as Xα4.更多还原