【作者】 赵晶；

【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， 2009， 博士

【摘要】 水稻白叶枯病是由黄单胞杆菌水稻致病变种(Xanthomonas oryzae pv.oryzae,Xoo)引起的一种细菌性维管束病害,是水稻生产上的重要病害之一。水稻是不仅是重要的粮食作物,也作为单子叶植物和谷类作物研究的一种模式植物。而随着多越来越多个水稻抗白叶枯病基因的克隆以及白叶枯菌基因组序列的完成,水稻抗白叶枯病的研究已经称为研究植物与微生物互作关系的一种模式。因此,不论从生产上还是从基础理论研究上,对水稻对白叶枯抗病机理进行深入和广泛的研究,从分子和遗传水平解释其机理都具有重要意义。Xa3/Xa26和Xa21是水稻中两个广谱的抗白叶枯病基因,它们都编码富亮氨酸重复类受体蛋白激酶,也都是属于基因家族成员。然而,它们具有不同的小种特异性,Xa21抗PXO99而Xa3/Xa26感PXO99。此外,Xa21的功能还受到不同生育期的影响。IRBB21对PXU99的抗性会随着植株的发育逐步增强,表现为从2叶期的感病到9-10叶期的完全抗病。而在牡丹江8中表达的Xa3/Xa26对PXO61表现全生育期抗性。我们构建了包含编码XA21与XA26不同结构域序列的嵌合基因,将它们以及Xa21都转到同样的品种牡丹江8中,在相同背景下考察它们的抗性,抗谱以及在不同生育期时的抗性,解释了该类蛋白各个结构与功能的关系。另外,利用酵母双杂交的方法,筛选与XA3/XA26激酶结构互作的蛋白,寻找参与XA3/XA26介导的抗病反应的蛋白。D56S编码产物包含XA3/XA26的富亮氨酸重复区和跨膜区以及XA21的激酶结构域;D57S编码产物包含XA21的富亮氨酸重复区和跨膜区以及XA3/XA26的激酶结构域。抗谱分析表明,D56S与D57S的抗谱分别与Xa3/Xa26和Xa21相似。与D56S或D57S只在胞内近膜区编码序列有差异的D52S和D92S则对所有试验菌株都不提供抗性。这些表明富亮氨酸重复区决定了XA21和XA3/XA26对白叶枯菌的小种特异性,而且XA21与XA3/XA26可能具有相同的下游抗病信号传导途径。另外,我们的结果也表明近膜区也能影响该类抗病蛋白的抗性特异性。在2叶期,4叶期,分蘖盛期,孕穗期以及始穗期对转基因材料和各种携带Xa3/Xa26或者Xa21的材料接种白叶枯菌PXO61,PXO99和PXO341。结果显示,在牡丹江8背景下,D56S,D57S,Xa21与Xa3/Xa26一样,对PXO61具有全生育期抗性。而在IR24背景或者明恢63背景下,Xa21和Xa3/Xa26只表现出成株期抗性。不同材料中Xa21和Xa3/Xa26对PXO99或PXO341的抗性也同样如此。这些都表明Xa21或Xa3/Xa26在不同生育期的抗性差异只是受到遗传背景的影响,而非他们的结构上的差异。实时定量PCR结果显示,在IRBB21中,Xa21的表达量随着植株生长逐渐增强,这与IRBB21从苗期到成株期对白叶枯菌的抗性变化相一致。与IRBB21相比,转Xa21基因牡丹江中Xa21的表达量在不同生育期都显著增强。此外,不论是转基因植株还是IRBB21,Xa21的表达量都在分蘖盛期最高,在此生育期是与Xa21在提供的抗性也最强,说明Xa21的功能具有剂量效应,转基因牡丹江8植株中高水平的Xa21也提供了对白叶枯菌的苗期抗性。用XA3/XA26的激酶区段筛选到3个在明恢63中与之互作的蛋白。其中,D4是mRNA剪接因子,C5是一个自吞噬相关蛋白,而C17是G蛋白的一个β亚基。推测它们可能参与Xa3/Xa26介导的抗病反应。更多还原

【Abstract】 Bacterial blight(BB),a vascular disease caused by Xanthomonas oryzae pv Oryza (Xoo),is one of the most serious disease of rice production.Rice is not only a staple in Asia,but also a model for monocots and cereal plant.With increasing resistance gene of BB and publication of Xoo genome sequence,study of BB has been a model of research on plant-microbe interactions.Hence,it is of important theoretical and practical significance to comprehensively and in-depth study of BB and elucidate the genetic and molecular mechanism of resistance.Xa21 and Xa3/Xa26 are two broad-spectrum resistance genes to Xoo.They both encode receptor like protein kinase and belong to a family.However,they are different in race-specificity.Xa21 is resisitant to PXO99 while Xa3/Xa26 is susceptible.In addition, function of Xa21 was affected by developmental stages of rice.The resistance to PXO99 gradually increased from susceptible 2-leaf stage to complete resistant 9/10-leaf stage with growth of IRBB21.Xa3/Xa26 expressing in Mudanjiang 8 is resistant to PXO61 at whole life.We constructed chimeric genes encoding diverse part of domain of XA21 and XA3/XA26 and transfer them to the same rice variety Mudanjiang 8.In order to understand the relationship of structure and function of each domain,the resistance, spectrum of resistance and resistance at different stages of chimeric genes and Xa21 as well as Xa3/Xa26 were then investigated.Additionally,we screen proteins interacting with kinase domain of XA3/XA26 by using yeast two hybrid technology.Chimeric gene D56S.which encodes product containing leucine-rich repeat domain with transmembrane domain of XA3/XA26 and protein kinase domain of XA21,and D57S encodes product containing eucine-rich repeat domain with transmembrane domain of XA21 and protein kinase domain of XA3/XA26.Resistance-spectrum analysis showed that resistance spectrums of D56S or D57S were similar to that of Xa3/Xa26 or Xa21 respectively.However,D52S and D92S,which were different with D57S or D56S only at encoding sequence of cytoplamtic juxtamembrane region,confered no resistance to PXO61.Above results suggested that leucine-rich repeat domain provided the race specificity of XA21 and XA3/XA26 and the important function of juxtamembrane region of XA21 and XA3/XA26.In addition,juxtamembrane region also affect the race specificity of XA21 and XA3/XA26.Transgenic lines and other rice materials carrying Xa21 or Xa3/Xa26 at 2-leaf stage, 4-leaf stage,maximum-tillering stage and heading stage were inoculated with Xoo strain PXO61,PXO99 and PXO341.Results show that in Mudanjiang 8,D56S,D57S and Xa21 confered resistance to PXO61 at all stages as well as Xa3/Xa26.While in background of IR24 or Minghui 63,Xa21 and Xa3/Xa26 provided resistance to PXO61 only at adult stage.Same results were achieved in experiment when inoculated with PXO99 and PXO341,which indicated that genetic background affected the performance of Xa21 and Xa3/Xa26 at diverse developmental stages.Relative expression level of Xa21 in transgenic Mudanjiang 8 was notable increased compared with in IRBB21.Additionally, at maximum-tillering stage,the expression level of Xa21 reach its top,this was consistent with its resistance.We concluded that gene dosage-effect is an important fact affecting function of Xa21.Three proteins interact with protein kinase domain of XA3/XA26 were obtained by yeast two hybrid screen.D4 is an mRNA splicing factor,C5 is a autophagy-related protein and C17 is a subunit of G protein.We speculated these proteins involved in Xa3/Xa2 6-mediated resistance.更多还原