导　　师： 周永力

学科专业： 090401

授予学位： 硕士

作　　者： ;

机构地区： 中国农业科学院

摘　　要： 水稻细菌性条斑病是我国重要的植物检疫性病害,在我国水稻生产上,尤其是杂交稻,造成极大的经济损失,目前在水稻中尚未发现抗细菌性条斑病的单个主效基因。水稻对细条病的抗性为数量性状,但目前经研究发现的多个细条病数量抗性基因效应较小,在抗病育种实践中不能广泛应用。 目前非寄主抗性基因的克隆及其在作物抗性改良中的作用备受关注。Rxo1是从玉米中鉴定、克隆的一个基因,携带该基因的转基因水稻植株对细条病菌的侵染能产生抗病反应,该发现在水稻细条病抗病育种实践中具有重大意义。鉴于人们对转基因植物生物安全性的日益重视,本研究利用双右边界双元载体,以期获得无选择标记、具有应用价值的水稻育种材料。同时利用反转录PCR及实时荧光定量PCR等技术,研究非寄主抗性基因Rxo1在水稻中对细菌性条斑病的抗性反应。 1.构建双右边界双元重组载体pMNDRBBin6-Rxo1,通过农杆菌介导法将非寄主抗性基因Rxo1导入我国杂交稻重要恢复系C418。经PCR检测,T0代共获得C418背景的Rxo1阳性株系11个。 2.对本实验室已经获得的转基因水稻纯合系接种水稻细条病菌,结果出现过敏性坏死反应的典型特征。通过RT-PCR技术初步分析水稻基因组中6个与过敏性坏死反应及水杨酸信号转导途径相关基因的表达谱变化,并通过实时荧光定量PCR对差异表达基因的表达谱进行验证,初步表明水杨酸信号传导途径参与了非寄主抗性基因Rxo1介导的水稻对细条病菌的抗病反应。 3.转录因子在植物防卫反应表达调控中发挥重要作用。利用RT-PCR检测WRKY转录因子家族在水稻受细条病菌侵染条件下的表达变化情况,发现水稻细条病菌的侵染诱导了若干WRKY基因的差异表达。在转基因水稻中检测到11个差异表达基因,其中7个基因参与水稻抗病反应信号水杨酸、茉莉酸转导途径,且调控方式多样。说明非寄主抗性基因Rxo1介导的防卫反应可能涉及SA、JA信号转导途径。利用生物信息学及系统发育分析可以为非寄主抗病机制及信号转导调控网络提供有用信息。 Bacterial leaf streak /(BLS/) in rice is an important quarantine disease of plant in China. BLS causes subtantial economic losses in rice production, esepecial in hybrid rice. No resistance controlled by single R gene has been identified in rice. The resistance of rice to BLS is a quantitative trait. As the effect of the quantitative trait loci for resistance to BLS locatied in rice is small, it is limited to be used widely in the breeding for disease resistance. It has been hot issues that clone non-host resistance genes and the usage in improving the resistance of crops. The Rxol gene which identified and cloned from maize was a nonhost gene resistant to BLS of rice, which mediated hypersensitive reaction in transgenic rice lines infected by Xanthomonas oryzae pv. oryzicola /(Xoc./). To overcome the safety concerns caused by selectable marker genes in transgenic plants, double right-border binary vector was used to cultivate marker-free breeding material. RT-PCR and real-time PCR were used to reveal the resistant mechanism of non-host resistance gene Rxol against Xoc. in rice. 1. The non-host resistance gene Rxol was transferred into C418, an important restorer line of japonica hybrid rice in China, using double right-border T-DNA binary vector pMNDRBBin6-Rxco1 through Agrobacterium-mediated transformation.11 transgenic lines of C418 were Rxol-positive through PCR analysis. 2. A homozygous T4 line containing a single copy of Rxol from transgenic 9804 plants were inoculated using a virulent Xoc strain. Hypersensitive reaction /(HR/) was triggered. The expression profiles of 6 genes related with HR and salicylic acid /(SA/) singal pathway were analyised by RT-PCR. The expression profiles were confirmed by real-time PCR. The results indicate that SA singal transduction pathway involved in the defence reaction induced by Rxol gene. 3. Transcription factors play an important role in regulating plant defence reaction. WRKY genes expression upon the inoculation of Xoc were analyised by RT-PCR. The results showed that 11 genes were expressed differently in transgenic rice. Among 11 genes, 7 genes involved in and regulated resistance reactions in different ways. The results showed that SA and JA singaling pathway may be involved in the non-host resistance reaction induced by Rxol gene. The information of bioinformatics and phylogenetic analyse will be significant to investigate the resistance mechanism and the networks consisted of singal transduction pathways.

关 键 词： 水稻细菌条斑病 非寄主抗性 基因 转录因子 反转录

分 类 号： [S511]

领　　域： [农业科学]