水稻细菌性条斑病菌三个Xop基因功能研究相关载体的构建
毕业论文关键词：水稻白叶枯病菌；水稻条斑病菌；XOP基因
Construction of vector for the functional study of three XOP genes in rice bacterial leaf streak
Abstract： Bacterial blight and bacterial leaf streak are two important diseases caused by Xanthomonas oryzae pv.oryzae (Xoo) and X.o.pv. oryzicola (Xoc),respectively. These two diseases threaten seriously on rice production. During the infection and pathogenesis, effectors secreted by Xanthomonas through three secretion system (T3SS) play very important roles in the interaction between bacteria and rice. These effectors are classified into Tal effector and non-Tal effector. Tal effector is characterized with conserved structure as transcription activator-like effector and non-tal effector named as XOP (Xanthomonas outer protein ) gene features in sequences without any identified conservation property. The most famous function of some Tal effectors is proved to promote disease development or induce resistance through regulation of host gene expression by binding the promoter of target gene in a sequence specific manner. And a few of Xop effectors are demonstrates to interfere bacteria-host interaction mainly through modulation of plant defense signal transduction in protein-protein interaction manner. There are more than 40 Xop candidate genes presented in each Xanthomonas genome, but only a very few of them have been investigated. In this paper, we constructed 19 vectors for three Xop genes contained specifically in Xoc strains. Those vectors assembled aimed to research the functions of Xops in the virulence and HR reaction, protein subcellular location, the secretion into the rice cell and the promoter function in answer to the plant factors. So this research is key to the subsequent functional research for the three Xop genes.
Key words: Xanthomonas oryzae pv. oryzae; X. o. pv. oryzicola; xop gene
目 录
摘要3
关键词3
Abstract3
Key words3
引言3
1材料与方法4
1.1PCR扩增目的条带 4
1.1.1引物的设计4
1.1.2 PCR体系5
1.1.3 PCR程序6
1.1.4电泳配胶6
1.1.5切胶回收6
1.1.6 TA克隆及质粒DNA的提取6
1.2目的片段连接至功能验证载体 8
1.2.1酶切8
1.2.2连接8
1.2.3转化8
1.2.4培养基9
1.2.5抗生素9
1.2.6 PCR验证9
2结果与分析10
2.1 PCR获得目的条带10
2.2目的片段酶切10
2.3连接至对应载体筛选出目的片段10
3讨论 13
致谢13
参考文献13
水稻细菌性条斑病菌三个Xop基因功能研究相关载体的构建
引言：植物在生长过程中会遭受各种生物和非生物的胁迫，其中病原微生物作为重要的生物胁迫受到科学家广泛地关注[1]。植物在与病原微生物的长期相互作用过程中进化出两套防御系统抵御病原物侵入[2]。其中第一层是由一些保守的病原/微生物相关分子模式（Pathogen/ Microbe-associated molecular patterns,PAMPs/MAMPs）所激发的防御反应[3]（PAMP- triggered immunity,PTI），植物识别细胞表面的细菌鞭毛蛋白、脂多糖等从而产生活性氧 (Reactive oxygen species,ROS)迸发[4]、丝裂原激活蛋白激酶（Motigen-activated protein kinase, MAPK）信号传导、病程相关基因（Pathogenesis-related gene,PR-gene）的表达以及胼胝质（callose depositions）的沉积以抵抗病原微生物的侵入[5]。第二层是由植物抗性（R）基因编码的 R 蛋白识别病原微生物分泌的无毒基因编码的效应蛋白（effector proteins）从而激发的防御反应（Effector-triggered immunity, ETI），在侵染位置产生快速的细胞死亡即超敏反应（Hypersensitive response,HR）以限制病原物的增殖[6]。植物通过 PTI 抵抗绝大部分病原菌的侵染，而革兰氏阴性病原菌为了克服植物的先天性屏障进化出三型分泌系统（Type Ⅲ secretion system, T3SS）,通过三型效应系统将三型效应蛋白 （Type Ⅲ effector proteins,T3Es）注入到寄主细胞中靶定到不同的细胞区室以干扰细胞活动从而促进其繁殖扩增[1]。植物为了应对三型效应蛋白对防御系统的干扰进化出可以识别效应蛋白的抗性（Resistance,R）蛋白，产生 HR 快速抑制病原物生长[7]。三型效应蛋白包括TAL 效应因子和 Non-TAL 效应因子，后者也叫做黄单胞菌外泌蛋白（Xanthomonas outer protein,XOP）。TAL 效应因子具有保守的转录激活因子的结构，而Non-TAL 效应因子一般具有各种酶活功能，但蛋白之间序列没有保守性[8]。这类效应因子广泛存在于革兰氏阴性病原菌，黄单胞菌中包含至少 40 类外蛋白（ Xanthomonas outer protein,Xop）,而大量的水稻黄单胞菌 Xop 功能未知[9]。水稻黄单胞白叶枯病菌和细菌性条斑病菌含有的non-TAL基因大多数是相同的，但在还发现细菌性条斑病菌中含三个特有的Xop基因，我们猜想这三个基因在细菌-水稻的互作中应该具有重要作用，因此我们设计构建基因的回补、基因编码的亚细胞定位、基因编码蛋白的泌出检测、互作蛋白的免疫共沉淀筛选以及启动子功能研究的相关载体，为后续研究准备了关键材料[10]。