【作者】 郑崇珂；

【导师】 赵开军；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2014， 博士

【摘要】 水稻(Oryza sativa L.)作为最重要的粮食作物之一，为全球超过半数的人口提供食物来源，因此对于粮食安全至关重要。然而由黄单胞杆菌水稻致病变种(Xanthomonas oryzae pv. oryzae，Xoo)引起的水稻白叶枯病(Bacterial blight)已经成为水稻生产中重要的病害之一，常常导致水稻减产。实践证明种植抗病品种是控制该病害最经济有效和环保的途径。因此发掘和利用抗白叶枯病基因对于水稻高产稳产尤为重要。近年来，随着对黄单胞菌TAL效应子研究的深入，TAL效应子与靶基因DNA序列之间识别的分子密码被破解，为TAL效应子的应用提供了理论基础。根据分子密码已经发展出了多种实验技术，包括人工组装的TAL效应子(designed TAL effector，dTALE)和TALENs核酸酶(TALE-based nucleases，TALENs)，用于基因功能研究和基因组定点编辑，并取得了显著成效。但TAL效应子最后半个重复(Last half repeat，LHR)的来源和功能尚待揭示。本论文主要包括两部分：第一部分对本课题组发掘的抗白叶枯病基因Xa32(t)进行精细定位；第二部分针对TAL效应子的保守结构，研究其重复序列中LHR的功能，为简化dTALE和TALENs载体的构建提供理论基础。获得的主要结果如下：关于抗白叶枯病基因Xa32(t)的精细定位：1.利用含有Xa32(t)基因的近等基因系Q143与感病材料JG30构建了含有6019个单株的F2分离群体，经过接种鉴定和遗传分析，该群体能够用于Xa32(t)基因的精细定位；2.新开发了5对在抗病材料Q143和感病亲本JG30之间有多态的分子标记，分别为K58、K57、Y72FF、Y77和Y99；通过分析F2分离群体的感病单株，上述5个多态标记与Xa32(t)基因紧密连锁，将Xa32(t)定位在水稻第11染色体长臂末端标记Y72FF和标记Y77之间0.16cM范围之内；3.利用Blast序列比对软件，将筛选到的多态标记与已测序的水稻日本晴基因组，和Tsuyuake、Kanto51的BAC克隆的序列进行比对构建了Xa32(t)基因位点的物理图谱，将Xa32(t)基因定位在水稻第11染色体长臂末端大约70kb范围之内；4.根据上述Xa32(t)定位区间，进行了Xa32(t)的候选基因预测，并通过Blast-P对预测基因的功能进行了分析。关于TAL效应子最后半个重复的功能研究：5.通过构建缺失最后半个重复的dTALEs及其报告载体，以GUS基因作为报告基因，利用烟草瞬时表达系统，根据GUS的定性和定量表达分析，证明TAL效应子最后半个重复在激活靶基因表达过程中是非必须的；进而设计了一个用于构建dTALEs的通用的重复区受体载体，命名为pSK-RAR-U；此载体的构建能够简化dTALEs载体的构建过程，节省时间和成本。6.利用水稻与Xoo互作系统，以xa27基因为报告基因，构建缺失最后半个重复的dTALEs。通过对IR24材料接种鉴定，重组菌的活性检测，以及xa27基因的表达量测定等实验进一步证实，缺失最后半个重复的dTALEs能够激活水稻基因组内天然靶基因的表达，再次证明TAL效应子最后半个重复在激活靶基因表达过程中是非必须的；结论：本研究将水稻抗白叶枯病基因Xa32(t)基因定位到第11染色体约70kb范围内，并进行了候选基因的预测分析；通过烟草瞬时表达实验和水稻感病基因表达实验证实TAL效应子最后半个重复在激活靶基因表达过程中是非必须的，并设计开发了一个通用的重复区受体载体pSK-RAR-U，简化了dTALEs载体的构建过程。更多还原

【Abstract】 Rice (Oryza sativa L.) as one of the most important food crops in the world, provides food sourceto more than half of the world’s population and constitutes an essential portion for the food security.However, bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), has become one ofthe most serious diseases of rice that often causes yield losses. In management of this disease, the mosteconomical and effective approach is cultivating resistant varieties. Therefore, the discovery andutilizition of bacterial blight resistance genes is particularly important for high and stable yield in riceproduction.In recent years, many important progresses have been achieved in studies on Xanthomonas TALeffectors. The molecular recognition codes between TAL effectors and host target genes have beendeciphered, which provides a theoretical basis for the application of TAL effectors. According to themolecular recognition codes, a series of experimental techniques including designed TAL effector(dTALE) and TALE-based nucleases (TALENs) have been developed and used to study gene functionand genome-specific editing, resulted in remarkable achievements.This dissertation consists of two parts. Part I: high-resolution genetic mapping and candidate geneprediction of bacterial blight resistance gene Xa32(t); Part II: investigation the function of last halfrepeat in the repeat region of TAL effectors, providing a theoretical basis for simplifying the design andconstruction of dTALE and TALENs. The main results are as follows:High-resolution genetic mapping of Xa32(t):1. An F2segregating population containing6019individuals was constructed by crossing thenear-isogenic line of Xa32(t) Q143with the susceptible parent JG30. Based on the inoculationidentification and genetic analysis, this population can be used for fine mapping of theresistance gene Xa32(t);2. Five new polymorphic markers, K58, K57, Y72FF, Y77and Y99, between Q143and JG30,were developed. Through analyzing all susceptible individuals of the F2population, the5polymorphic markers were proven in linkage with Xa32(t). The resistance gene Xa32(t) wasmapped within a0.16cM region of the rice chromosome11, between markers Y72FF andY77.3. The linked markers were anchored on the Nipponbare genome and sequenced BAC clonesfrom rice line Tsuyuake and Kanto51by sequence Blast analysis and the physical map wasconstructed. As a result, the resistance gene Xa32(t) was finally mapped within about70kbon the long arm of rice chromosome11between Y72FF and Y77.4. Based on the targeted region of Xa32(t) locus, candidate genes of Xa32(t) were identified andputative functions for these genes were analyzed by Blast-P.Investagation on the last half repeat of TAL effectors:5. dTALEs lacking the last-half-repeat and reporter plasmids in which the GUS gene was used as a reporter were constructed. Transient expression assays in N. benthamiana leaves wereperformed and the results showed that the last half repeat of TAL effectors was dispensablefor its DNA-binding and gene activation. Based on the finding, a universal repeat arrayreceptor plasmid named pSK-RAR-U was constructed. With this plasmid, the dTALEconstruction process was simplified.6. By constructing the last-half-repeat lacking dTALE targets the xa27gene in rice genome ofIR24, the rice-Xoo interaction assays were performed and the results confirmed that thelast-half-repeat lacking dTALE can activate the expression of xa27and resulted in bacterialblight resistance. The results proved once again that the last half repeat of TAL effectors wasdispensable for its DNA-binding and gene activation.Conclusion: In this study, bacterial blight resistance gene Xa32(t) gene was mapped within about70kb range on the rice chromosome11. The candidates of Xa32(t) were predicted; Through tobaccotransient expression experiments and gene expression experiments in susceptible rice revealed that thelast half repeat of TAL effector is dispensable in the activation of target gene expression. Based on thisfinding, a universal repeat array receptor plasmid named pSK-RAR-U was developed to simplfy theconstruction of dTALEs or TALENs.更多还原