【作者】 侯文倩；

【导师】 孔令让；

【作者基本信息】 山东农业大学 ， 作物遗传育种， 2014， 博士

【摘要】 赤霉病是由禾谷镰刀菌为主引起的一种小麦真菌病害，主要发生在世界温暖湿润和半湿润地区，在我国主要发生在长江中下游地区。近年来随着气候的变暖，小麦赤霉病有向黄淮海麦区蔓延的趋势。小麦赤霉病的抗源相对狭窄，目前生产上所用的抗源主要是苏麦3号及其衍生系，由于其抗性强而且稳定，所以是进行小麦赤霉病分子标记和遗传育种研究的重要材料。随着生物技术、基因测序技术和基因操作技术的发展，在基因组学、蛋白质组学层面上对小麦赤霉病抗性相关基因的克隆和功能验证工作取得了较大进展。本研究对在感/抗赤霉病的小麦近等基因系Apogee/Apogee73S2转录组测序数据中挖掘出的差异表达基因进行生物信息学分析；同时利用大麦条纹花叶病毒介导的病毒基因沉默（BSMV-VIGS）技术对候选基因进行功能验证，利用分子生物学方法分析候选基因的生物学特征。获得以下主要结果：1.利用BSMV-VIGS技术在小麦品种宁7840叶片和穗部分别建立了基因沉默体系，成功沉默了小麦TaPDS基因，得到了目的基因沉默后的白化表型，并且该白化表型在叶部可以持续21天以上，在穗部可以持续30-40天直至小麦成熟，为利用BSMV-VIGS技术研究目的基因功能建立了良好的试验体系；同时本试验还建立了小麦叶片离体培养技术鉴定小麦赤霉病抗性的体系，为小麦赤霉病的研究提供了更加快捷、方便的方法。2.利用小麦赤霉病感/抗赤霉病近等基因系Apogee/Apogee73S2的转录组数据，发掘了3个与小麦赤霉病抗性相关的基因，分别是编码多聚半乳糖醛酸酶抑制蛋白的基因TaPGIP、编码ABC转运蛋白的多药抗性基因TaPDR7和编码钙/钙调素蛋白激酶的基因TaCBRLK。利用实时荧光定量PCR技术对TaPGIP、TaPDR7和TaCBRLK基因进行禾谷镰刀菌和脱氧雪腐镰刀菌烯醇（DON）处理条件下的基因表达模式分析，结果显示上述3个候选基因均受到禾谷镰刀菌和DON处理的上调表达，说明其参与禾谷镰刀菌侵染的响应，可能是小麦中具有赤霉病抗性的候选基因。3.通过叶部BSMV-VIGS基因沉默体系分别沉默了TaPGIP、TaPDR7和TaCBRLK基因，利用叶片离体培养方法鉴定基因沉默后小麦对赤霉病的抗性反应，结果显示，沉默目的基因之后，禾谷镰刀菌在基因沉默叶片上的生长和蔓延速度明显快于其在接种病毒空载体的叶片和野生型叶片上的生长速度；结合台盼蓝染色法鉴定禾谷镰刀菌侵染小麦叶片产生的腐生斑大小，结果显示，禾谷镰刀菌在基因沉默叶片产生的腐生斑明显大于其在接种病毒空载体的叶片和野生型叶片上产生的腐生斑。上述结果表明，分别沉默基因TaPGIP、TaPDR7和TaCBRLK后，减弱了小麦叶片对赤霉病的抗性，说明TaPGIP、TaPDR7和TaCBRLK基因可能是小麦中具有赤霉病抗性的候选基因。进一步对TaPGIP基因在小麦穗部进行基因沉默，同时接种禾谷镰刀菌鉴定目的基因沉默植株对赤霉病的抗病反应，结果显示，在小麦穗部沉默TaPGIP基因之后，禾谷镰刀菌在小麦穗部的蔓延速度明显快于其在接种病毒空载体的麦穗和野生型麦穗上的蔓延速度，产生的枯死小穗数目也明显多于接种病毒空载体的植株和野生型植株，说明在小麦穗部沉默TaPGIP基因之后，有利于赤霉菌侵染和发生，进一步证明TaPGIP基因是小麦中具有赤霉病抗性的一个候选基因。4.利用烟草和洋葱表皮细胞进行亚细胞定位分析，结果表明，TaPGIP被定位在烟草和洋葱表皮细胞的细胞壁上，TaPDR7和TaCBRLK被定位在烟草表皮细胞的细胞膜上。结合三个基因在植物细胞中的生理功能，可以得出上述三个候选基因抵抗禾谷镰刀菌入侵的模型：TaPGIP在感受到禾谷镰刀菌入侵的信号之后高效表达，翻译产生大量的PGIP蛋白，该蛋白与禾谷镰刀菌在入侵小麦过程中分泌的多聚半乳糖醛酸酶（PGs）结合，抑制PGs的活性，以抑制禾谷镰刀菌对小麦细胞的破坏，进而阻止禾谷镰刀菌对小麦的入侵；当禾谷镰刀菌到达小麦细胞膜并进入细胞质时，TaPDR7感受到禾谷镰刀菌的入侵信号，高效表达翻译产生具有转运功能的ABC转运蛋白，将禾谷镰刀菌入侵小麦后产生的DON运输到细胞外，以维持小麦细胞的健康状态，减轻禾谷镰刀菌的侵染对小麦细胞造成的毒害；同时， TaCBRLK也会感受禾谷镰刀菌的入侵并高效表达，翻译产生具有信号转导作用的蛋白激酶，启动植物体内的信号转导通路，从而进行信号的传递，启动植物体内的病原防御机制，以抵抗真菌病原的入侵。利用实时荧光定量PCR对在植物激素诱导条件下目的基因的表达模式进行分析，结果显示，上述三个基因可能均受到生长素（IAA）的诱导上调表达。更多还原

【Abstract】 Fusarium head blight (FHB) was mainly caused by Fusarium graminearum Schwabe. Itwas prevailed mainly in the warm humid and subhumid regions around the world. In China,FHB had been occurred in Yangtse River Valley and along with the global warming it had atendency of spreading to Huanghuaihai region. However, the resistant resources of FHB werenarrow, Sumai3and its derived lines were major resistant resources applied in wheat genomicsstudy. Also, because of their strong and stable resistance to FHB, the cultivars were widely usedto locate major QTLs and for wheat breeding. With the development of biochemical,sequencing and gene manipulating technology, identification and functional analysis of theFHB resistance genes had achieved great progress in genomics and proteomics research.In this article, we combined transcriptome sequencing and bioinformatics analysis toexplore the resistance relevant genes in scab sensitive and resistance near-isogenic linesApogee and Apogee73S2infected with Fusarium graminearum. Barely stripe mosic virusinduced by gene silencing (BSMV-VIGS) was practiced to verify the candidate genesdiscovered in the transcriptome database and their biochemical characteristics were ensured inthe present study. The results were as follows:1. BSMV-VIGS system had been successfully established in wheat leaves and spikes usingTaPDS as a report gene. The photobleaching phenotype could last more than21days in wheatleaf and30to40days in spike, which made a good establishment to study the target genesresistance to other diseases. Meanwhile, we also established the detached leaf culture systemwhich made the identification of FHB easier and faster.2. Three FHB resistance candidate genes, i.e. TaPGIP3encoding a polygalacturonaseinhibiting protein, TaPDR7encoding a pleiotropic drug resistance protein and TaCBRLKencoding a CaM binding protein kinase were identified in FHB sensitive and resistancenear-isogenic lines Apogee and Apogee73S2. Real time PCR was used to analyze theirexpression patterns under the treatment of Fusarium graminearum and trichothecenedeoxynivalenol (DON), the results showed that all of the three genes were up-regulated after treatments of either Fusarium graminearum or DON, which indicated that they might be thecandidate genes in the FHB resistance.3. The FHB resistance of the three target genes had been verified using the detached leafculture after silencing each of the genes by BSMV-VIGS appraoch. The results showed thatgrowth and spread of Fusarium graminearum were much faster compared to the leaves infectedwith BSMV::00and wild type after silencing the genes in wheat leaves. Typan blue wasadopted to check the scab spots make by the invasion of Fusarium graminearum, it was testedthat, scab spots produced in the gene silencing leaves were much larger than those in the leavesinfected with BSMV::00and wild type. All of the findings accounted for the FHB resistance ofthe three genes. In the experiment of silencing TaPGIP in wheat spikes, after infecting withFusarium graminearum, the hypha grew faster in the TaPGIP silencing spike comparing to thespikes inoculated with BSMV::00and wild type, besides, the dried-up spikelets were more thanspikelets inoculated with BSMV::00and wild type. All of the results conferred that TaPGIPmight be a candidate FHB resistance gene in wheat.4. Sub-cellular location with tobacco and onion epidermis cells showed that TaPGIP waslocated on the cell wall of tobacco and onion epidermis cells, while TaPDR7and TaCBRLKwere located on the plasmid membrane of tobacco epidermis cells. According to thephysiological function of the three genes and their locations in plant cells, we drew a model onhow the three genes worked during the invasion of Fusarium graminearum: TaPGIP which waslocated on the cell wall was able to percept stimulus of Fusarium graminearum and then washighly expressed to translate plenty of PGIP proteins to combine with PGs produced byFusarium graminearum in order to inhibit the invasion of Fusarium graminearum; whenFusarium graminearum reached to the membrane, however, TaPDR7could be up-regulted byFusarium graminearum and translated enough ABC transporters to transport DON outside ofthe cell to keep the stage of cell health, then mitigated damage caused by Fusariumgraminearum invasion; in the mean time, TaCBRLK could be up-regulated to translate huge ofCaM binding protein kinases to transduce signals inside plant cells to activate the antiviraldefense systems in plants to defend against Fusarium graminearum. Real time PCR waspracticed to analysis the expression patterns of the three genes under the treatment of planthormones, the results implied that all of the three genes were up-regulated by indole acetic acid(IAA).更多还原