【作者】 李艳；

【导师】 吴云锋；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2015， 博士

【摘要】 目前已报道的植原体病害已有1000多种,其中多种病害尤其木本植物上的都是致死性的。小麦蓝矮病是目前仅在我国有报道的一种小麦上的植原体病害,主要分布在陕西、宁夏、甘肃和内蒙古等西北干旱和半干旱的冬麦区,由介体条沙叶蝉（Psammotettix striatus L.）传播。小麦蓝矮病影响小麦植株的生长,影响小麦的抽穗和结实,造成小麦的大幅度减产,严重威胁小麦生产。目前,已开展了小麦蓝矮植原体基因组学及致病性等方面的研究,但是对寄主与小麦蓝矮植原体之间的互作机理还知之甚少。从转录水平了解寄主与小麦蓝矮植原体互作的基因表达特征,揭示寄主在感染小麦蓝矮植原体后在代谢、病害防御等方面的作用机制,将为研究小麦蓝矮植原体的致病性及其抗病品种的选育提供理论依据。因此,本研究以植原体的模式植物长春花为材料,采用c DNA-AFLP技术对感染小麦蓝矮植原体的长春花进行差异基因表达谱分析,并通过荧光定量PCR技术验证了c DNA-AFLP表达谱的准确性;利用RACE方法克隆病程相关蛋白基因及防御相关的基因全长,并通过生物信息学分析编码蛋白的基本特征;在转录水平分析这些基因在长春花与小麦蓝矮植原体互作过程中的表达模式。主要研究结果如下:1.以长春花为材料,采用c DNA-AFLP技术对感病植株中表现花绿变的花组织与健康对照的花组织进行差异基因表达谱分析,共分离到258条TDFs,其中201条TDFs的表达受到小麦蓝矮植原体侵染的诱导,而57条TDFs的表达受到抑制。通过与Gen Bank的nr数据库进行相似性比对,125条TDFs有明确的基因功能,参与了代谢、能量、信号转导、致病和防御等多条生物代谢途径。因此,这些基因的分离对深入了解寄主与植原体之间的互作机理有重要作用。2.从得到的TDFs中选择了8个TDFs,对其进行荧光定量PCR分析,除了一个基因的表达量变化与c DNA-AFLP的结果相悖外,其他基因的表达量变化与c DNA-AFLP得到的结果一致,说明c DNA-AFLP结果准确可靠。3.通过c DNA-AFLP分离到的TDFs的功能注释,找到两个基因W₈-35和W₁-23可能与花瓣的绿变相关。通过荧光定量PCR分析两个基因在花褪色、花绿变、花变叶及健康花组织中的表达量变化,结果显示W₁-23在花变态的各个阶段表达量均高于健康长春花;而W₈-35在花褪色样品中的表达量上升,在花绿变和花变叶中表达量低于健康花组织,说明两个基因在花变态的过程中发挥了作用。4.对长春花的10个内参基因在小麦蓝矮植原体侵染过程中的表达稳定性进行评价,通过软件ge Norm和Norm Finder得到的结果显示,Cr L23在整个小麦蓝矮植原体侵染的过程中表达相对最稳定;在长春花表现不同程度的花变态样品中,Cr TUB和Cr TUA是最稳定的内参基因;在长春花的不同组织样品中,Cr ACT在两个软件得到结果中表现最稳定。5.通过RACE方法,以c DNA-AFLP得到的基因片段为基础,克隆到两个长春花的PR蛋白基因全长,分别为Cr PR5和Cr PR6基因。Cr PR5基因全长1049bp,ORF为750bp,编码249个氨基酸。Cr PR6基因全长1682bp,ORF长1170bp,编码389个氨基酸。生物信息学分析结果显示Cr PR5蛋白序列N端有信号肽,无跨膜区;Cr PR6没有信号肽也没有跨膜区,可能定位在过氧化物酶体上。6.对长春花的PR蛋白基因Cr PR1a,Cr PR2,Cr PR4,Cr PR10a,Cr PR5,Cr PR6,Cr PR13和Cr PR14在感染小麦蓝矮和枣疯植原体的长春花中的表达量进行q RT-PCR分析,结果显示同一PR基因在两种植原体侵染的长春花中表达模式不同。大部分基因在嫁接后5天即受到WBD的诱导表达,而受到JWB诱导表达的时间点则相对滞后。同时以上结果也表明不同的PR蛋白在两种植原体与长春花的互作体系中发挥的作用不同。7.通过RACE方法,克隆到长春花的CBSX3基因,该基因全长1051bp,ORF为618bp,编码205个氨基酸。对Cr CBSX3基因在小麦蓝矮植原体的侵染过程中及不同组织中的表达量进行分析,结果显示该基因在长春花花中的表达量最高,在根中的表达量最低;在嫁接小麦蓝矮植原体10天后即受到强烈诱导表达。在本氏烟叶片中的亚细胞定位结果显示该基因编码的蛋白定位在细胞质;瞬时表达结果显示Cr CBSX3在本氏烟叶片中能够抑制BAX诱导的细胞坏死。以上实验结果说明该基因在侵染早期参与了长春花与小麦蓝矮植原体之间的互作。更多还原

【Abstract】 Phytoplasmas are associated with diseases in over one thousand of plant species, especially are lethal in some woody plants. To date, Wheat blue dwarf（WBD） disease, caused by wheat blue dwarf phytoplasma, has only been reported in China. It is transmitted by leafhopper Psammotettix striatus L., and mostly distributes in arid and semiarid areas of northwestern China, including Shaanxi, Ningxia, Gansu and Inner Mongolia. The growth of infected wheat was depressed, and the wheat head sprouting and fructification were influenced. The disease could reduce the wheat yield, and threat wheat production. Recently, the researches involved in WBD phytoplasma genomes and pathogenicity have been carried out. However, the mechanisms of the interaction between host plant and phytoplasma were obscure. Consequently, understanding the expression profiling of plant-phytoplasma interaction at the transcriptome level, so as to elucidate the mechanisms involved in metabolism and defense after host plant infected by WBD phytoplasma, will be greatly important for WBD pathogencity research and resistant breeding. In the present study, c DNA-AFLP technique was used to analyze the differential expression profile of WBD phytoplasma-infected periwinkle（Catharanthus roseus）. Quantitative real-time PCR were carried out to verify the expression patterns of TDFs derived from c DNA-AFLP analysis. Pathogenesis-related protein（PRs） genes and some genes related with defense were full-length cloned using RACE method, following by bioinformatics characterization. Finally, the expression patterns of these genes were analyzed during the interaction between periwinkle and WBD phytoplasma. The main results are as follows:1. Using periwinkle as the material, the differential expression profile of WBD phytoplasma-infected flowers with virescence were analyzed by c DNA-AFLP technique. Two hundred and fifty-eight reliable TDFs were obtained after selective amplification and sequencing. Of the 258 transcripts, 201 TDFs were up-regulated during phytoplasma infection, while 57 were repressed. Meantime, 125 had relatively clear functions in various categories（eg. metabolism, energy, signal translocation and disease/defense and so on） when searching the non-redundant protein database. Therefore, these genes can be valuable resources for understanding molecular changes in the plant-phytoplasma interaction.2. Eight TDFs were selected for q RT-PCR assays. The same expression patterns of all TDFs were found as observed in the c DNA-AFLP analysis, except for one TDF which was appeared down-regulated on the gel, turned out to be up-regulated in q RT-PCR assays. The results revealed that the c DNA-AFLP technique was efficient and reliable in identifying expressed genes in host plant induced by phytoplasma infection.3. Among the isolated TDFs, two genes（W₁-23 and W₈-35） were speculated to be involved in flower virescence. The expression patterns of these two genes were detected in the flowers with discorloration, virescence, phyllody and healthy ones. The results showed that the expression levels of W₁-23 in different stages of flower malformation were higher than that in healthy control, while the expression of W₈-35 was up-regulated in flowers with discorloration, and down-regulated in flowers with virescence and phyllody. These indicated that W₁-23 and W₈-35 have a role during flower malformation.4. Ten candidated reference genes were selected, and their expression stability during WBD phytoplasma infection were evaluated using the algorithms ge Norm and Norm Finder program. The results showed that Cr L23 performed well when all samples were considered. Cr TUB and Cr TUA were the most stable reference genes for flower malformation during WBD phytoplasma infection. In addition, Cr ACT was the most stable reference gene for samples from different tissues of periwinkle.5. According to the sequences derived from c DNA-AFLP analysis, two PR genes were cloned with RACE method. The full length of Cr PR5 was 1049 bp,and the ORF was 750 bp which encoded 249 aa. While Cr PR6 was 1682 bp in length, and the ORF was 1170 bp, which encoded 389 aa. Cr PR5 was predicted to have a signal peptide within its N terminal residues and lack the transmembrane. However, Cr PR6 had no signal peptide or transmemberane region, and was predicted to be localized on peroxisome.6. q RT-PCR analysis was carried out for the expression of Cr PR1 a, Cr PR2, Cr PR4, Cr PR10 a, Cr PR5, Cr PR6, Cr PR13 and Cr PR14 during WBD and Jujube Witches’ Broom（JWB） phytoplasma infection, respectively. The expression patterns were different for the same gene in two phytoplasma-infected periwinkles. Most of the genes were induced by WBD phytoplasma after grafted 5 days, while the time point of being induced by JWB phytoplasma was later. The results indicated that PRs have different roles in these two periwinkle-phytoplasma systems.7. The full length of Cr CBSX3 was cloned using the RACE method. The ORF of Cr CBSX3（1051bp） was 618 bp, and encoded 205 aa. Expression profiles analysis showed that Cr CBSX3 expressed with the highest level in the flower, and the lowest one in the root. In addition, Cr CBSX3 was strongly induced after WBD phytoplasma grafted 10 days during the periwinkle-WBD interactions. Subcellular localization in Nicotiana benthamiana leaves showed that Cr CBSX3 localized in the cytoplasm. Transient expression analysis indicated that Cr CBSX3 could suppress the cell death caused by BAX in tobacco cells, which indicated that Cr CBSX3 had a vital role in the early stage of this interaction system.更多还原