【作者】 王磊；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2017， 博士

【摘要】 欧洲葡萄(Vitis vinifera L.)是世界上主要的栽培种,但是对白粉病抗性差;葡萄白粉病是严重影响欧洲葡萄生长发育和经济价值的主要因素之一。本研究课题组前期根据白河-35-1高抗白粉病的特性,接种白粉菌构建了cDNA文库。本研究以cDNA文库中获得的EST序列(GR883881)为基础,克隆了VpRH2的基因全长及其启动子序列,筛选获得了VpRH2的互作蛋白VpGRP2A,进行了VpRH2和VpGRP2A的亚细胞定位分析和互作位置分析,将VpRH2在欧洲葡萄无核白中过表达,并进行了抗病性分析,主要研究结果如下:1.通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends,RACE)获得VpRH2基因全长,其开放阅读框(Open reading frame,ORF)为1164 bp,编码387个氨基酸。经蛋白质结构分析,VpRH2含有RING-H2和跨膜结构域,属于ATL家族,与欧洲葡萄ATL20同源,氨基酸同源性为93.02%,其中154位氨基酸残基的天冬氨酸为插入突变;在葡萄基因组网站预测发现,VpRH2定位在第二条染色体上。2.通过对抗病葡萄白河-35-1和感病品种无核白接种白粉病进行定量PCR分析表明,在抗病葡萄中VpRH2表达量升高,感病葡萄中Vv RH2表达量降低。亚细胞定位分析得出VpRH2定位在细胞膜、细胞质,在ER、TGN/EE和内膜组分中也有分布。ABA处理后,VpRH2的表达表现出下调趋势,经SA和Me JA处理后表现出上调的趋势。3.根据公布的葡萄基因组序列同源克隆获得了VpRH2启动子序列,胁迫相关的顺式作用元件ABRE、Box-W1、TC-rich repeats、TCA-element和TGACG-element等存在于启动子的各个区域,与欧洲葡萄Vv RH2启动子相比,存在较长的3段缺失突变和一段插入突变。将VpRH2和Vv RH2的启动子构建GUS融合载体,瞬时转化欧洲葡萄佳利酿叶片,经接种白粉菌、喷施Me JA、SA、ABA以及高温和低温等不同胁迫处理后进行GUS染色和GUS蛋白定量,分析表明:经白粉菌、Me JA和SA诱导处理后,VpRH2启动子与Vv RH2启动子启动活性均表现出了增长的趋势,而VpRH2启动子受诱导更为显著;在高温处理后VpRH2和Vv RH2启动子都表现出了增长的趋势,但Vv RH2启动子活性高于VpRH2启动子;两个启动子启动活性在ABA处理后表现出下调的趋势而对低温胁迫处理不敏感。4.通过酵母双杂交技术获得VpRH2的互作蛋白VpGRP2A(Glycine-rich RNA-binding protein 2A),并通过双分子荧光互补实验(Bimolecular fluorescencecomplementation,Bi FC)验证得出互作发生在细胞膜和细胞质中。VpRH2和VpGRP2A通过26S蛋白酶体途径降解,但是VpGRP2A的蛋白降解没有受到VpRH2的明显促进。VpGRP2A能够促进VpRH2的表达,同时VpGRP2A的表达受到VpRH2的抑制。5.中国野生华东葡萄VpGRP2A与欧洲葡萄Vv GRP2A表现出100%的同源性;通过对抗病葡萄白河-35-1和感病品种无核白接种白粉病进行定量PCR分析表明,VpGRP2A和Vv GRP2A表现出相似的表达模式,均为接种白粉病后6~24 h内表现出下调的趋势,而后趋于稳定;VpGRP2A具有细胞核定位,在内质网、TGN/EE和内膜组分中也有分布。VpGRP2A在ABA处理后上调表达,在SA和Me JA处理后表现出下调的趋势。6.转基因OERH2葡萄表现出对白粉病增强的抗性,生长初期表现出加速生长的表型,同时在OERH2转基因葡萄海绵组织细胞内部有结构变化,经超薄切片观察确认为定位于液泡和液泡膜上高密度物质。转基因OERH2拟南芥表现出对拟南芥白粉菌增强的抗性和外源ABA增强的耐受性。综上所述,中国野生华东葡萄泛素连接酶基因VpRH2具有抗葡萄白粉病菌的功能。更多还原

【Abstract】 European cultivated grape species have the best fruit quality but show almost no resistance to powdery mildew(PM).PM caused by Uncinula necator is a harmful disease that has a significant impact on the economic value of the grape crop;the Chinese wild grapevine V.pseudoreticulata accession Baihe-35-1 was selected as our research target because of its resistance to multiple fungi,particularly U.necator.The cDNA library of V.pseudoreticulata accession Baihe-35-1 inoculated with U.necator contained EST sequence(GR883881),named VpRH2,the full length cDNA and the promoter were obtained;VpGRP2A was selected out as the interacting protein of VpRH2,the potential links between VpRH2 and VpGRP2 A were determined by subcellular localizations and BiFC(bimolecular fluorescence complementation),over-expression of VpRH2 in transgenic Thompson Seedless was used to survey the responding to U.necator,the conclusions are as follows:1.The full length cDNA of VpRH2 was obtained by RACE(rapid-amplification of cDNA ends).The ORF(open reading frame)of VpRH2 was 1,164 bp and encoded 387 amino acids,covers a RING-H2 and transmembrane.VpRH2 belonged to the ATL subfamily and was the homologous gene of ATL20 in V.vinifera,which shared 93.02% amino acid identity;residue 154 is an aspartic acid,which is an insertion mutation compared to VvRH2;VpRH2was predicted to be located on chromosome 2 using the Grape Genome Browser.2.The expression of VpRH2 was clearly induced by U.necator inoculation compared with its homologous gene VvRH2 in a PM-susceptible grapevine V.vinifera cv.Thompson Seedless.VpRH2 was located at the cell membrane,cytoplasm,ER,TGN/EE,and endormembrane compartments.The expression of VpRH2 was inhibited by ABA treatment,but reduced by SA and MeJA treatments.3.The promoter of VpRH2 was cloned and several stress-regulated elements,such as ABRE,Box-W1,TC-rich repeats,TCA-element and TGACG-motif were found in the VpRH2promoter;compared to the VvRH2 promoter,three sections of sequences were deleted in the VpRH2 promoter,and one was inserted.The VpRH2 promoter and VvRH2 promoter were transiently transformed into grapevine V.vinifera Carignane leaves;powdery mildewinoculation,MeJA,SA,ABA,heat stress and cold stress treatments were used,as a result VpRH2 promoter activity was up-regulated by pathogen infection,SA and MeJA treatments comparing with VvRH2 promoter;the activity of VpRH2 and VvRH2 was induced by heat treatment,and the increased activity of VvRH2 promoter was higher than that of VpRH2promoter;the activity of two promoters were inhibited by ABA treatment and no obviously changed by cold treatment.4.VpGRP2 A was selected out by Y2 H as the interacting protein,and BiFC indicated that VpRH2 physically interacted with VpGRP2 A on the cell membrane and cytoplasm.The degradation of VpRH2 and VpGRP2 A was via UPS,but when VpRH2 increased,the protein level of VpGRP2 A did not obviously declined.VpGRP2 A enhanced the expression of VpRH2 but VpRH2 suppressed the expression of VpGRP2 A.5.The amino acid sequence of VpGRP2 A shared 100% similarity to VvGRP2A;the expression of VpGRP2 A and VvGRP2 A after U.necator inoculation showed a similar trend in the two grapes,clearly decreased from 6 hpi to 24 hpi;VpGRP2A was located at the nuclear,ER,TGN/EE,and endormembrane compartments.The expression of VpGRP2 A was inhibited by SA and MeJA treatments,but the expression was up-regulated by ABA treatments.6.OERH2-positive grape plants showed enhanced resistance to powdery mildew compared with the wild-type,displayed an accelerated growth phenotype,semi-thin sections of OERH2 showed that polymerized macromolecules was found in the OERH2 plants’ spongy parenchyma.In the ultra-thin section,polymerized macromolecules were found in the large central vacuole and tonoplast.OERH2 Arabidopsis plants showed enchanced tolerance to powdery mildew and ABA treatment.In summary,the ubiquitin ligase gene VpRH2 from Chinese wild grape V.pseudoreticulata has the function in resistance to powdery mildew.更多还原