【作者】 余义和；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2013， 博士

【摘要】 葡萄白粉病菌（Uncinula necator [Schw.] Burr.）是危害葡萄生产的重要真菌病害之一。目前生产上栽培的主要是欧洲葡萄（Vitis vinifera. L）品种，这些品种品质优良且产量高，但抗病性差。我国具有丰富的野生葡萄种质资源，并携带大量的抗病基因。本研究以中国野生华东葡萄高抗白粉病株系白河35-1（Vitis pseudoreticulata Baihe35-1accession）为材料，克隆了泛素连接酶基因UIRP1基因及其启动子；研究了UIRP1基因及其启动子对病原菌的响应模式；分析了UIRP1基因的泛素连接酶活性及其亚细胞定位情况；筛选到一个与UIRP1相互作用的转录因子基因VpWRKY11；进一步分析了二者相互作用模式，并探讨了二者在植物抗病反应中的作用。主要获得了以下的研究结果：1、在中国野生华东葡萄白河35-1株系中克隆了UIRP1基因，该基因全长1068bp，ORF（开放阅读框）为723bp，编码240个氨基酸，其GenBank登录号为JF502034；生物信息学分析发现UIRP1基因N端存在一个C3HC4-type RING finger保守结构域，C端具有一个跨膜结构域。采用同源克隆的方法克隆了UIRP1基因启动子，长度为1182bp，预测分析发现其含有基本启动子元件和多个与抗病相关的顺式作用元件。2、对中国野生华东葡萄白河35-1株系接种白粉病诱导，发现UIRP1的表达量在接种病原菌后的12h、48h各呈现一次表达高峰，说明UIRP1的表达是受葡萄白粉病诱导的。将UIRP1基因启动子与GUS基因融合后瞬时转化葡萄叶片，发现UIRP1基因启动子的活性可以受葡萄白粉菌的诱导。3、中国野生华东葡萄UIRP1基因在大肠杆菌中表达，纯化后获得融合蛋白，采用体外泛素化试验分析是否具有泛素连接酶活性。结果发现UIRP1能与E1、E2以及泛素分子发生泛素化反应，UIRP1基因的RING finger结构域的保守氨基酸位点突变导致不能发生泛素化，如UIRP1-C30S、UIRP1-H47A，非保守氨基酸位点发生突变则不影响泛素化反应，如UIRP1-A66S。4、以中国野生华东葡萄泛素连接酶基因UIRP1为诱饵筛选文库，发现其与VpWRKY11相互作用；亚细胞定位试验发现中国野生华东葡萄泛素连接酶基因UIRP1定位在细胞核、细胞质与细胞膜上；双分子荧光互补试验再一次验证了泛素连接酶基因UIRP1与VpWRKY11相互作用，并且发现其相互作用发生在细胞核上。5、经在酵母体内与中国野生华东葡萄泛素连接酶基因UIRP1相互作用的VpWRKY11转录激活分析证明，它不具有转录激活活性，但其在植物活体内可以结合W-box顺式作用元件，具有转录激活活性；亚细胞定位试验表明中国野生华东葡萄VpWRKY11定位在细胞核；对中国野生华东葡萄白河35-1株系接种白粉病诱导，发现中国野生华东葡萄VpWRKY11是受葡萄白粉病诱导表达的基因。6、中国野生华东葡萄泛素连接酶基因UIRP1作用在VpWRKY11转录因子基因的上游，并以VpWRKY11转录因子为底物，依赖于26S蛋白酶体介导VpWRKY11转录因子的降解。中国野生华东葡萄泛素连接酶基因UIRP1降解负调控抗病因子VpWRKY11，导致其下游NPR1、AOS以及LOX2等抗病相关基因的高表达，最终提高植物的抗病性。综上所述，中国野生华东葡萄泛素连接酶基因具有抗葡萄白粉病的功能。更多还原

【Abstract】 Grapevine powdery mildew (Uncinula necator [Schw.] Burr.) is one of the mostimportant fungal diseases that damages grapevine production. Vitis vinifera is currently onemajor species, which possesses good quality and high yield. However, most of them aresensitive to diseases. China has abundant wild grape species that contain some valuableresistant-gene resources. In this study, one powdery mildew-resistant accession Baihe35-1ofChinese wild Vitis pseudoreticulata was used as the material to clone Chinese wild Vitispseudoreticulata UIRP1gene and its promoter. The responsive pattern of UIRP1gene and itspromoter to pathogen have been analyzed, respectively. The ubiquitin ligase activity and thesubcellular localization of the UIRP1gene have also been investigated. A transcription factorgene VpWRKY11that interacts with UIRP1has been screened, and their interactive modelwere further analyzed, as well as their roles in plant disease-resistant reaction were alsoexplored. The main results are described as followings.1. UIRP1gene was cloned from Chinese Wild V. pseudoreticulata accession Baihe35-1.The gene is1068bp in length with723bp opening read frame (ORF), encoding240aminoacids, and its GenBank accession number is JF502034. Bioinformatics analysis showedUIRP1gene possesses a C3HC4-type RING finger conserved motif at N-terminus and atransmembrane domain at C-terminus. The promoter of UIRP1gene was cloned usinghomology cloning method, in which is1182bp in length. Prediction analysis demonstratedthat UIRP1gene promoter contains the basic promoter elements and a plurality of cis-elements related to disease resistance.2. Inoculation with powdery mildew in Chinese Wild V. pseudoreticulata accessionBaihe35-1displayed the expression of UIRP1present two peaks in12hpi and48hpi,respectively, suggesting the expression of UIRP1is induced by powdery mildew. Thepromoter of UIRP1was fused with GUS, and transiently transform into grapevine leaves, theresults showed the activity of UIRP1promoter is induced by powdery mildew.3. The Chinese Wild V. pseudoreticulata UIRP1gene was expressed in Escherichia coli, and further purified to obtain the fusion protein. In vitro ubiquitination assay were conductedto analyze whether or not it possess ubiquitin ligase activity. The results showed UIRP1hasthe ubiquitin ligase activity, and mutation in conserved amino acid of the RING finger motifcould not occur ubiquitination. In contrast, mutation in non-conserved amino acid of theRING finger motif could not interfer the ubiquitination.4. Chinese Wild V. pseudoreticulata ubiquitin ligase gene UIRP1was used as the bait toscreen the library, and obtain its interactive protein VpWRKY11. Subcellular localizationdisplayed that UIRP1gene localized in nucleus, cytoplasm and cell membrane. Bimolecularfluorescence complementation (BiFC) assays further showed UIRP1interaction withVpWRKY11in the nucleus.5. The activation activity of VpWRKY11, which interacts with Chinese Wild V.pseudoreticulata ubiquitin ligase gene UIRP1, has no transcription in yeast. But VpWRKY11can combine the W-box cis-element, and has the transcription activation activity in planta.Result from subcellular localization assay showed VpWRKY11gene localized in nucleus.Inoculation with powdery mildew in Chinese Wild V. pseudoreticulata accession Baihe35-1showed the expression of VpWRKY11was induced by powdery mildew.6. The Chinese Wild V. pseudoreticulata ubiquitin ligase gene UIRP1acts in theupstream of the VpWRKY11, and UIRP1degrades VpWRKY11protein, and this degraderely on26S protease. The Chinese Wild V. pseudoreticulata ubiquitin ligase gene UIRP1negatively degrades and regulated VpWRKY11, and resulted in high expression of NPR1、AOS and LOX2disease-resistant related genes, therefore improving plant resistance.In conclusion, the ubiquitin ligase gene of Chinese Wild Vitis pseudoreticulata possessthe grape powdery mildew resistant function.更多还原