【作者】 朱自果；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2012， 博士

【摘要】 葡萄白粉病(Erysiphe necator (Schw.) Burr.)遍布世界葡萄主要栽培区，给葡萄生产造成很大损失。在生产上广泛栽培的品种主要来自欧洲葡萄，但大多数欧洲葡萄不抗白粉病。现代果树育种的理论和实践证明，利用抗病的葡萄近缘野生种进行抗病育种是可行的。中国野生葡萄是抗病育种的宝贵资源，其中华东葡萄（Vitis pseudoreticulata）株系白河-35-1表现出对白粉病的高抗性，但对其抗病的机制还不清楚。植物对病原菌的响应是通过一系列信号转导途径来完成的，转录因子在其中发挥了重要作用。葡萄基因组含有2000多个转录因子，但是对其功能研究甚少。本研究通过测序研究白粉菌诱导下中国野生华东葡萄抗病相关基因的种类，对分离获得的转录因子（ERF基因和NAC基因）进行表达及功能方面的研究，取得的主要结果如下：1、对前期构建的白粉病诱导的华东葡萄株系白河-35-1的cDNA文库进行大规模测序。3500个克隆被随机测序，去除低质量的序列并经过聚类分析获得1727条高质量的EST （Expressed sequence tag）序列并登录GenBank，登录号为GR882788~GR884514。利用InterProScan和GO数据库进行功能注释，983个EST序列被功能注释，占全部序列的56.9%。基于GO分类法，进行细胞组分,生物进程和分子功能等3个层次的分类。分子功能分类法中，结合功能占48.2%；生物进程分类中，参与代谢和细胞进程的占60.1%和56.2%；细胞组分分类中，细胞和细胞部分均占42.7%。2、筛选了四类与抗病相关的EST序列：转录因子、防御基因、R基因和活性氧代谢基因。半定量PCR分析表明这些基因可能参与中国野生华东葡萄抵御白粉病的过程。利用RACE（Rapid-amplification of cDNA ends）技术获得22条抗白粉病相关的cDNA全长序列，分别为VpERF1、VpERF2、VpERF3、VpNAC1、VpWRKY3、VpHsf1、VpMYB1、VpMYB2、VpDRE1、VpDRE2,、VpB-Zip1、VpPR1、VpPR1.2、VpPR4、VpPR10.2、VpPR14、VpDRP1、VpMLO1、VpUSP1、VpSGT1、VpTBE1，VpGST1。登录号为：GU393310，GU393312，GU393311，GU393316，JF500755，GU393313，GU393314，GU393315，GU393308，GU393309，GU393307，GU269633，GU269634，JN977472，HQ634186，JQ814381， JF500756，JQ814382，GU946975，JQ814383，JQ814369，JQ814380。3、中国野生华东葡萄三个乙烯响应因子基因(Ethylene responsive factor)VpERF1，VpERF2和VpERF3分属于ERF家族的B3、B2和B3亚族，相互之间蛋白序列相似度较低，仅有21.9%，在AP2/ERF区域的相似度却高达82.49%；与欧洲葡萄ERF基因具有较高同源性，但存在氨基酸片段的缺失和突变，表明所获的三个ERF基因为葡萄ERF家族新成员。4、葡萄白粉菌接种情况下，三个ERF基因均受诱导表达，但在抗病株系和感病株系中的表达模式有所不同，VpERF1在抗、感株系中的表达无规律；VpERF2和VpERF3在抗病株系中的最大诱导量要高于感病株系。在非生物胁迫处理下，VpERF1受干旱和高温的诱导；VpERF2受干旱、低温和高温的诱导；VpERF3受低温和高温，但诱导程度较小。此外VpERF1和VpERF3可能参与水杨酸(Salicylic acid，SA)、茉莉酸甲酯(Methyl jasmonate，MeJA)和乙烯（Ethylene，ET）等3种信号转导途径，VpERF2可能参与MeJA和ET等两种信号转导途径。5、中国野生华东葡萄三个ERF基因含有AP2/ERF保守结构域、核定位信号区域和酸性激活区域。亚细胞定位试验表明，三个ERF基因均定位在细胞核内。酵母杂交和葡萄叶片瞬时转化试验表明，VpERF1和VpERF2在酵母中具有转录激活活性，VpERF3未表现，但三个ERF基因在葡萄中能够结合GCC盒来激活下游GUS基因的表达，说明三个ERF基因均具转录激活活性。6、葡萄叶片瞬时转染试验表明，VpERF2和VpERF3能够阻碍葡萄白粉菌菌丝的生长，但VpERF1对其没有影响。在烟草中过量表达VpERF2和VpERF3能够提高对烟草青枯菌（Ralstonia solanacearum）和疫霉菌（Phytophtora parasitica var．nicotianae Tucker）的抗性，但VpERF1却增加对这两种病原菌的敏感性。半定量PCR分析表明， VpERF2和VpERF3能够上调病程相关蛋白（pathogenesis related protein，PR）基因PR1，PR2，PR4和PR5的表达，但VpERF1仅能上调部分PR基因的表达。7、利用同源序列克隆的办法获得了1616bp的VpERF3基因的启动子，序列分析其含有根特异性元件和保卫细胞特异性元件。VpERF3基因启动子转基因拟南芥的组织化学染色分析表明，VpERF3基因的启动子具有根和保卫细胞特异性。8、中国野生华东葡萄VpNAC1属于TERN亚家族，定位于细胞核且具有转录激活功能。葡萄白粉菌诱导下，VpNAC1基因的表达先上升后减弱，但最大诱导量在抗病株系要高于感病株系。VpNAC1受MeJA处理的强烈诱导，SA和ET处理虽也能诱导VpNAC1基因的表达但较弱，表明VpNAC1可能主要参与MeJA信号转导途径。VpNAC1受干旱、低温和高温的诱导，特别是干旱，最高诱导量比对照高40多倍。在烟草中过量表达VpNAC1增强对烟草白粉病(Erysiphe cichoracearum)和疫霉病的抗性，同时能够上调PR基因的表达。更多还原

【Abstract】 Powdery mildew (Erysiphe necator.(schw.) Burr.) causes great losse to the grapeproduction in the world. The most worldwide cultivated varieties are from Vitis vinifera whichwas generally susceptible to E. necator. The modern fruit breeding theory and practice provedthat it is feasible to breed resistant cultivars using wild grape. Chinese wild grape is preciousresource for resistance breeding, among which Vitis pseudoreticulata accession Baihe-35-1showed high resistance to E. necator, but the disease resistant mechanism is not understood.Transcription factor plays an important role in response to pathogen. The grapevine genomecontains more than2000transcription factors, but little is known to the function. Thisresearch aims to understand the types of the defense related gene of Chinese wild Vitispseudoreticulata through large-scale sequencing, and study the expression and function of thetranscription factors (ERF and NAC gene). Mainly results obtained are as follows:1. Large-scale sequencing of the cDNA library from V. pseudoreticulata accessionBaihe-35-1inoculated with E. necator.3500clones were randomly sequenced,1727ESTswere obtained by removal of low quality sequences and cluster analysis. All the ESTs weredeposited at the NCBI GenBank database under the accession numbers GR882788-GR884514. The ESTs were submitted to the public databases (InterProScan and GO) forsimilarity analysis and assigned GO categories,56.9%(983) ESTs could be annotated. For thecategory of molecular function, there contain ‘binding’48.2%; for the category of biologicalprocess, there contain ‘metabolic process’60.1%and ‘cellular process’56.2%; for thecategory of cellular component, there contain ‘cell’42.7%and ‘cell part’42.7%.2. Four disease resistance related genes (Transcription factor, defense genes, R gene andactive oxygen metabolism) were identified from the annotated ESTs. The semi-RT-PCRanalysis indicated that these disease resistance related genes may be involved in the prosess ofChinese wild Vitis pseudoreticulata Baihe-35-1against powdery mildew. Twenty twofull-length cDNAs related to disease resistance were cloned by rapid-amplification of cDNAends technique. They are VpERF1, VpERF2, VpERF3, VpNAC1, VpWRKY3, VpHsf1,VpMYB1, VpMYB2, VpDRE1, VpDRE2, VpB-Zip1, VpPR1, VpPR1.2, VpPR4, VpPR10.2,VpPR14, VpDRP1, VpMLO1, VpUSP1, VpSGT1, VpTBE1, VpGST1. The GenBank numbers are: GU393310, GU393312, GU393311, GU393316, JF500755, GU393313, GU393314,GU393315, GU393308, GU393309, GU393307, GU269633, GU269634, JN977472,HQ634186, JQ814381, JF500756, JQ814382, GU946975, JQ814383, JQ814369, JQ814380.3. Chinese wild Vitis pseudoreticulata VpERF1, VpERF2and VpERF3belong to the B3,B2and B3subfamily of ERF family, respectively. There is a low similarity (21.9%) betweenthe three ERF proteins, but they possess a conserved AP2/ERF domain with the similarity82.49%. Although three VpERFs proteins have high similarity with V. vinifera ERF proteins,but they exist difference in protein sequence because of the protein fragment deletion andsingle amino acid mutation, indicating the three VpERFs are novel ERF genes.4. VpERF1, VpERF2and VpERF3were induced by E. necator, but the expressionpatterns are different. The maximum induction of VpERF2and VpERF3in E.necator-resistant grapevine was higher than that in E. necator-susceptible grapevine butVpERF1. Under the abiotic stress, VpERF1was induced by drought and heat stresses;VpERF2was induced by drought, cold and heat stresses; VpERF3was induced by cold andheat stresses, but the induction was very low. Moreover, VpERF1and VpERF3were involvedin SA, MeJA and ET signal pathways, VpERF2was involved in MeJA and ET signalpathways.5. VpERF1, VpERF2and VpERF3contain AP2/ERF domain, nuclear localization signaland trans-activation domain. The sub-cellular localization indicated that three ERF geneslocalized to nucleus. Yeast hybrids indicated that VpERF1and VpERF2functioned as atranscriptional activator but VpERF3. Transient transformation in grapevine indicated thatthree ERF genes function as transcriptional activators by binding to the GCC box cis-element.6. Transient transfection in grapevine indicated that VpERF2and VpERF3could hinderthe development of hypha but VpERF1. Overexpression of VpERF2and VpERF3in tobaccoincreased the resistance to bacterial pathogen Ralstonia solanacearum and fungal pathogenPhytophtora parasitica var．nicotianae Tucker, while VpERF1increased the susceptible tothese two pathogens. In addition, VpERF2and VpERF3resulted in the accumulation of fourPR genes expression in transgenic tobacco plants. But, only part of PR gene transcripts wereup-regulated by VpERF17. The1616bp VpERF3promoter was obtained by homologous cloning. Sequenceanalysis indicated that VpERF3promoter contains root specific cis-acting element and guardcell specific cis-acting element. Chemical histological staining in transgenic Arabidopsisindicated that VpERF3specially expressed in root and guard cell, indicating VpERF3is atissue-specific promoter.8. VpNAC1belongs to the TERN subgroup. The sub-cellular localization and trans-activation activity assays indicated that VpNAC1is a nuclear targeting protein andfunctions as a transcriptional activator. After inoculation with E. necator, expression ofVpNAC1showed the pattern with increased first and then declined, but the maximuminduction in E. necator-resistant grapevine was higher than that in E. necator-susceptiblegrapevine. VpNAC1was strongly induced by methyl jasmonate, while induction level ofsalicylic acid and ethylene was low. VpNAC1was induced by drought, cold and heat stresses,especially the induction level of drought treatment was more40fold than control.Over-expression of VpNAC1in tobacco plants enhanced their resistance to Erysiphecichoracearum and Phytophtora parasitica var． nicotianae Tucker, and regualted theexpression of PR genes as a positive regulator.更多还原