【作者】 张今今；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2003， 博士

【摘要】 中国葡萄属野生种对真菌病害具有丰富多样的抗性。如何有效地利用这些种质中的抗病基因，改良现有的欧洲葡萄栽培品种，实现抗病与品质结合，是葡萄育种家长期攻克的目标，也是我们从事的重要攻关课题。 本研究在田间葡萄白粉病发病盛期，对中国葡萄属野生种华东葡萄株系白河-35-1、感病的欧洲葡萄品种佳利酿及其F₁代感病单株6-12-3，用病叶压片法人工接种葡萄白粉病病原菌，诱导抗病性表达。在不同时期分别提取叶片总RNA，利用mRNA差异显示技术，经DDRT-PCR、测序胶电泳、银染、Northern杂交、序列测定及Blast检索。获得了与葡萄抗白粉病基因相关的差异表达cDNA片段及其序列，并对序列进行了同源性比较分析。取得的主要的研究结果是： 1．建立了适于葡萄材料总RNA提取的改进SDS／酚法。在改进SDS／酚法中，向提取缓冲液加入16％水溶性PVP和1％β-巯基乙醇，有效抑制了酚类物质的影响；在匀浆上清液中加入1／3体积5mol·L^-1乙酸钾（pH4.8），可以去除多糖的干扰。因此，改进的SDS／酚法能有效抑制酚类物质和多糖对总RNA提取的影响，获得质量高、完整性好的总RNA，28S rRNA亮度约为18S rRNA的2倍，A260／280介于1.8～2.0之间，A260／A230大于2.0，幼叶总RNA产率为261.20μg／g，成熟叶产率为191.40μg／g，完熟果皮产率为31.50μg／g。 2．建立了葡萄抗白粉病基因DDRT-PCR体系。筛选了DDRT-PCR体系中各反应因素的浓度，优化后25μL体系中各组份浓度为：cDNA第一链产物1.0μL（RT反应体系中总RNA用量1.5μg）、dNTPs浓度为0.25mmol／L、MgCl₂浓度为1.5mmol／L、锚定引物浓度为2.5mmol／L、随机引物浓度为2.5mmol／L、Taq酶用量为1.0U。该反应体系的产物经测序胶电泳和银染，可以获得数量适宜、带型清晰、重复性好的cDNA片段。 3．应用mRNA差异显示技术研究中国葡萄属野生种华东葡萄株系白河-35-1在白粉病病原菌侵染诱导下，抗白粉病基因的表达。获得了抗白粉病基因差异表达的cDNA片段19个，经二次PCR扩增和Northern杂交分析，最终获得了抗白粉病基因的5个cDNA片段：T11CA／B0322-280（Vprdrf4）、T11GC／S11-428（Vprdrf7）、T11CA／B0313-410（Vptual）、T11CA／B0315-360（Vprdrf3）、T11CA／B0304-474（Vprdrf2），分别由280bp、428bp、410bp、360bp和474bp的碱基序列组成。GenBank中的登录号依次为:CD347664，CD347666，CD662166，CD347663，CD347662。 4.研究了感病的欧洲葡萄品种佳利酿和白河一35一IX佳利酿的Fl代感病单株6一12一3在白粉菌侵染条件下，基因差异表达的情况，获得了感病相关cDNA片段6个:TI IGC/B0316一220、TllGC/B 0316一450、TllCA/BO314一960、TllGC/B 0316一440、TI IC刀BO304一420、TI IC户JB0320一210;感病亲本佳利酿中特异表达的eDNA片段5个:TllGC/B 0316一410、TllGC/B 0316一250、TllC户JB0304一420、TllCA/B0340一720、TllCA/B0316一1000;感病后代6一12一3中特异表达的eDNA片段12个:T 1 1 CA/B0304一550、TllGC/B 0316一265、TllGC旧0316一430、TllC户JB0320一390、T 1 1 CA/B0304一390、TllCA/B0313一290、TllCA了B0313一340、TllCA/BO313一380、TI ICA/BO313一450、TllCA/B 0313一650、TllCA/B0313.780、TllCA/B0304一470。 5.测序结果在基因数据库及蛋白质数据库中的同源性对比分析表明，vPtual为编码葡萄叶片a一微管蛋白基因的部分序列，长度为410 bp。分析了VPtual在葡萄抗白粉病过程中表达调控的变化。该基因的表达水平随侵染时间延续逐渐减小并消失，为负调控基因片段。Vprd麟为编码葡萄叶片核糖体RNA基因的部分序列，长度为280饰。VPrdrf4在葡萄抗白粉病过程中，表达水平随侵染时间延续逐渐增高，为正调控基因片段。更多还原

【Abstract】 There is abundant resistance in Chinese wild Vitis species. Utilizing these excellent germplasm to breed new grape varieties, which have not only good cultivated characters but also disease resistance, is a long-time target o f disease resistance breeding in grape. In order to gain insight into the molecular mechanism of the pathogeny-induced resistance and to investigate the expression of resistant gene related cDNAs, we initiated a study for isolation of the powdery mildew resistance gene or related cDNAs of Vitis using mRNA differential display PCR. This technique provided a molecular foundation for disease resistance breeding and also holded promise for the study for differential gene expression in fruits.In the vineyard, Uncinula necator was inoculated on the leaves of the grape materials: Vitis pseudoreticulata Baihe-35-1, Vitis vinifera Carignane and FI 6-12-3 to induce the expression of disease resistance. With thereinafter steps: total RNA extraction from leaves at different sampling periods, cDNAs reverse transcription, sequencing electrophoresis, silver staining, Northern blot, sequencing and Blast searches alignment in DNA database, we gained these results:1. To gain the high purity and integrity RNA, the modified SDS/phenol, guanidinum thiocyanate and commercial total RNA extraction kit were employed. These methods all can obtain total RNA from young leaf of grapevine, but only the modified SDS/phenol method can extract undegraded total RNA from mature leaf and ripe fruit skin. Following this efficient procedure, we routinely obtained 191.40μg/g ~261.20μg/g total RNA from leaf and 31.50 μg/g from ripe fruit skin. The ratio of A260/A280=1.8-2.0. The RNA is highly stable and sufficiently pure for DDRT-PCR, RACE, Northern hybridization and cDNA library construction.2. By comparing key factors in DDRT-PCR, the DDRT-PCR reaction system for Vitis was established. The optimizing reaction system contained 1.0uL cDNA, 0.25mmol-L-1 dNTPs, 1.5mmol-L-1 MgCb, 2.5mmol-L-1 anchored primer, 2.5mmol-L-1 arbitrary primer, and 1.0 U Taq. This DDRT-PCR system can be a useful tool to study fruits developmental7processes at the RNA level.3. By researching the expression of powdery mildew resistance gene after infection in Baihe-35-1 using DDRT-PCR. Five cDNA fragments related to resistance powdery mildew were identified using this approach. T11CA/B0313-410 (Vptual) is 410bp, T11CA/B0304-474 (Vprdrf 2) is 474bp, T11CA/B0315-360 (VprdrfS ). is 360bp, T11CA/B0322-280 (Vprdrf4) is 280bp, TilGC/S11-428 (Vprdrf7) is 428bp. Vprdrf 7, which has no alignment in DNA database, is a new gene of powdery mildew resistance. The accession numbers of GenBank database are CD662166, CD347662, CD347663, CD347664, CD347666.4. By comparing the differential expression cDNAs of Carignane and 6-12-3, 6 sensitive disease reduced cDNA fragments, T11GC/B0316-220, T11GC/B0316-450, T11CA/B0314-960, T11GC/B0316-440, T11CA/B0304-420 , T11GC/B0316-220 , T11CA/B0320-210 were gained. All of these fragments displayed similarly on Carignane and 6-12-3 after the pathogeny infection, were screened using this technique. Besides, 5 specific expression cDNA fragments of Carignane, T11GC/B0316-410 , T11GC/B0316-250, T11CA/B0304-420, T11CA/B0340-720, T11CA/B0316-1000 and 12 specific expression cDNA fragments of 6-12-3, T11GC/B0316-265 , T11GC/B0316-430, T11CA/B0320-390 , T11CA/B0304-390 , T11CA/B0304-550 , T11CA/B0313-290, Tl 1CA/B0313-340 , Tl 1CA/B0313-380 , Tl 1CA/B0313-450 , T11CA/B0313-650 , T11CA/B0313-780, T11CA/B0304-470, were also obtained.5. Sequencing and alignment analysis shows: Vptual, which is 410 bp, encodes the leaf a-tubulin gene of Vitis. The transcription of Vptual is depressed in the process of inducing. It is a down-regulated cDNA. Vprdrf4,partial sequence of leaf ribosomal protein of Vitis, the length of Vprdrf4 is 280 bp. By reason of the expression of it is enhancing during the course after fungi infected, Vprdrf4 is up-regulated cDNA.更多还原