【摘 要】
：
葡萄白粉病(Uncinula necator)是严重危害欧洲葡萄的真菌病害之一。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
【出 处】
：
西北农林科技大学 西北农林科技大学
论文部分内容阅读
葡萄白粉病(Uncinula necator)是严重危害欧洲葡萄的真菌病害之一。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。为了研究中国野生葡萄抗白粉病基因,本课题组采用mRNA差异显示技术(mRNA differential display reverse transcription-PCR, DDRT-PCR)进行了中国野生葡萄高抗葡萄白粉病的华东葡萄(V. pseudoreticulata)白河-35-1在白粉病病原菌侵染诱导下抗病基因克隆的研究,获得了抗白粉病基因差异表达的cDNA片段10个, BLAST分析表明,在白粉病病原菌侵染后表达而对照不表达的cDNA片段T11GG/B0324-292 (GenBank登录号:DT725418)与拟南芥SBP (SQUAMOSA promoter binding protein, SBP)基因有较高的同源性,说明获得的cDNA片段T11GG/B0324-292是中国野生葡萄SBP基因的一段cDNA序列。本研究就是在获得中国野生葡萄SBP基因cDNA片段T11GG/B0324-292的基础上,采用cDNA末端快速扩增技术(RACE)并通过生物学信息软件分析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。主要研究结果:1.中国野生葡萄白河-35-1 SBP基因3’端cDNA克隆与序列分析:在获得中国野生葡萄SBP基因cDNA片段T11GG/B0324-292的基础上,通过RACE技术,获得了华东葡萄白河-35-1VpSBP基因的3’端cDNA序列,1177bp,同源性分析表明,VpSBP 3’cDNA核苷酸序列与欧洲葡萄基因组高度同源,与拟南芥启动子结合蛋白家族基因如SPL1、SPL2有较高的同源性。2.中国野生葡萄白河-35-1 SBP基因cDNA全长序列的克隆与分析表明:在获得中国野生葡萄SBP基因cDNA片段T11GG/B0324-292的基础上,设计特异引物,通过RACE技术,获得了华东葡萄白河-35-1VpSBP基因的完整cDNA序列,全长2566bp组成,命名为VpSBP。具有完整序列的开放阅读框架,编码640个氨基酸,核苷酸与氨基酸序列分析表明,VpSBP基因其它植物SBP基因核苷酸有74%—87%的同源性,其中与拟南芥核苷酸与氨基酸序列均在70%以上。3.葡萄总RNA的提取。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,28SRNA亮度约为18SRNA的1-2倍,适于DDRT-PCR、RACE等研究。
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