【作者】 杨进孝；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2004， 硕士

【摘要】 本研究通过两年的田间和试验室工作，围绕葡萄优质抗病育种的中心目标，以中国葡萄属野生种白藜芦醇高产株系商南－24、抗白粉病株系白河-35-1 和欧洲葡萄感炭疽病品种凤凰－51 为主要材料，利用 RT-PCR 技术就白藜芦醇次生代谢过程调控和抗病基因克隆新策略研究方面作了一些尝试性的探索，主要取得以下几个方面的研究结果： 1.通过抗病基因克隆新策略和定量－简并 PCR 技术，得到与中国葡萄属野生种抗白粉病株系白河-35-1 抗病可能有密切关系的基因片段三条：9－2、7＃、15＃,它们的大小分别为 200bp、565bp、211bp。这几个基因片段的序列在 GeneBank 的登录号为分别为：AY656730、 AY656725、 AY656731。9－2 基因片段与从欧洲葡萄逆境胁迫的 cDNA文库中的多条 EST 序列同源；进一步分析表明该基因片段可能是葡萄抗病和次生代谢过程中涉及基因调控的某一反式作用因子的编码基因的一部分，在反式作用因子与葡萄抗病过程中信号的识别、传递、放大或防卫基因的转录都可能有关。7＃基因片段与欧洲葡萄受逆境胁迫的 cDNA 文库和多种植物微丝形成期的 cDNA 文库中的多条 EST 序列存在着同源关系，但具体功能未知；定量 PCR 结果表明，该基因片段在白河－35－1 受白粉病侵染时转录量有较大的变化，因此该基因片段可能与植物的纤维性物质合成及相应的葡萄结构性抗病诱导有关。15＃基因片段在生物信息学数据库中未找到任何同源序列，说明该基因片段可能是一条新基因，该基因片段在定量－简并 PCR 中的欧亚种葡萄感白粉病品种佳利酿的 cDNA 扩增结果中表现为低水平的本底表达，但在白河－35-1 中随病原菌入侵增幅明显，这暗示着该基因片段可能是一条首次发现的在葡萄抗病中有特殊意义的新基因。2.在葡萄PAL 途径抗病相关基因克隆研究方面。通过增加提取缓冲液中PVP的含量、减小样品量与总缓冲液比值，在提高总 RNA 提取纯度的同时提高了相对产率。从中国葡萄属野生种白藜芦醇高产株系商南-24 mRNA 中经 RT-PCR 克隆到与葡萄白藜芦醇合成和葡萄抗病有密切关系的 STS 基因家族的三个成员的 14＃、40－1、41－1 基因片段，它<WP=7>们的大小分别为：772bp、720bp、283bp。这几个基因片段的序列目前已等录 GeneBank，登录号为：AY656724、 AY656725、 AY656726。定量 PCR 同时获得有类似作用的 CHS基因（果实与叶片中各得到一条）、PPO 基因（叶片中特异表达）的三个基因片断。从商南-24 DNA 中经 PCR 扩增得到具有相同作用的苯丙氨酸裂解酶基因片断一个，它们的大小分别为 460bp、340bp、267bp．这几个基因片段的序列目前也已等录 GeneBank，登录号为：AY656727、 AY656728、 AY656729、AY656733。 3.在葡萄重要植保素物质白藜芦醇高产机制研究方面，从商-24 DNA 中经 PCR 扩增得到两个准 STS 启动子，其中一个上面可能有病原诱导应答元件。将白藜芦醇产生机制的差异细化到不同葡萄株系的不同基因家族成员的转录活性的不同及其单核苷酸位点多态性上。 4. 在抗病基因克隆中，通过定量 PCR 技术研究了受白粉病病原菌侵染不同时间的中国葡萄属野生种抗白粉病株系白河-35-1 中部分本实验中得到的防卫基因的转录活性变化情况，研究了欧亚种葡萄感炭疽病品系凤凰－51 果皮中防卫基因 CHS 转录量与炭疽病侵染面积的关系。在一定程度上验证并丰富了防卫基因与葡萄抗病性的关系的理论，为研究葡萄抗病基因克隆提供了方便新颖的采样思路。，通过对生物信息学分析方法—Motif 分析的反向思维，运用 PCR 原理建立了定量-简并 PCR 技术体系，并经过了初步验证。 5.建立了确定在本地气候和本试验条件下葡萄抗病基因表达时间的方法，并根据本试验方法确定了中国葡萄属野生种抗白粉病株系白河-35-1 抗白粉病基因的大致表达时间为 5～8ｄ。为有的放矢地克隆葡萄抗病基因提供了可能。更多还原

【Abstract】 In this vineyard Some Chinese Wild Vitis specis including Shangnan-24 (in whichresveratrol content is very high) and Baihe-35-1 (which is resistant to Powdery Mildew) andsome Vitis vinifera specis (the primary material is Fenghuang-51) were studied. By thetechnology of RT-PCR ,we explored some aspects in this direction of modern breeding, andsome results of our research are as follows: 1. By the new strategy for cloning disease resistance gene and the new efficienttechnology, we got three gene fragments from baihe-35-1 which is one resistant stain toPowdery Mildew in Chinese wild Vitis species, These three gene fragments are named 9-2,7#and15#,with the length of 200bp、565bp、211bp respectively.and the accession number ofthem in GeneBank are: AY656730, AY656725, AY656731. 9-2 gene fragment shows highhomlogous to those ESTs from abiotic stressed leaves of Vitis vinifera var. Another anylysismethod shows that it is homlogous to Vitis vinifera VvmybA1 gene for myb-relatedtranscription factor, this indicates this gene fragment may be a part of the gene of a transcriptfact, and it may play an important role in the resistance to disease .The homlogous anylysisindicated that 7# gene fragment may be a part of some gene which take part in the regulationof the fiber synthesis, which result in the induced resistance of structure. By this way, thisgene fragment shows high homlogous to those ESTs from abiotic stress expressed sequencetag database for abiotic stressed leaves of Vitis vinifera var. too.15# gene fragment has nohomlogous sequence in any bioinfomatic database in the internet, this indicated that it is anew gene, which may have some special function. 2. By the technology of RT-PCR, we cloned three members of stilbene synthase genefamily from the total RNA of Chinese wild Vitis species Shangnan-24, which can producemore Resveratrol in the same condition. with the length of772bp、720bp、283bp respectively,and the accession number of them in GeneBank are: The accession number of them inGeneBank are：AY656724, AY656725, AY656726. Besides these gene fragments, we gotanother four gene fragments by the same method, they are parts of the genes as follows:chalcone synthase gene, polyphenol oxidase gene, and phenylalanine ammonia-lyase gene. In<WP=9>these gene fragments, two fragments of chalcone synthase gene are gotten from leaves andfruit peel of shangnan-24 respectively, and the fragment of phenylalanine ammonia-lyasegene is special because it is amplified from DNA of shangnan-24.The length are 460bp、340bp、267bp respectively, and the accession number of them in GeneBank are：AY656727、AY656728、 AY656729、AY656733。 3. The research of the mechanism of the different display in the different strain isinteresting. Now, we have gotten two stilbene synthase gene promoter similar sequences,considered that the fragment we have cloned and some results of RT-PCR, we have gottensome new solution about the mechanism of the different display in different strain. Forexample, the different activity of different members in gene family and SNP in different strainmay contribute a lot to the different display in different strain. 4.In the field of mechanism of disease resistance, we researched the activity of stilbenesynthase gene after the different time of inoculation of Powdery Mildew, whichcontributed to the research of cloning of resistance gene, and it helps us know more about themechanism of different strains. 6.A more efficient technology to isolate total RNA has been developed ,what’s more, wedeveloped a new method to ascertain the period in which the disease resistance genetranscript, which is a basis to clone new disease resistance gene. How to clone diseaseresistance gene is a difficult problem in this research field. After many research in this field,we decided to use a new technology, which was proved to be very efficient. 7. In our research, we applied the gene engineering of secondary更多还原