【作者】 曹江玲；

【导师】 王跃进；

【作者基本信息】 西北农林科技大学 ， 果树学， 2012， 硕士

【摘要】 葡萄（Vitis spp.）是世界性重要的经济作物，常遭受真菌病的危害。葡萄白藜芦醇是一种重要的植保素，在植物对病原菌的防御反应中起重要作用，同时又对人体健康具有消炎、抗癌、预防心脑血管疾病等营养保健作用。芪合成酶是在葡萄中催化合成白藜芦醇的关键酶。本研究克隆了中国野生华东葡萄株系‘白河-35-1’（Vitis pseudoreticulataaccession Baihe-35-1）抗白粉病芪合成酶基因，克隆了野生毛葡萄株系‘丹凤-2’（Vitisquinquangularis accession Danfeng-2）抗白粉病芪合成酶基因，并对其进行了生物信息学分析及转化研究。取得的结果如下：1．通过RACE技术克隆获得在病原菌诱导下的中国野生华东葡萄株系‘白河-35-1’芪合成酶基因家族成员53条，其中4条序列编码区有缺失，为假基因。将获得的芪合成酶基因序列登入GenBank，登录号为：JQ886584至JQ886636。欧洲葡萄和中国野生华东葡萄株系‘白河-35-1’芪合成酶基因部分序列同源最高达98%，但聚类亲缘关系相对较远。2．采用RT-PCR技术克隆中国野生毛葡萄株系‘丹凤-2’芪合成酶基因VqDSTS1，并进行了序列及表达模式分析。结果表明，VqDSTS1基因cDNA编码区全长为1179bp，GenBank登录号为JQ342086,编码392个氨基酸；氨基酸序列分析表明，VqDSTS1含有芪合成酶基因家族的特征识别序列‘IPNSAGAIAGN’和‘GVLFGFGPGLT’；序列比对显示VqDSTS1与其他葡萄种质的芪合成酶氨基酸序列一致性在95.2%⁹8.7%之间；半定量RT-PCR分析表明，VqDSTS1受白粉病诱导表达，呈双峰模式。为进一步研究中国野生毛葡萄株系‘丹凤-2’芪合成酶基因家族的表达及功能分析提供了基础。3．在欧洲葡萄胚状体再生体系中，‘红地球’花药的最佳诱导培养基为NN69+CH0.5g·L^-1+PVP1g·L^-1＋2,4-D0.5mg·L^-1+BAP4.0mg·L^-1；子房的最佳诱导培养基为NN69+CH0.5g·L^-1+PVP1g·L^-1＋2,4-D1.0mg·L^-1+BAP4.0mg·L^-1。赤霞珠的花器官胚性愈伤组织的诱导效率要明显高于无核白。NN69培养基的诱导率最高（花药29.3%，子房32.5%），其次是B5培养基（花药4.5%，子房9.1%），MS培养基诱导率最低（花药4.0%，子房6.3%）。细胞悬浮培养的方法可迅速扩繁胚性愈伤组织，且能够获得均一的体细胞胚。4．农杆菌介导的遗传转化中，潮霉素对愈伤组织的最低致死浓度为13mg·L^-1；采用此浓度对胚性愈伤组织进行抗性筛选。GV3101菌株转化率明显高于LBA4404菌株。转化后抗性愈伤组织随着继代次数的增加，报告基因GFP的表达逐渐增强。更多还原

【Abstract】 The grapevine is one of the most important fruit crops in the world, which are generallysusceptible to Erysiphe necator and Plasmopara viticola. Stilbene synthase （STS） is the keyenzyme for producing the resveratrol. Grape resveratrol is a phytoalexin, plays an importantrole in plant defense responses to pathogens, and yet also shows significant health-promotingeffects including anti-cancer, prevention of cardiovascular disease and neuroprotectiveproperties. In this study, stilbene synthase genes were cloned from Chinese wild V.pseudoreticulata accession ‘Baihe-35-1’ which showed high resistance to powdery mildewand downy mildew. A stilbene synthase gene was clone from Chinese wild V. quinquangularisaccession ‘Danfeng-2’ which showed high resistance to powdery mildew and high content ofresveratrol in mature berries; Bioinformatics analysis and the study of transformation wereperformed in this study. The main results are described as followings.1．STS sequences covered all coding region and3’ UTR were cloned from E. necatorinoculated-leaves of Chinese wild V. pseudoreticulata accession ‘Baihe-35-1’ by RACEamplification. RT-PCR products were analyzed by gel electrophoresis, cloned and more than800individual clones were sequenced. Sequence analysis identified that ‘Baihe-35-1’ have53single STS genes, four of which were pseudogene which do not encode stilbene synthasecorrectly. GenBank Accession numbers of STS from V. pseudoreticulata were from JQ886584to JQ886636. STS sequences from V. vinifera and V. pseudoreticulata sequences werehomologies, which the highest level can reach98%, but most of them cannot be grouped in asingle cluster.2．VqDSTS1, was isolated by reverse transcription-polymerase chain reaction （RT-PCR）from Chinese wild Vitis quinquangularis accession ’Danfeng-2’, and then analysed thesequence and expression model. The results suggested the full length cDNA sequence ofVqDSTS1was1179bp, and the accession number of VqDSTS1was JQ342086, and encoded392amino acid residues. Detailed analysis of the amino acid sequence revealed thatVqDSTS1contains "IPNSAGAIAGN" and "GVLFGFGPGLT" which can be found in allstilbene synthase gene family. With alignment of amino acid sequences, VqDSTS1shared highly identity between95.2%⁹8.7%with the stilbene synthase from other Vitis. From theanalysis of semi-quantitative, it was found that the expression model of VqDSTS1hadtwin-peak model. This work would make a foundation to investigate the function of stilbenesynthase gene family from Chinese wild Vitis quinquangularis accession ’Danfeng-2’.3．During the construct process of the system of somatic embryos from Vitis vinifera, itwas found that the best induction medium was NN69+CH0.5g·L^-1+PVP1g·L^-1＋2,4-D0.5mg·L^-1+BAP4.0mg·L^-1from anthers of ‘Red Globe’; for the overies of ‘Red Globe’,NN69+CH0.5g·L^-1+PVP1g·L^-1＋2,4-D1.0mg·L^-1+BAP4.0mg·L^-1. The embryo callusinduction rate from ‘Cabernet Sauvignon’ was obviously higher than the from ‘ThompsonSeedless’. The induction rate of NN69basic medium was the highest （anthers29.3%, overies32.5%）; B5for the second （anthers4.5%, overies9.1%）; MS the lowest （anthers4.0%, overies6.3%）. The method of cell suspension culture could quickly proliferate the embryo callus cells,and then get the same somatic embryos.4．During the agrobacterium-mediated transformation, the lethal concentration of Hygwas mg·L^-1, efficiently when using this concentration of Hyg in the process of selecting theresistant callus. The tansformation rate of GV3101was obviously higher than LBA4404. Theexpression of report gene GFP was ever-increasing, when the resistant callus was sub-culturedfor several times.更多还原