【作者】 彭少兵；

【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】 葡萄作为世界重要经济水果，病害是引起葡萄减产的重要原因，用化学防治的方法增加生产成本，降低葡萄的品质。利用抗病资源培育抗病品种是解决这一问题的有效途径。我国野生葡萄资源对主要葡萄病害有较强的抗性。利用中国野生葡萄资源进行抗病育种具有重要意义。本研究一方面针对中国野生葡萄再生率低的问题，进行了茎尖培养及再生的研究；另一方面，在前期研究的基础上，分析乙二醛氧化酶基因的功能，并利用同源克隆等方法克隆分析了病程相关蛋白基因以及热激转录因子的功能，旨在于更好保护利用中国野生葡萄资源，进一步揭示中国野生葡萄抗病机制，研究取得以下主要研究结果。1、从初代培养、增殖培养、生根培养三个阶段研究了影响华东葡萄株系‘白河-35-1’茎尖组织培养的因素，重点研究了不同盐浓度、不同生长调节物质种类与浓度、糖浓度、不同抗氧化剂及接种材料对组培‘白河-35-1’生根培养的影响。结果表明：较适宜的初代培养基为MS+0.2mg.L^-1IBA+0.5～1mg.L^-1BAP；增殖过程中用MS+1.0mg.L^-1BAP+0.1mg.L^-1NAA取得较的增殖效果；生根培养较好的培养基为：1/2MS+琼脂6g.L^-1+IBA0.2mg.L^-1+活性碳1g.L^-1。2、用‘白河-35-1’的叶片等材料为外植体进行了器官再生的研究，结果表明：用叶片为外植体的不定芽再生率比叶柄和茎段为外植体的再生率高，在接种后10d出现愈伤组织，40d出现再生芽，再生芽多为丛生芽。‘白河-35-1’叶片再生适宜的基本培养基为MS培养基，适宜的分裂素为TDZ，浓度为2.0㎎.L^-1，适宜的生长素为NAA，浓度为0.1㎎.L^-1。经过液体培养的材料，其叶片再生率比一般继代培养的叶片高，再生诱导时先形成为胚性愈伤，进而形成叶状结构，最后得到再生芽。3、用了8种中国野生葡萄10个株系以及2种欧洲葡萄雄蕊和雌蕊进行了胚状途径再生的研究，其中华东葡萄V.pseudoreticulata W.T.Wang的2个株系‘白河-35-2’和‘广西-2’，刺葡萄V. davidii株系‘宁强-6’，及欧洲葡萄Vitis vinifera L2个品种‘雷司令’和‘梅尔诺’获得了胚状体，雄蕊为外植体时胚状体诱导率比雌蕊为外植体高，MSC培养基胚状体诱导率比其他3种培养基效果好，愈伤组织诱导率的高低与胚状体诱导率高低没有直接关系。NN69为基本培养基、用植物凝胶为凝固剂有利于中国野生葡萄胚状体诱导。4、在前期研究的基础之上，进一步分析了乙二醛氧化酶基因（VpGLOX）的功能，用qRT-PCR分析了VpGLOX在抗病株系‘白河-35-1’以及感病株系‘佳利酿’中受白粉病诱导表达的情况，在2种葡萄基因型中VpGLOX的表达均受葡萄白粉病的诱导，在抗病基因型‘白河-35-1’中接种后基因表达量变化量大于感病基因型，表达量比同时期感病型基因高。构建了该基因的表达载体和干扰载体，进行烟草遗传转化，获得了4株过量表达的转基因烟草，转基因烟草中乙二醛氧化酶含量以及H2O2含量增加，转基因烟草对烟草疫霉病的抗性增加。用瞬时转化的方法，对转入的VpGLOX进行干扰处理，干扰处理后的植株乙二醛氧化酶含量下降，但H2O2含量无明显变化。5、采取同源克隆的方法，从‘白河-35-1’叶片cDNA中克隆得到葡萄病程相关蛋白VpPR17（GenBank登录号：JQ248075），并用生物信息学的方法分析了该基因编码氨基酸序列的一些特点。该基因开放阅读框有681个碱基，编码226个氨基酸，等电点（PI）为：6.30，分子量为25.41kDa，有信号肽。VpPR17编码蛋白序列与马铃薯、烟草的病程相关蛋白27相似性分别为70%和71%，亚细胞定位结果表是明：VpPR17在细胞质中表达，有的细胞器中多些。qRT-PCR分析表明，在抗病株系‘白河-35-1’中VpPR17的表达基本不受白粉病诱导而在感病品种‘佳利酿’中白粉病接种后变化很明显。将VpPR17连入原核表达载体pGEX-6T-1，转入E.coli BL21中，经IPTG诱导后重组菌在约50kDa的位置出现一特异条带，说明VpRFP1基因在表达菌E.coli BL21中得到表达，其蛋白粗提液有抑菌活性。通过农杆菌瞬时转化法将VpPR17转入‘白河-35-1’叶片，后接种霜霉菌，经转化的叶片在接种后的3、5、9d的叶片菌丝的发展速度明显少于对照，说明VpPR17与植物抗病相关。6、利用RACE技术从中国野生葡萄华东葡萄cDNA中克隆得到1个葡萄热激转录因子，命名为VpHsf1（GenBank登录号为：GU393313），cDNA全长1428bp，开放阅读框为918bp，VpHsf1编码305个氨基酸，预测蛋白质分子约33.7kD和估算等电点为5.09，同源对比表明：VpHsf1属于HSF B类家族，VpHsf1具有核定位功能。在葡萄中，VpHsf1的表达受病原菌、高温和干旱所诱导。qRT-PCR分析表明，在抗病株系‘白河-35-1’以及感病株系‘佳利酿’中VpHsf1的表达均受葡萄白粉病的诱导，与感病品种不同是，在抗病基因型中，表达量较低。烟草中过量表达VpHsf1降低了烟草的耐热性，而增加了获得耐热性，转基因烟草对烟草黑胫病更为敏感，也降低了烟草抵抗渗透胁迫的能力。总之，VpHsf1与葡萄的生物和非生物胁迫有关。更多还原

【Abstract】 Grape is the most economically important worldwide fruit. Plant disease is the mainreason leads to production reduction. Chemical control methods increase the production costs,and reduce the quality of grapes. Using the disease resistance germplasm resources tocultivate resistant varieties are effective ways to solve this problem. Chinese wild vitis havestrong resistance on the main diseases of grape. It is great significance to take advantage ofChinese Wild grapes for disease-resistant breeding. This research not only aims at the Chinesewild vitis’ low regeneration rate, but also clone and characterizes the disease resistance relatedgenes such as, VpGLOX, VpPR17and VpHsf1. We aimed at better protection and utilization ofChinese wild vitis, further revealing the mechanism of disease resistance of Chinese wild vitis.The novel finding were as follow:1. This research focus on some factors which influence the tissue culture of stem apexof Vitis.pseudoreticulata Baihe-35-1in three stages: initially culture, propagation, rooting,especially the salt concentration, types and concentration of growth regulators, sugar,antioxidants and the material. It show that，the suitable medium as follow: MS+0.2mg.L^-1IBA+0.5～1mg.L^-1BA for initially culture, MS+1.0mg.L^-1BA+0.1mg.L^-1NAA for propagation,1/2MS+Ager6g.L^-1+IBA0.2mg.L^-1+VC1g.L^-1for rooting。2. We study the organogenesis using the leaves and others from Baihe-35-1as explants.It showed that: the regeneration rates from leaves are higher than that from the petioles andstems. The callus appears from10days after inoculation, and shoot appears in40days, mostof the shoot are caespitose shoots. MS was suitable basic medium for regeneration ofBaihe-35-1, TDZ was suitable cytokinins for regeneration of Baihe-35-1, NAA was suitableauxin for it, the appropriate concentration was2.0㎎.L^-1and0.1㎎.L^-1 respectively. Theregeneration rates much higher after the liquid culture, It fromed embryogenic callus at first,then Leaf-like structures, shoot appeared at last.3. We study the somatic embryogensis from10lines of8species of Chinese wild vitisand2Vitis vinifera varieties, And got somatic embryos from two lines ‘Baihe-35-1’ and‘Guanxi-2’ belongs to V.pseudoreticulata W.T.Wang,‘Ningqiang-6’（V. davidii） and ‘Merlot’, ‘White Riesling’（Vitis vinifera L） among them. The embryoid induction rate using thestamens as are higher than the pistil. MSC is better than other3type medium, There is nodirect relationship between the callus induction rate and the somatic embryos induction rate.Itwas suitable for somatic embryogensis from Chinese Wild grapes using the NN69as the basicmedium and Phytagel.4. Based on the preliminary studies, we analysis the function of glyoxal oxidase relatedgenes（VpGLOX）. qRT-PCR are used to analyse the expression of VpGLOX inBaihe-35-1（powdery mildew resistant） and Carignane （susceptible strains）, The expression ofVpGLOX in the two kinds of genotypes are both induced by the grape powdery mildew,Genesexpression after inoculation in Baihe-35-1changes greater than in susceptible genotype,Theexpression vector and interference vector of the gene are constructed,4over expressiontransgenic tobacco are obtained through tobacco genetic transformation, glyoxal oxidase andH2O2in the transgenic tobacco was increased and transgenic tobacco added the resistance toPhytophtora parasitica var. nicotianae Tucker. The VpGLOX in transgenic tobacco wasinterfered with agro-infiltration,, the glyoxal oxidase in the plants after interferencedecreased and but no significant changes with H2O2.5. Grape pathogenesis-related protein VpPR17（GenBank accession no. JQ248075） areobtained from Baihe-35-1leaves cDNA by homology cloning methods, and some of thefeatures of the gene encoding acid sequence are analyzed by bioinformatics methods. Thegene has681bp，encoding226amino acids, isoelectric point （PI）:6.30，molecular weightof25.41kDa，contain signal peptide. The similarity of VpPR17encoded protein sequence andpotato, tobacco pathogenesis-related proteins27are70%and71%, respectively, Thesubcellular localization shows that VpPR17expresses in the cytoplasm and some shows morein the organelles. qRT-PCR analysis shows that expression of VpPR17in Baihe-35-1arenot induced by powdery mildew inoculation,but it changes obviously after powdery mildewinoculation in Carignane.VpPR17are transferred to prokaryotic expression vectorpGEX-6T-1,then into E.coli BL21, recombinant protein in the position of about50kDa afterIPTG induction shows a specific band, it shows that VpPR17gene expressed in the E.coliBL21, the protein crude extracts have antibacterial activity. VpPR17are transferred toBaihe-35-1leaves by agro-infiltration, and then inoculated with downy mildew, the myceliumon the transgenic leaves at3、5、9d after inoculation was significantly less than that ofcontrol, indicating that VpPR17was related to grape disease resistance.6. An heat shock transcription factor（Hsf）, designated as VpHsf1（GenBank accession no.GU393313）, was isolated from Chinese wild Vitis pseudoreticulata for the first time using therapid amplification of cDNA ends （RACE）. Its full-length cDNA is1428bp, encoding an Hsf protein of306amino acids with a calculated molecular mass of33.96kDa. Multiplesequence alignment and phylogenetic analyses showed that VpHsf1was a novel member ofthe Hsf class B2family. Nuclear localization of the protein of VpHsf1was detected in onionepidermal cells. VpHsf1expression was induced by heat and drought，as well as pathogenErysiphe necator. After the infection of E. Necator, VpHsf11was also induced in twograpevine genotypes, and the induction of VpHsf1kept in a low level in E. necator-resistantgrapevine genotypes different from susceptible ones. VpHsf1overexpression in tobaccoreduced the plant’s basal thermotolerance, increased its acquired thermotolerance, andenhanced its susceptibility to osmotic stress and pathogen Phytophtora parasitica var.nicotianae Tucker. Taken together, the results indicated that grapevine VpHsf1is involved inbiotic and abiotic stresses.更多还原