华东葡萄泛素连接酶基因VpUIRP2和VpUIRP3的抗白粉病特性分析  

Characteristic analysis of powdery mildew resistance of ubiquitin ligases VpUIRP2 and VpUIRP3 from Chinese wild Vitis pseudoreticulata

作　　者：王辰[1,2,3] 姚文孔 谢小青[1,2,3] 童威霍 王磊[1,2,3] 王杰 王跃进[1,2,3] 

机构地区：[1]西北农林科技大学园艺学院,陕西杨凌712100 [2]旱区作物逆境生物学国家重点实验室,陕西杨凌712100 [3]农业部西北地区园艺作物生物学与种质创制重点实验室,陕西杨凌712100

出　　处：《果树学报》2016年第12期1477-1491,共15页Journal of Fruit Science

基　　金：国家自然科学基金(No.31171924)

摘　　要：【目的】分析华东葡萄抗白粉病株系‘白河-35-1’的Ring型泛素连接酶基因VpUIRP2和VpUIRP3的转基因植株对葡萄白粉病的抗性,研究这2个基因的抗白粉病特性,为改良欧洲葡萄抗病性提供可用基因。【方法】利用同源克隆的方法获得中国野生华东葡萄‘白河-35-1’泛素连接酶基因VpUIRP2和VpUIRP3的编码区序列(CDS);通过实时荧光定量PCR方法检测其在白粉菌诱导和水杨酸处理后的葡萄叶片中的表达;使用聚乙二醇(PEG)介导法转化拟南芥原生质体进行亚细胞定位;通过农杆菌介导法获得转基因‘无核白’和‘红地球’植株;经过苯胺蓝染色,利用血球计数板计数分析白粉菌在转基因植株叶片上的生长情况,鉴定转基因株系的抗病性。【结果】中国野生华东葡萄‘白河-35-1’泛素连接酶基因VpUIRP2和VpUIRP3响应白粉菌和水杨酸处理,表达模式明显区别于感病对照‘赤霞珠’。中国野生华东葡萄‘白河-35-1’VpUIRP2蛋白定位在细胞质,VpUIRP3蛋白定位无特异性。通过农杆菌介导的遗传转化获得4个VpUIRP2的转基因‘红地球’株系和1个‘无核白’株系,转基因株系对白粉病表现出抗性。【结论】中国野生华东葡萄‘白河-35-1’泛素连接酶基因VpUIRP2能增强葡萄对白粉病的抗性,可以作为抗病基因用于改良欧洲葡萄的抗病性。【Objective】We cloned ubiquitin ligases VpUIRP2 and VpUIRP3 from highly resistant acces-sion of Chinese wild V. pseudoreticulata‘Baihe-35-1＇and studied their expression characteristics in or-der to provide available genes for improving disease-resistance of European grape（Vitis vinifera L.）. Thegenes of VpUIRP2 and VpUIRP3 were introduced into European grape, and the disease resistance of trans-genic plantlets against grapevine powdery mildew（Uncinula necator） was analyzed.【Methods】The patho-gen-related genes were screened in a PM-inoculated c DNA library of‘Baihe-35-1＇. Among the ETSsidentified, two ETSs were predicted to encode a putative RING finger protein, belonging to the E3 ligasefamily. Then homologous cloning was employed to obtain coding sequences of VpUIRP2 and VpUIRP3.The bioinformatics methods were used to analyze conversed domains, chromosome localization informa-tion and deductive protein properties. In order to study genes＇ expression pattern, pathogen inoculationand phytohormone treatment were conducted. Uncinula necator were inoculated on the leaves of‘Baihe-35-1＇and‘Cabernet Sauvignon＇（V. vinifera‘Cabernet Sauvignon＇）by friction extrusion method, whileleaves treated with dd H2 O were used as control. Each sample was randomly collected with three leaves inthe positions of third or fourth knot from the shoot tip at 0, 6, 12, 24, 36, 48, 60 and 72 h post inoculation.Phytohormone treatment was conducted as follows： 1 mmol·L-1salicylic acid（SA） solution was sprayed onthe leaves of‘Baihe-35-1＇and‘Cabernet Sauvignon＇and the samples were collected at 0, 1, 3, 6, 9, 12,24 and 36 h post treatment. Genes＇ responses to Uncinula necator and salicylic acid were identified viaq RT-PCR method. Besides, their subcellular localizations were showed by PEG-mediated transformationof Arabidopsis protoplasts. The coding sequences（CDSs） of VpUIRP2 and VpUIRP3 were cloned intop BI221-GFP to construct the subcellular localization vectors, and subcellular loca

关 键 词：中国野生华东葡萄 泛素连接酶 白粉病 遗传转化 表达分析 

分 类 号：S663.1[农业科学—果树学]