中国野生毛葡萄芪合酶基因VqSTS11和VqSTS23调控抗白粉病的研究

Research on Control of Disease Resistance by Stilbene Synthase Genes Derived from Chinese wild Vitis quinquangularis

利用同源克隆法得到抗白粉病的中国野生毛葡萄‘丹凤–2’芪合酶基因VqSTS11和VqSTS23的开放阅读框,并构建过表达载体;通过器官发生途径诱导不抗白粉病的欧洲葡萄‘无核白’分生愈伤组织并采用农杆菌介导法对其进行转化;经过PCR检测和Westernblot鉴定,获得了5株VqSTS11超量表达植株和3株VqSTS23超量表达植株;人工接种白粉菌发现,转基因植株中白粉菌菌丝生长速度慢,孢子萌发受到一定的抑制,并且转基因植株中芪合酶基因相对表达量升高,抗病相关基因表达上调,芪类物质含量积累增多。本研究结果进一步证明中国野生毛葡萄‘丹凤–2’芪合酶基因VqSTS11和VqSTS23对白粉菌具有抗性,‘丹凤–2’可以为改良欧洲葡萄品种抗病性提供抗病基因,作为抗病育种的资源。

Two complete open reading frames of VqSTS11 and VqSTS23,were respectively isolated from Vitis quinquangularis’Danfeng-2’using homologous cloning technique and further for overexpression vector construction.Meristematic bulk(MB)of cultivated Vitis vinifera’Thompson Seedless’,were induced through organogenesis and further for Agrobacterium-mediated transformation to obtain transgenic plants.After PCR detection and Western blot identification,5 transgenic lines of VqSTS11 and 3 transgenic lines of VqSTS23 were verified,respectively.Transgenic plants and non-transgenic plants were artificially inoculated with powdery mildew.Microscopic observation demonstrated that the hyphae growth in transgenic lines were slower than those in non-transgenic plants and spore germination was inhibited in transgenic plants.The disease resistance of transgenic plants was further analyzed by qRT-PCR and HPLC.The results displayed that not only the expression of STSs in transgenic plants increased,and the expression of disease resistance-related genes was up-regulated,but also stilbene compounds in the transgenic plants were accumulated.The expression product of’Danfeng-2’VqSTS11 and VqSTS23 could tolerate powdery mildew.Therefore,’Danfeng-2’could be used to improve the disease tolerance and provide disease-tolerant candidate genes for European grape cultivars,which could be served as a breeding resources for disease-tolerant breeding.