Gambino Giorgio, Gribaudo Ivana, Leopold Stephan, Schartl Angelica, Laimer Margit
Istituto Virologia Vegetale C.N.R.-Unità staccata Viticoltura-Grugliasco, Via Leonardo da Vinci 44, I-10095 Grugliasco (Turin), Italy.
Plant Cell Rep. 2005 Dec;24(11):655-62. doi: 10.1007/s00299-005-0006-4. Epub 2005 Oct 21.
The Grapevine FanLeaf Virus-Coat Protein (GFLV CP) gene was inserted through Agrobacterium-mediated transformation in Vitis vinifera "Nebbiolo", "Lumassina" and "Blaufränkisch". Two plasmids were used: pGA-CP+ (full-length GFLV CP gene with an introduced start codon) and pGA-AS (same gene in antisense orientation). Forty-three transgenic lines were regenerated. As several lines in Southern blots share same hybridization patterns, eight independent line groups resulted for "Nebbiolo", one for "Lumassina", and two for "Blaufränkisch". Inserted T-DNA copies ranged from one to three; one line probably contains an incomplete copy of T-DNA. Except for one "Nebbiolo" line, no evidence for methylation of the transgene at cytosine residues was found by Southern analyses. Specific mRNA was present at variable expression levels; some lines accumulated the coat protein while in others the protein was not detectable by ELISA.
通过农杆菌介导的转化,将葡萄扇叶病毒外壳蛋白(GFLV CP)基因导入酿酒葡萄“内比奥罗”、“卢马西纳”和“蓝佛朗克西”。使用了两种质粒:pGA-CP+(带有引入的起始密码子的全长GFLV CP基因)和pGA-AS(反义方向的相同基因)。再生了43个转基因株系。由于Southern杂交中几个株系具有相同的杂交模式,“内比奥罗”产生了8个独立的株系组,“卢马西纳”产生了1个,“蓝佛朗克西”产生了2个。插入的T-DNA拷贝数从1到3不等;一个株系可能含有不完整的T-DNA拷贝。除了一个“内比奥罗”株系外,Southern分析未发现转基因在胞嘧啶残基处发生甲基化的证据。特定的mRNA以可变的表达水平存在;一些株系积累了外壳蛋白,而在其他株系中通过ELISA检测不到该蛋白。