USDA-ARS Appalachian Fruit Research Station, 45 Wiltshire Rd., 25430, Kearneysville, WV, USA.
Plant Cell Rep. 1994 Nov;14(1):18-22. doi: 10.1007/BF00233291.
Plum hypocotyl slices were transformed with the coat protein (CP) gene of plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPVCP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and β-glucuronidase genes. Integration and expression of the transferred genes into regenerated plum plants was verified through kan resistance, GUS assays, and PCR amplification of the PPV-CP gene. Twenty-two transgenic clones were identified from approximately 1800 hypocotyl slices. DNA, mRNA, and protein analyses of five transgenic plants confirmed the integration of the engineered CP gene, the accumulation of CP mRNA and of PPV-CP-immunoreactive protein. CP mRNA levels ranged from high to undetectable levels, apparently correlated with gene structure, as indicated by DNA blot analysis. Western analysis showed that transgenic plants produced amounts of CP which generally correlated with amounts of detected mRNA.
李胚轴切片通过与含有质粒 pGA482GG/PPVCP-33 的根癌农杆菌共培养,转化李痘病毒(PPV-CP)的外壳蛋白(CP)基因。该双元载体携带 PPV-CP 基因构建体,以及嵌合新霉素磷酸转移酶和β-葡萄糖醛酸酶基因。通过卡那抗性、GUS 分析和 PPV-CP 基因的 PCR 扩增,验证了转移基因在再生李植株中的整合和表达。从大约 1800 个胚轴切片中鉴定出 22 个转基因克隆。对五株转基因植物的 DNA、mRNA 和蛋白质分析证实了工程 CP 基因的整合、CP mRNA 和 PPV-CP 免疫反应性蛋白的积累。CP mRNA 水平从高到检测不到的水平不等,显然与基因结构相关,如 DNA 印迹分析所示。Western 分析表明,转基因植物产生的 CP 量通常与检测到的 mRNA 量相关。