摘要
将改良的绿色荧光蛋白（ＧＦＰ）基因，插入到植物表达载体中，构建双ＣａＭＶ３５Ｓ启动子驱动下的植物表达载体ｐＢＩＧＦＰ，在Ｋａｎ浓度为２０ｍｇ／Ｌ的筛选培养基上，用含有ｐＢＩＧＦＰ质粒的根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ（ＳｍｉｔｈｅｔＴｏｗｎｓｅｎｄ）Ｃｏｎｎ）ＬＢＡ４４０４感染青蒿（ＡｒｔｅｍｉｓｉａａｎｎｕａＬ．）叶片，获得５个抗Ｋａｎ阳性丛生芽系。Ｓｏｕｔｈｅｒｎｂｌｏｔｔｉｎｇ分析表明，外源ＧＦＰ基因已整合到青蒿转基因芽Ｇ１系的基因组中。在ＯＬＹＭＰＵＳ＿ＢＨ２型荧光显微镜下，观察到转基因芽中有较强的绿色荧光。表明绿色荧光蛋白基因在青蒿转基因芽中业已表达。从而建立以绿色荧光蛋白基因为报告基因的、根癌农杆菌介导的青蒿转基因系统。
A plant expression vector pBIGFP containing a modified green fluorescent protein (GFP) gene was constructed, in which the GFP gene was placed under double CaMV 35S promoter. Leaf explants were infected with Agrobacterium tumefaciens (Smith et Townsend) Conn LBA4404 containing GFP expression vector to induce shoot clusters on selected medium. A concentration of 20 mg/L Kan was used to select Kan resistant shoot clusters. Five Kan resistant shoot cluster lines were established. Southern blot analysis showed that the foreign gene had been intergrated into the genome of Artemisia annua. Bright green fluorescence was observed when transgenic shoots were excited with blue light in fluorescent microscopy (OLYMPUS_BH2 model). The results suggested that an efficient transgenic system of Artemisia annua via Agrobacterium tumefaciens mediated transformation was established using GFP gene as the report gene, and the GFP gene was expressed in Artemisia annua.
基金
国家"九.五"科技攻关
国家自然科学基金
关键词
青蒿
绿色荧光蛋白
转基因丛生芽
Artemisia annua , Green fluorescent protein, Transgenic shoot clusters
