洋桔梗的转基因研究  

Study of Transformation Eustoma russellianum

作　　者：陈奇[1] 王阁奇[1] 马莉[1] 陈丽梅[1] 

机构地区：[1]昆明理工大学生物技术研究中心,昆明650224

出　　处：《生物技术通报》2010年第3期109-113,共5页Biotechnology Bulletin

基　　金：国家自然科学基金(30670163);教育部留学回国人员科研启动基金(2005-55);云南省中青年学术与技术带头人培养费(2004PY01-5);昆明理工大学人才培养费

摘　　要：以叶盘为外植体,通过农杆菌菌株C58Cl(pPMP90)介导,开展了用植物表达载体pK2-35S-GFP(含GFP基因)转化洋桔梗(Eustoma russellianum)的研究。洋桔梗叶盘用农杆菌浸染15-20min,在共培养基上与农杆菌共培养2d后,转移到分化培养基上诱导愈伤组织。10d后选取部分叶盘在紫外灯下观察GFP的荧光亮点,结果80%以上的叶盘都有GFP的荧光亮点,说明农杆菌对叶盘的感染效率很高。在分化培养基上红花洋桔梗叶盘不定芽分化频率约为30%,把分化培养基上形成的小苗转移到含有Km(20mg/L)的生根培养基上,进行生根诱导筛选具有Km抗性的转基因植株,统计结果说明红花洋桔梗的转化效率为2.4%。随机选取4株具有Km抗性的洋桔梗植株进行PCR检测,结果均为阳性,证明GFP基因已整合到洋桔梗基因组中,选取经PCR检测为阳性的植株叶片,用激光共聚焦显微镜观察,结果发现有GFP的绿色荧光出现在叶片细胞质中,说明叶片中有GFP蛋白的表达,转化试验成功。Transformation study was conducted in Lisianthus, Eustoma russellianum, with a plant expression vector pK2-35S-GFP （containing GFP gene） by Agrobacterium-mediated method using leaf discs as the explants. The Eustoma russellianum leaf discs were incubated with the Agrobacterium for 15 -20 rain. After co-cultured with the Agrobacterium for 2 days, the leaf discs were transferred to callus-induced medium. Ten days later, a part of the leaf discs were selected and subjected to GFP spot observation under a UV-tamp. The results showed that 80% of the selected leaf discs displayed GFP spots, indicating high infection efficiency of the leaf discs by the Agrobacterium. The efficiency of the adventitious bud induction was 30% on the shoot-induction medium. The shoots were transferred to root induction medium with Km（20 mg/L）for selection of Km-resistance plants. Statistics analysis indicated that the transformation efficiency of Eustoma russellianum was 2.4%. Of the Km-resistance plants, 4 plants was randomly selected for PCR analysis. The positive results inidicated that the GFP gene was integrated into the genome of Eustoma russellianum. The leaves were selected from the plants comfirmed by PCR analysis and subjected to GFP expression observation under a laser scanning confocal microscope. The results showed that GFP green fluorescent appeared in the cytoplasm of leaf cells, indicating the GFP protein expression in the leaves and the success of the transformation experiments.

关 键 词：洋桔梗 农杆菌介导 转基因 GFP基因 

分 类 号：S681.9[农业科学—观赏园艺] Q943.2[农业科学—园艺学]