农杆菌介导的CyMV-CP基因对石斛遗传转化研究  

Study on Genetic Transformation of CyMV-CP Gene into Dendrobium through Agrobacterium

作　　者：张振华[1,2] 王涛[2] 陈喜蓉[3] 李潞滨[2] 杨凯[4] 朱靖杰[1] 

机构地区：[1]海南大学园艺园林学院,海南海口571100 [2]中国林业科学研究院林业研究所国家林业局林木培育重点实验室,中国北京100091 [3]海南省林业科学研究所,海南海口571100 [4]北京农学院农业应用新技术北京市重点实验室,中国北京102206

出　　处：《热带作物学报》2011年第3期463-467,共5页Chinese Journal of Tropical Crops

基　　金：海南省重大科技研发专项(080102)

摘　　要：以本实验室构建的含有建兰花叶病毒外壳蛋白基因(CyMV-CP)的pBI121为表达载体,以继代培养2～3次的石斛类原球茎为转化受体,用农杆菌介导法转化石斛,建立并优化了石斛的遗传转化体系。结果表明:类原球茎经农杆菌菌液(OD600值为1.0)侵染30 min后,24℃培养4 d,用头孢霉素(500 mg/L)脱菌后转接到卡那霉素(150 mg/L)筛选培养基中进行筛选培养;对获得的转CyMV-CP石斛进行PCR和实时荧光定量RT-PCR检测,表明目的基因已经整合到石斛基因组中,并得到表达。所建立的转化体系,受体材料来源丰富、转化过程简单,可重复性强、转化效率高、假阳性少,适合于石斛的遗传转化研究。The genetic transformation system using the vector pBI 121 containing CyMV-CP gene as the expression vector,the subcultured PLBs for 2～3 times as the recipient materials was constructed and optimized through the agrobacterium-mediated gene transformation method to transform target genes into Dendrobium.Specifically,Agrobacterium tumefaciens suspension（OD600=1.0）was infected 30 min,followed by co-cultured at 24 ℃ for 4 d.The successfully infected PLBs using the screening medium which contained 150 mg/L Kana and 500 mg/L Cef were determined.PCR detection and real time quantity RT-PCR analysis demonstrated that the target gene was successfully integrated into the genome of Dendrobium.The transformation system with many advantages,such as recipent materials readily available,the transformation process simple and repeatable,in addition to high transformation efficiency and less false positive was suitable for genetic transformation research of Dendrobium.

关 键 词：石斛 CyMV-CP基因 农杆菌 转化 

分 类 号：S682.31[农业科学—观赏园艺]