含gfp植物转基因表达载体的构建及在矮牵牛转基因不定根中的高效表达.pdf

HEREDITAS (Beijing) 2008 8 , 30(8): 1069―1074 ISSN 0253-9772 研究报告 DOI: 10.3724/SP.J.1005.2008.01069 gfp 徐纪明, 向太和 , 310036 摘要：利用pBIN19 、pGFP 和pCHS 质粒, 成功构建了CaMV 35S 启动子驱动的gfp 基因的植物转基因表达载 体 pBIN-35S-GFP, 并导入野生型发根农杆菌 K599 。矮牵牛的转化实验表明, 矮牵牛离体叶片被发根农杆菌 K599( 带pBIN-35S-GFP 质粒)感染生根率达45% 。对诱导的不定根基因组DNA 的PCR 检测表明, 不定根基因 组中含有发根农杆菌K599 Ri 质粒中的rolB 基因和外源gfp 基因;转基因不定根在蓝色光激发下能发出强烈的 绿色荧光, 表明构建的转基因载体pBIN-35S-GFP 能实现gfp 基因的高效表达。该载体在 CaMV 35S 启动子的 两端各有一个多克隆位点, 可以方便地进行启动子替换, 用于研究不同启动子的功能。此外, 该载体在gfp 基因 的5′端含有多克隆位点, 在 3′端含有EcoR Ⅰ和Bsm Ⅰ两个单一酶切位点, 可以方便地在 5′端连接上目标基因, 表达含 GFP 的融合蛋白, 进行目标基因编码蛋白的亚细胞定位; 也可以方便地切除gfp 基因, 连上需要的目的 基因进行转化。 ：绿色荧光蛋白基因; 表达载体; 构建; 转基因; 不定根 Construction of a novel vector harboring green fluorescence protein gene (gfp) and high expression of gfp in transformed roots of Petunia hybrida XU Ji-Ming, XIANG Tai-He College of Life and Environment Sciences, Hangzhou Normal University, Hangzhou 310036, China Abstract: A novel vector pBIN-35S-GFP was constructed from the plasmids of pBIN 19, pGFP, and pCHS, which included gfp gene driven by the CaMV 35S promoter. The hairy roots of Petunia hybrida were induced by wild-type Agrobacterium rhizogenes K599 harboring pBIN-35S-GFP with the frequency of 45%. The PCR results showed that rolB from K599 Ri plasmid and gfp from pBIN-35S-GFP were co-transformed into the genome of P. hybrida . The high activity of green fluo- rescence protein was detected by fluorescence microscopy. In particularly, the vector carries multiple cloning sites at both 5′ and 3′ of the CaMV 35S promoter, which allows easy exchange 35S promoter to study other promoter functions. In addition, there are multiple cloni