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

Construction of a novel vector harboring green fluorescence protein gene (gfp) and high expression of gfp in transformed roots of Petunia hybrida

利用pBIN19、pGFP和pCHS质粒,成功构建了CaMV35S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K599Ri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CaMV35S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EcoRⅠ和BsmⅠ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。

A novel vector pBIN-35S-GFP was constructed from the plasmids of pBIN19, 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 cloning sites at 5＇ end and one-sites of EcoR I and Bsm I sites at 3＇ end of gfp. Therefore, it supports to fusion target genes to expression fusion protein and can be replaced with any other genes of interest for genetic transformation.