摘要
通过转白藜芦醇合酶（resveratrol synthase，RS）基因提高虎杖毛状根中主要活性成分——白藜芦醇（resveratrol，RE）和白藜芦醇苷（polydatin，PD）的含量。用改进的高盐低pH法提取葡萄基因组DNA，通过PCR扩增获得RS基因序列，构建表达载体pCAMBIA1300—35S—RS，然后采用冻融法转化发根农杆菌ATCC11325，继而浸染划伤的虎杖无菌幼苗叶片，PCR和RT—PCR法鉴定RS基因在虎杖毛状根中的整合与表达，高效液相色谱（highly effective liquid chromatography，HPLC）法测定转基因毛状根中RE及PD的含量。首次成功诱导获得转RS基因虎杖毛状根，经鉴定该基因已在虎杖毛状根中得到整合与表达，RE和PD的含量分别为17～187μg·g^-1DW和836～1970μg·g^-1 DW，而未转基因虎杖毛状根中RE和PD的含量分别为0～130μg·g^-1 DW和190～320μg·g^-1 DW。在所选取的不同根系中，转基因毛状根PD的含量均得到显著提高，最高含量是未转基因毛状根的5倍，而RE含量提高不显著。
To increase the content of active constituent - RE and PD of Polygonum cuspidatum hairy root, through Ri-mediated gene transformation technology, modified high salt low pH method was used to distill genome DNA of grapevine （ Vitis raparia）. Primer was designed according to sequence of Genebank （ AF128861 ）. Through PCR amplification obtain RS gene sequence was obtained. Binary vector pCAMBIA1300-35S-RS was constructed. Frost thawing method was used to transform Agrobacterium rhizogenes ATCCl1325. Scratched aseptic seedling leaf of Polygonum cuspidatum was contaminated subsequently. DNA conformity and mRNA expression of RS gene were investigated by PCR and RT-PCR respectively. RE and PD in transgenic hairy root were determined by HPLC. For the first time successfully inducement acquires transformed RS gene hairy root of Polygonum cuspidatum. Content of active constituents -- RE and PD were 17 - 187μg·g^-1 DW and 836 ～ 1 970 μg·g^-1 DW, respectively, the non-transgenic hairy root was 0 -130μg·g^-1 DW and 190 ～320 μg·g^-1 DW. In the different root selected, the content of PD was much higher than that in non-transformed hairy roots of Polygonum cuspidatum, the highest content is 5 times, but the content of RE has not increased apparently.
出处
《药学学报》
CAS
CSCD
北大核心
2007年第9期995-999,共5页
Acta Pharmaceutica Sinica
基金
河北省自然科学基金资助项目(C2005000741).
