摘要
为研究三七细胞中SS、DS共超表达对皂苷合成的影响,构建了三七SS超表达载体,并以已超表达了DS的愈伤组织为侵染材料,借助农杆菌LBA4404将SS导入并整合到三七基因组中,采用QRT-PCR法检测转基因株系中SS、DS的表达水平,香草醛-冰醋酸-高氯酸显色法检测皂苷含量。结果显示：三株同时转SS、DS细胞系（T-4、T-5、T-9）中,SS的相对表达量分别为仅转DS株系的5.66、7.37、7.46倍;DS的相对表达量分别为仅转DS株系的1.39、2、1.41倍;总皂苷含量为仅转DS株系的1.7倍,普通细胞的2.4倍;5种单体皂苷：R1、Rg1、Re、Rb1、Rd含量总和分别为仅转DS株系的1.33、1.61、1.71倍,普通细胞的5.62、6.81、7.26倍;此外,Re含量出现大幅增加,达到三七药材的5-6倍。表明SS、DS在皂苷生物合成途径中均起正调控作用,双基因共超表达能进一步提高皂苷含量,揭示途径中两个甚至更多基因间可能存在正协同调控效应。
To study the effects of the co-overexpression of the squalene synthase（SS） and dammarenediol-II synthase（DS） genes in Panax notoginseng cells on saponin synthesis, an overexpression vector of SS derived from P. notoginseng was constructed and integrated into the P. notoginseng genome by culturing with Agrobacterium tumefaciens LBA4404. Expression levels of SS and DS in transgenic cell lines were detected using quantitative reverse transcription-polymerase chain reaction（QRT-PCR）, and the saponin contents were determined by the vanillin-glacial acetic acid-perchloric acid colorimetric method. The results showed that for the three strains（T-4, T-5, T-9） in which SS and DS were overexpressed simultaneously, the relative SS expression levels were 5.66, 7.37, and 7.46 times greater than that of the strain with only DS overexpression, and the relative DS expression levels were 1.39, 2.00, and 1.41 times greater than that of the strain overexpressing only DS. The total saponin content of P. notoginseng（PNS） in the three strains was 1.7 times that in the strain overexpressing only DS, and 2.4 times of that observed in normal cells. The total monomer saponin content, including R1, Rg1, Re, Rb1, and Rd, in the three strains was 1.33, 1.61, and 1.71 times greater than that in the strain overexpressing only DS, and 5.62, 6.81, and 7.26 times greater than that in normal cells. In addition, the Re content was significantly higher and reached 5–6 times that observed in P. notoginseng medicinal materials. The results indicated that both SS and DS genes played positive regulatory roles in the saponin biosynthetic pathway, and overexpression of these two genes could further enhance the saponin content, demonstrating that synergistic regulatory effects might exist for two or more genes in the biosynthetic pathway.
出处
《现代食品科技》
EI
CAS
北大核心
2015年第2期7-13,共7页
Modern Food Science and Technology
基金
国家自然科学基金资助(31260070
31060044)
关键词
三七
鲨烯合酶
达玛烯二醇合酶
三七总皂苷
超表达
Panax notoginseng
squalene synthase
dammarenediol-II synthase
total saponin
overexpression
