三七细胞中过表达FPS基因对皂苷合成的影响

Effect of Over-expressing Farnesyl Pyrophosphate Synthase(FPS) Gene of Panax notoginseng Cell on Saponin Synthesis

为研究三七细胞中过表达法呢基焦磷酸合酶(farnesyl pyrophasphate synthase,FPS)基因对皂苷合成的影响,构建了FPS过表达载体,借助农杆菌将FPS整合到三七细胞基因组中,利用q RT-PCR检测阳性细胞系中FPS的相对表达量,香草醛-高氯酸-冰乙酸显色法测定总皂苷含量,HPLC法测定单体皂苷含量。结果显示:四株转FPS阳性细胞系中,FPS的相对表达量高于未转基因对照,最高为对照的3.01倍;总皂苷含量高于未转基因对照,最高为对照的2.09倍;四种重要单体皂苷(Rh1、Rg1、Re和Rd)含量均高于未转基因对照,其中,Re含量的增加幅度最为明显,最高为对照的4.35倍;Rh1、Rd和Rg1含量也有一定幅度增加,最高分别为对照的2.66倍、2.90倍和1.76倍。结果表明:FPS是三七皂苷合成途径中的一个限速酶,在三七细胞中过表达FPS可提高皂苷含量,为进一步利用代谢工程手段实现三七皂苷的同源或异源高效表达奠定基础。

To study the effect of over-expressing farnesyl pyrophosphate synthase(FPS) in Panax notoginseng(P. notoginseng) cells on saponin synthesis, an FPS over-expression vector was constructed and integrated into the genome of P. notoginseng cells via Agrobacterium tumefaciens. Relative expression levels of FPS in the positive cell lines were analyzed by quantitative real-time polymerase chain reaction(q RT-PCR). The contents of Panax notoginseng saponins(PNSs) and monomer ginsenosides were estimated by vanillin-perchloric acid-glacial acetic acid colorimetric method and high performance liquid chromatography(HPLC), respectively. The results showed that the relative FPS expression levels and PNS content were both higher in the four transgenic FPS-positive cell lines, than those in non-transgenic control, with highest levels at 3.01-times and 2.09-times higher, respectively. Additionally, the content of important monomer ginsenosides(Rh1, Rg1, Re, and Rd) were all higher in the four transgenic FPS-positive cell lines than those in the non-transgenic control. Among these, the increase in Re content was the most significant, with highest level was up to 4.35 times as much as that of control. Moreover, the content of Rh1, Rd, and Rg1 were increased to some extent as well, with highest levels at 2.66-, 2.90-, 1.76-times that of the control, respectively. These results suggest that FPS is one of the rate-limiting enzymes in the PNS biosynthetic pathway. Thus, over-expressing FPS in P. notoginseng cells could enhance PNS content, which has potential applications in metabolic engineering to realize homologous or heterologous expression of PNS.