| Panax notoginseng saponins (PNS), which belongs to triterpenoid saponins of dammarane type, are the main active components of Chinese traditional valuable medicinal herb. P. notoginseng. Studies have shown that PNS have good physiological activities in central nervous system, cardio-cerebrovascular system, blood system, immune system, anti-fibrosis, anti-aging, anti-tumor, etc. As making deep studies of biological activities for PNS and part monomer saponins, P. notoginseng has been used more and more widely, and market demand was also expanded. Because of the harsh growth environment, plant diseases and pests, crop rotation needing, low land-capability and difficult to popularize large cultivation of P. notoginseng, the gap between supply and demand has been broadened significantly. In recent years, gene regulation of plant secondary metabolism engineering has become the hot and difficult research of molecular biology. Saponins are known as one of the important components in plant secondary metabolism, which yield and composition were determined by the key enzymes of biosynthesis. With the deep study of saponins biosynthetic pathway, the control of PNS biosynthesis through metabolic engineering has been gradually become possible. The technology of molecular biology was used in this project to research the roles of SS, DS in P. notoginseng saponins biosynthetic pathway, provide a theoretical basis and technical basis for the future of PNS homologous or heterologous efficient expression system.First, the callus induction and the cell proliferation of P. notoginseng were preliminary studied; and the stable callus cell lines were established as experimental materials for the later study. The results indicated that the highest inductivity of cotyledons callus was35%at25℃in dark when2.0mg/L2,4-D and1.0mg/L KT were added into MS basic medium, and moreover, under such conditions the callus grew the fastest.SS and DS full-length cDN A were cloned from the leaves of P. notoginseng, and the amino acid sequences of SS, DS for homology analysis were carried out. The alignment results revealed that the similarities of the amino acid sequences of P. notogensing SS compared with those of P. quinquefolius, P. ginseng, Centella asiatica, and Artemisia carvifolia are99%,98%,90% and81%. The homology of DS amino acid sequences between P. notogensing and P. ginseng was99%, and such homology between P. notogensing and Centella asiatica was74%. The constitutive plant expression vectors pCAMBIA2300S-SS and pCAMBIA2300S-DS were constructed by DNA recombination technique, and transferred into Agrobacterium (LBA4404and EHA105) by freeze-thawed method. Agrobactrium mediated transformation of P. notoginseng cotyledons callus was carried out. Eighteen transgenic cell lines were screened by PCR of genomic DNA, which were numbered by TE1, TE2, TE3, TE6, TE7, TE8, TE9, TE10, TE18, TE21, TE24, TE26, TL1,TL2, TL3, TL5, TL6and TL7, respectively. Nine cell lines growing fast were chose to study SS expression. The results of real-time PCR show that SS expression level had increased in eight genetically modified cell lines, and SS mRNA expression level in TE10was up to3.25-fold higher than that of wild-type cell line.In order to define the role of SS in P. notoginseng saponins biosynthetic pathway, we selected four genetically modified cell lines which grew more quickly and detected their PNS and monomer saponins contents. PNS and monomer saponins contents of TE1, TE8and TE10were higher than the control, but those contents of TE9were lower than the control. The highest monomer saponins content was TE10, which was about2.59times higher than the control. The highest PNS content was TE8, which was about1.27times higher than the control. the results of experiment agree with expect results. These results preliminarily showed that raising the transcription level of SS is conducive to accumulate saponins, which indicated that SS plays an important role in saponins biosynthetic pathway. |
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