| Panax ginseng has been used as tonics for several thousand years in China. Previous studies have suggested that the main effective components of ginseng are ginsenosides, which possess many pharmacological activities including anti-cancer, anti-fatigue, anti-aging, and anti-diabetic. Up to now, ginsenosides are mainly produced from cultured ginseng. Ginseng is a perennial plant, sensitive to soil conditions and grows slowly. In our country, the fields for ginseng culture are mainly obtained through cutting forest, while the fields which have been used for ginseng culture can not be reused in 20-30 years, resulting in gradually-reduced fields available for ginseng culture and gradually-decreased economic returns of ginseng culture. Since 1980's, scientists from several countries have tried to produce ginsenosides by biotechnologies including ginseng cell culture and callus culture; however, productivity obtained by these means is low due to low growth rate. Ginseng hairy roots, which are induced by Agrobacterium rhizogenes and characterized as fast hormone-independent growth, genetic and biosynthetc stability, therefore are thought as the most potential alternative for ginsenoside production. However, compared with ginseng cultured in fields, the ginsenoside contents of ginseng hairy roots are relatively low. This is one of the main bottle-necks for industrialization of ginseng hairy roots. It is an available strategy to improve ginsenoside biosynthesis of ginseng hairy roots by metabolic engineering based on ginsenoside biosynthetic pathway.Previous studies have suggested that ginsenosides and phytosterols share the same precursor, 2,3-oxidosqualene. The enzymes cycloartenol synthase (CS) and dammarenediol synthase (DS) are the key enzymes responsible for ginsenoside and phytosterol biosynthesis from 2,3-oxidosqualene, respectively. CS catalyzes the transformation from 2,3-oxidosqualene to cycloartenol, which is further transformed into phytosterols through serial biochemical reactions. Under the catalysis of DS, 2,3-oxidosqualene is transformed into dammarenediol, which provides primary molecular skeleton for ginsenosides biosynthesis.In this study, suppression of phytosterol biosynthesis in P. ginseng hairy roots, which was achieved by antisense-RNA suppression of CS, led to more precursors available for ginsenosides biosynthesis, therefore resulted in enhanced ginsenoside levels. The main contents and conclusions of this paper are as follows.A 901bp CS gene fragment was amplified through RT-PCR with primers designed based on the P. ginseng CS sequence in GeneBank. The 901bp fragment was cloned into a vector pMD18-T, and transformed into Escherichia coli JM109. Then the target fragment was subcloned into a plant expression vector pBI121 containing a selection marker of kanamycin resistance, in the antisense orientation. The pBI121 vector harboring antisense-CS construct was introduced into Agrobacterium rhizogenes A4 by a conventional method. Antisense-CS ginseng hairy roots were obtained by infecting the root discs of 3-year old P. ginseng with A. rhizogenes containing antisense-CS vector. The positive hairy root lines were screened on MS medium with kanamycin. Northern blot analyses were performed with 3 randomly-selected control hairy root lines (hairy root lines with empty pBI121 vector: C-2, C-4, C-7) and 5 antisense-CS hairy root lines (A-16, A-25, A-28, A-33, A-38). The results indicated that the antisense-CS hairy root lines can transcribe abundant antisense CS-RNA, while there were almost no antisense CS-RNA in control lines. Furthermore the sense CS-RNA levels of antisense lines were remarkably lower than those of control lines.According to our observations, all antisense-CS lines and control lines presented the typical traits of hairy roots and had no morphological difference. This suggested that antisense suppression of CS had no effect on morphous of ginseng hairy roots. However, in liquid MS medium, the antisense lines exhibited a slower growth than control lines at early stage; but there was no obvious difference in the final biomass. This implied that antisense-CS manipulation had deleterious effects on hairy root growth during early days of the culture cycle; therefore it is persuasive that phytosterols play an important role in ginseng hairy roots growth during early stage of the culture cycle.Moreover, total phytosterol, 2,3-oxidosqualene and total ginsenoside levels of antisense-CS hairy root lines and control lines cultured in MS liquid medium, were analyzed and compared at appropriate time points of the culture cycle. Total ginsenoside levels in all lines increased rapidly after day 15 and reached a plateau after day 25, suggesting that ginsenosides are primarily synthesized during the later stage of the culture cycle. Before day 15, total ginsenoside levels were comparable in antisense-CS lines and control lines. However, after day 20, except A-33 line, all antisense-CS lines exhibited higher total ginsenoside levels than control lines. It is noteworthy that the total ginsenoside levels were different among antisense-CS lines, of which the A-16 line had the highest total ginsenoside level (about 200% of that of control lines); however there was no obvious difference in total ginsenoside levels of control lines. The total phytosterol levels of antisense-CS lines were lower than those of control lines, implying that phytosterol biosynthesis was suppressed by antisense suppression of CS. In addition, the total phytosterol levels of all lines were relatively stable all through the culture cycle. The 2,3-oxidosqualene levels of control lines were stable during the culture cycle, while the 2,3-oxidosqualene levels of antisense lines were obviously higher at early stage than at the later stage of the culture cycle. The 2,3-oxidosqualene levels of antisense lines were higher than those of control lines at early stage of the culture cycle, but comparable at the later stage.To further dissect the mechanism of the enhanced ginsenoside levels in antisense-CS lines, DS and CS enzyme activities in antisense and control lines were determined at day 20 of the culture cycle. The results indicated that all antisense lines, except A-33, exhibited higher DS enzyme activities than control lines, while the CS enzyme activities of antisense lines were lower than control lines. As DS and CS are the key enzymes for phytosterol and ginsenoside biosynthesis, respectively; the decrease of CS enzyme activity and increase of DS enzyme activity in antisense lines could primarily account for the decreased phytosterol levels and enhanced ginsenoside levels in antisense lines. The decreased CS enzyme activities in antisense lines should be caused by antisense suppression of CS; while the increased DS activities in antisense lines were possibly induced by the higher 2,3-oxidosqualene level at early stage during the culture cycle.We can conclude from above analysis that antisense suppression of CS in P. ginseng hairy roots can lead to decreased CS enzyme activity, thus inhibite the biosynthesis of cycloartenol, from which phytosterols derive, and further suppress phytosterol biosynthesis from 2,3-oxidosqualene. As phytosterols and ginsenosides share the same precursor, 2,3-oxidosqualene, suppression of phytosterol synthesis led to more precursor available for ginsenoside biosynthesis, therefore resulted in enhanced ginsenoside levels in antisense-CS P. ginseng hairy roots. Additionally, antisense suppression of CS also led to higher enzyme activity of DS, the key enzyme for ginsenoside biosynthesis. This might be caused by the higher levels of 2,3-oxidosqualene, which is the substrate of DS, during the early stage of the culture cycle of ginseng hairy roots. Antisense suppression of CS had no effects on the morphous of P. ginseng hairy roots, while led to a slow growth rate of P. ginseng hairy roots during early stage of the culture cycle. This study is significant for further investigation of ginsenoside biosynthesis pathway and ginsenoside biosynthesis regulation.
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