| Tropane alkaloids, including hyoscyamine, anisodamine, scopolamine and anisodine, are produced exclusively by several Solanaceae species plants such as Datura and Hyoscyamus. These compounds are widely used as parasympatholytics that competitively antagonize acetylcholine with great commercial demands. Hyoscyamine and scopolamine are two of the most common anticholinergic and antispasmodic agents. Anisodamine and anisodine possess analogical therapeutic properties since first isolated from Anisodus tanguticus.Anisodus acutangulus, a traditional Solanaceous perennial medicinal herb, has a long history of use in China. It has been used as an analgesic medicine in Yunnan Province and classified as an endangered species. The total plant alkaloids reach above 1.0% in wild A. acutangulus, which is much higher than common Solanaceae species as an attractive resource plant species for the production of tropane alkaloids.The effects of different physical and chemical factors such as types of medium, carbon sources, nitrogen sources and PH on hairy root growth and alkaloids production were studied in suspension culture of A. acutangulus hairy root. The results showed that N6 medium was best not only for hairy root growth but also for the accumulation of alkaloids. Sucrose as carbon source was best for growth and alkaloids accumulation in the four carbon sources. The hairy root increment and alkaloids production was highest when the nitrogen concentration was 90mmol/l. PH 6.5 was suitable for hairy root suspension culture.The effects of methyl jasmonate (MeJA), salicylic acid (SA), absolute ethyl alcohol (EtOH) and Ag+ on the growth and tropane alkaloids accumulation of in A.acutangulus hairy roots were studied. The result demonstrated that four kinds of elicitors had a negative effect on the growth of hairy roots, in which MeJA most obvious. The highest enhancement of tropane alkaloids was achieved with EtOH followed by MeJA and SA. EtOH was added showed a drastically increase of alkaloid content, particularly scopolamine was significantly increased compared with the control cultures. SA reduced the amount of alkaloid accumulation in the 24h and beginning to gradually rise in the 48h. However, Ag+ played a limited role in the biosynthesis of alkaloids in A.acutangulus hairy roots.In this experiment the vector pCAMBIA1304~++AaPMT1+AaH6H were constructed. As well, the single gene transformation and co-transformation of A.acutangulus were mediated by Agrobacterium rhizogenes. Analyze gene expression and determination the tropine alkaloids indicated that AaPMT1 gene expression levels of AaPMT1-single transformed hairy roots were higher than control. At the same time, the contents of hyoscyamine and anisodamine in the transgenic hairy roots were higher than which in control. Hyoscyamine and anisodamine of AaH6H-single transformed hairy roots no significant increase comparison with control cultures. As AaH6H gene over-expression, scopolamine and anisodine had a remarkable enhancement. The alkaloid content of AaPMT1+AaH6H transgenic hairy roots had a significant improvement. The maximum concentration of scopolamine increased 70-fold and anisodine increased 26-fold compared with the control cultures. It showed that two key enzyme genes in the metabolic pathway played coordinating role to promote the target production of the metabolic flux.
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