Studies On Biotransformation Among Different Compounds Of Tropane Alkaloids In Anisodus Actangulus Hairy Roots

Tropane alkaloids (TA) including anisodamine, anisodine, hyoscyamine and scopolamine exclusively exist in Solanaceous plants. They are widely used as anticholinergic agents, which act on the parasympathetic nervous system. They have been widely used for a long time, especially for hyoscyamine and scopolamine. Furthermore, with the development of science and technology, the effects of anisodamine and anisodine such as improving the microcirculation, treating glaucoma except anticholinergic agents have been found gradually. Moreover, anisodamine and anisodine also showed protective effects on acute lung injury induced by oleic acid and microvascular injury of acute renal failure. Most importantly, anisodamine is not as toxic as atropine and has less negative effects on the central nervous system than scopolamine. Therefore, TA all have a significant medical and commercial value.A. acutangulus is a Chinese native medicinal plant with higher content of total alkaloids and it has been considered as an attractive plant resource for TA yield. But the natural amounts of anisodamine, anisodine and scopolamine are not very high in A. acutangulus. Therefore, it is important to improve their production by genetic engineering.The first research project is to improve the yields of tropane alkaloids in Anisodus acutangulus hairy root by bio-engineering technology. The vector pCAMBIA1304~++AaPMT+AaTRI including genes encoding Putrescine N-methyltransferase (PMT) and tropinone reductase I (TRI) involved in tropane alkaloids biosynthesis were simultaneously introduced into Anisodus acutangulus hairy roots using Agrobacterium-mediated gene transfer technology in order to gain transgenic hairy roots. RT-PCR and HPLC analysis were used to determine the expression levels of AaPMT, AaTRI and the contents of tropane alkaloids (anisodamine, anisodine, hyoscyamine and scopolamine), respectively. Transgenic hairy root lines expressing both AaPMT and AaTRI produced higher levels of alkaloids was 8.7-fold higher than control. Especially for scopolamine and anisodine, their contents were 49.8-fold and19.6-fold higher than the amounts in the control line, respectively. Therefore, transgenic hairy root culture system is a promising approach for improvement and large-scale production of TA in the future.The second research project is to detect the effect of AaTRI gene on the yields of tropane alkaloids in Anisodus acutangulus hairy root by bio-engineering technology. The vector pCAMBIA1304~++AaTRI (sense) and the vector pCAMBIA1304~++AaTRI (antisense) were simultaneously introduced into Anisodus acutangulus hairy roots using Agrobacterium-mediated gene transfer technology in order to gain transgenic hairy roots. Transgenic hairy root lines expressing AaTRI (sense) produced higher levels of tropane alkaloids compared with cobtrol. In line T12 (sense), the content of tropane alkaloids was 2.8-fold higher than control. On the contrary, transgenic hairy root lines expressing AaTRI (antisense) produced lower levels of tropane alkaloids compared with cobtrol. In line T21 (antisense), the production just accounted for 11.6% as compared to control. From the result above, we can draw a conclusion that transgenic hairy root lines expressing AaTRI (sense) could help to accumulate the production of tropane alkaloids while the lines expressing AaTRI (antisense) could restrict the accumulation.The third research project is the study on biotransformation among different compounds of tropane alkaloids in anisodus acutangulus hairy roots. After co-cultivation, RT-PCR and HPLC analysis were used to determine the expression levels of genes in the pathway and the contents of tropane alkaloids (anisodamine, anisodine, hyoscyamine and scopolamine), respectively. The results showed that the contents of anisodamine, anisodine and scopolaminee in the extraction of the hairy roots were significantly raised as compared to blank control when the standerds of tropane alkaloids were put into media. Especially when 10μM scopolamine was put into media, the contents of anisodine and scopolamine were 525.87, 523.06μg g DW-1, which was 7.2-fold, 8.1-fold higher than the content of control, respectively. Moreover, through the analysis of enzyme gene expression and the changes of the contents of four compounds of TA after bioconversion, we defined the possible metabolic pathway of anisodine. Therefore, this project not only provided a new method to stimulate A. acutangulus hairy roots to produce more tropane alkaloids except transgenic method, but also defined the possible metabolic pathway between scopolamine and anisodine.