Metabolic Engineering Of Tropane Alkaloids In Anisodus Acutangulus

A few genera of the plant family Solanaceae including Anisodus, Atropa, Datura, Duboisia, Hyoscyamus and Scopolia are able to produce biologically active tropane alkaloids simultaneously. Tropane alkaloids have been established drugs for medical treatment since many years before. The tropane alkaloids hyoscyamine and scopolamine are used medicinally as anticholinergic agents that act on the parasympathetic nerve system. So they are used in medicine to treat spasms, to sedate patients and for dilatation (mydriasis) of the pupil.Anisodus acutangulus is a Solanaceous perennial plant that is endemic to China and classified as an endangered species in China. As it contains tropane alkaloids, it has been used as common folk herbal anaesthetic medicine in Yunnan Province for several hundred of years. It is reported that total plant alkaloids reached above 1.2% dw in wild A. acutangulus, which is much higher than the common Solanaceae species such as Atropa belladonna, Datura stramonium and Hyoscyamus niger, so it has been an attractive resource plant specie of production of tropane alkaloids in China.Here we report for the first time the cloning and characterization of two full-length cDNAs encoding AatrI (GenBank Accession No. EU424321) and AatrII (GenBank Accession No. EU424322) from Solanaceous plant A. acutangulus by rapid amplification of cDNA ends (RACE). The cloned full-length cDNA of AatrI (GenBank Accession No. EU424321) was 1188 bp, which contained a 822bp ORF encoding a deduced protein of 273 amino acid residues. The cloned full-length cDNA of AatrII (GenBank Accession No. EU424322) was 1116 bp, which contained a 783bp ORF encoding a deduced protein of 260 amino acid residues. Sequence comparison results indicated AatrI and AatrII had high homology with other tropinone reductases from H. niger, D. stramonium etc, but AatrI and AatrII only showed identity of 60.8%. Phylogenetic tree analysis results showed that AatrI and AatrII belonged to different clusters and have the closest relationship with TRI and TRII from H. niger, respectively. Expression pattern analysis founded that AatrI and AatrII can express in all the tested tissues including root, stem and leaf, but transcript level of AatrI was much lower than AatrII. Expression of AatrI and AatrII could be enhanced by methyl jasmonate, with weak effect for AatrI and strong effect for AatrII.The vector pCAMBIA1304+ contained the single gene AatrI or two genes AatrI &Aah6h were constructed. Disarmed Agrobacterium tumefaciens strain C58C1 was used for plant transformation. AatrI-transformed hairy root lines was accompanied by 1.87-fold of hyoscyamine and 8-fold scopolamine compared with control roots. The AatrI&Aah6h covalently transgenic hairy roots was accompanied by 1.81-fold of hyoscyamine and 19-fold scopolamine compared with control roots. All these facts indicated AatrI and Aah6h are promising target sites of genetic engineering to increase the tropane alkaloids in A. acutangulus.