Molecular cloning and characterization of two tropinone reductases in Anisodus acutangulus and enhancement of tropane alkaloid production in AaTRI-transformed hairy roots

摘要

Tropane alkaloids are used medicinally as anticholinergic agents with increasing market demand, so the improvement and production of active components from medicinal plants using molecular biotechnology show great potential for applications that should benefit human healthcare. Two tropinone reductases constitute a branching point in the biosynthesis of tropane alkaloids. In the present paper, we report for the first time the cloning and characterization of two full-length cDNAs encoding TRI (tropinone reductase I) (GenBank (R) accession number EU424321) and TRII (tropinone reductase II) (GenBank (R) accession number EU424322) from the solanaceous plant Anisodus acutangulus by rapid amplification of cDNA ends. Sequence comparison indicated that AaTRI (A. acutangulus TRI) and AaTRII (A. acutangulus TRII) had high homology with other tropinone reductases from Hyoscyamus niger, Datura stramonium etc., but AaTRI and AaTRII showed identity of only 60.8%. Phylogenetic-tree analysis showed that AaTRI and AaTRII belong to different clusters and have the closest relationship with H. niger TRI and TRII respectively. Expression-pattern analysis showed that AaTRI and AaTRII were expressed in all tissues tested, including root, stem and leaf, but the transcript level of AaTRI was much lower than AaTRII. Expression of AaTRI and AaTRII could be enhanced by methyl jasmonate, with a weak effect for AaTRI and a strong effect for AaTRII. AaTRI-transformed hairy-root lines were accompanied by a mean 1.87-fold higher level of hyoscyamine and a mean 8-fold higher level of scopolamine compared with control roots, indicating that AaTRI is a promising target for genetic engineering to increase tropane alkaloid in A. acutangulus.