title: Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots.
abstract: BACKGROUND
  Tropane alkaloids (TA) including anisodamine, anisodine, hyoscyamine and scopolamine are a group of important anticholinergic drugs with rapidly increasing market demand, so it is significant to improve TA production by biotechnological approaches. Putrescine N-methyltransferase (PMT) was considered as the first rate-limiting upstream enzyme while tropinone reductase I (TRI) was an important branch-controlling enzyme involved in TA biosynthesis. However, there is no report on simultaneous introduction of PMT and TRI genes into any TA-producing plant including Anisodus acutangulus (A. acutangulus), which is a Solanaceous perennial plant that is endemic to China and is an attractive resource plant for production of TA.
  
  
  RESULTS
  In this study, 21 AaPMT and AaTRI double gene transformed lines (PT lines), 9 AaPMT single gene transformed lines (P lines) and 5 AaTRI single gene transformed lines (T lines) were generated. RT-PCR and real-time fluorescence quantitative analysis results revealed that total AaPMT (AaPMT T) and total AaTRI (AaTRI T) gene transcripts in transgenic PT, P and T lines showed higher expression levels than native AaPMT (AaPMT E) and AaTRI (AaTRI E) gene transcripts. As compared to the control and single gene transformed lines (P or T lines), PT transgenic hairy root lines produced significantly higher levels of TA. The highest yield of TA was detected as 8.104 mg/g dw in line PT18, which was 8.66, 4.04, and 3.11-times higher than those of the control (0.935 mg/g dw), P3 (highest in P lines, 2.004 mg/g dw) and T12 (highest in T lines, 2.604 mg/g dw), respectively. All the tested samples were found to possess strong radical scavenging capacity, which were similar to control.
  
  
  CONCLUSIONS
  In the present study, the co-expression of AaPMT and AaTRI genes in A. acutangulus hairy roots significantly improved the yields of TA and showed higher antioxidant activity than control because of higher total TA content, which is the first report on simultaneous introduction of PMT and TRI genes into TA-producing plant by biotechnological approaches.
authors.affiliation: Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai, Normal University, Shanghai 200234, China. gykai@yahoo.com.cn
authors.forename: Guoyin
authors.initials: G
authors.lastname: Kai
authors.forename: Sheng
authors.initials: S
authors.lastname: Yang
authors.forename: Xiuqin
authors.initials: X
authors.lastname: Luo
authors.forename: Wentao
authors.initials: W
authors.lastname: Zhou
authors.forename: Xueqing
authors.initials: X
authors.lastname: Fu
authors.forename: Ang
authors.initials: A
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Jianbo
authors.initials: J
authors.lastname: Xiao
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology; D030821:Plants, Genetically Modified / Q000201:enzymology; D016133:Polymerase Chain Reaction; D018749:RNA, Plant / Q000378:metabolism; D019657:Solanaceae / Q000201:enzymology* / Q000235:genetics; D014326:Tropanes / Q000032:analysis / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D018749:RNA, Plant; D014326:Tropanes; D000429:Alcohol Oxidoreductases; C091007:tropine dehydrogenase; D008780:Methyltransferases; C112598:putrescine N-methyltransferase