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Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots.

Kai G, Yang S, Luo X, Zhou W, Fu X, Zhang A, Zhang Y, Xiao J - BMC Biotechnol. (2011)

Bottom Line: 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.All the tested samples were found to possess strong radical scavenging capacity, which were similar to control.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai, Normal University, Shanghai 200234, China. gykai@yahoo.com.cn

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.

Conclusion: 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.

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Schematic biosynthetic pathway of tropane alkaloid in A. acutangulus. From the picture, we know that anisodine comes from the change of scopolamine (result is not present).
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Figure 1: Schematic biosynthetic pathway of tropane alkaloid in A. acutangulus. From the picture, we know that anisodine comes from the change of scopolamine (result is not present).

Mentions: In TA biosynthetic pathway, Putrescine N-methyltransferase (PMT) was considered as the first rate-limiting upstream enzyme while tropinone reductase I (TRI) was an important branch-controlling enzyme (Figure 1). Hence, these two genes are promising target sites for genetic engineering to increase TA in A. acutangulus hairy root cultures. Recently, the cDNAs of Putrescine N-methyltransferase (AaPMT) and tropinone reductase I (AaTRI) of A. acutangulus have been successfully cloned by our laboratory [3,19]. However, there is no report on simultaneous introduction of PMT and TRI into any TA-producing plant including A. acutangulus. In this paper, AaPMT and AaTRI genes were simultaneously introduced into A. acutangulus hairy roots for the first time.


Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots.

Kai G, Yang S, Luo X, Zhou W, Fu X, Zhang A, Zhang Y, Xiao J - BMC Biotechnol. (2011)

Schematic biosynthetic pathway of tropane alkaloid in A. acutangulus. From the picture, we know that anisodine comes from the change of scopolamine (result is not present).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111346&req=5

Figure 1: Schematic biosynthetic pathway of tropane alkaloid in A. acutangulus. From the picture, we know that anisodine comes from the change of scopolamine (result is not present).
Mentions: In TA biosynthetic pathway, Putrescine N-methyltransferase (PMT) was considered as the first rate-limiting upstream enzyme while tropinone reductase I (TRI) was an important branch-controlling enzyme (Figure 1). Hence, these two genes are promising target sites for genetic engineering to increase TA in A. acutangulus hairy root cultures. Recently, the cDNAs of Putrescine N-methyltransferase (AaPMT) and tropinone reductase I (AaTRI) of A. acutangulus have been successfully cloned by our laboratory [3,19]. However, there is no report on simultaneous introduction of PMT and TRI into any TA-producing plant including A. acutangulus. In this paper, AaPMT and AaTRI genes were simultaneously introduced into A. acutangulus hairy roots for the first time.

Bottom Line: 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.All the tested samples were found to possess strong radical scavenging capacity, which were similar to control.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Plant Biotechnology, College of Life and Environment Sciences, Shanghai, Normal University, Shanghai 200234, China. gykai@yahoo.com.cn

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus