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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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The explant used for hairy root induction.
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Figure 8: The explant used for hairy root induction.

Mentions: The disarmed A. tumefaciens strain C58C1 harbouring both the A. rhizogenes Ri plasmid pRiA4 [19] and each of the four plasmids constructed above, were used for plant genetic transformation. Different explants including leaf blades, petioles or stems were isolated from 4-week-old in vitro grown sterile seedlings of A. acutangulus (Figure 8). The isolated explants were cut into small pieces (about 1 cm, 20 pieces per dish) followed by pre-incubation on hormone-free MS containing 8.0 g/L agar plates in the dark for 2 days, and then inoculated with C58C1 bacterial strain for 15 minutes. The explants were dried by blotting with sterile filter paper and then placed back on their original culture plates for cocultivation in the dark for 2 days. Then, the explants were rinsed with sterile water, and transferred onto B5 medium supplemented with 500 mg/L cefotaxime sodium to eliminate the residual Agrobacterium, 0.5 mg/L hygromycin to select positive hairy roots. The apical tips of hairy root induced from transformed explants were excised and sub-cultured on B5 solid medium with 500 mg/L cefotaxime at 2-week intervals, the concentration of cefotaxime was gradually lowered and finally omitted after 3 months. When cultures had been cleared of Agrobacterium, hairy roots were transferred onto hormone-free B5 solid medium without cefotaxime [19,28].


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)

The explant used for hairy root induction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: The explant used for hairy root induction.
Mentions: The disarmed A. tumefaciens strain C58C1 harbouring both the A. rhizogenes Ri plasmid pRiA4 [19] and each of the four plasmids constructed above, were used for plant genetic transformation. Different explants including leaf blades, petioles or stems were isolated from 4-week-old in vitro grown sterile seedlings of A. acutangulus (Figure 8). The isolated explants were cut into small pieces (about 1 cm, 20 pieces per dish) followed by pre-incubation on hormone-free MS containing 8.0 g/L agar plates in the dark for 2 days, and then inoculated with C58C1 bacterial strain for 15 minutes. The explants were dried by blotting with sterile filter paper and then placed back on their original culture plates for cocultivation in the dark for 2 days. Then, the explants were rinsed with sterile water, and transferred onto B5 medium supplemented with 500 mg/L cefotaxime sodium to eliminate the residual Agrobacterium, 0.5 mg/L hygromycin to select positive hairy roots. The apical tips of hairy root induced from transformed explants were excised and sub-cultured on B5 solid medium with 500 mg/L cefotaxime at 2-week intervals, the concentration of cefotaxime was gradually lowered and finally omitted after 3 months. When cultures had been cleared of Agrobacterium, hairy roots were transferred onto hormone-free B5 solid medium without cefotaxime [19,28].

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