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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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TA production analyzed by HPLC. A) TA content in transgenic P lines. B) TA content in transgenic T lines. C) TA content in transgenic PT lines. BC, control hairy root cultures generate from blank-vector transformation. The values are means ± S.D of triplicate analyses. *, **, and *** Significant difference at P < 0.05, 0.01, and 0.001 respectively.
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Figure 4: TA production analyzed by HPLC. A) TA content in transgenic P lines. B) TA content in transgenic T lines. C) TA content in transgenic PT lines. BC, control hairy root cultures generate from blank-vector transformation. The values are means ± S.D of triplicate analyses. *, **, and *** Significant difference at P < 0.05, 0.01, and 0.001 respectively.

Mentions: The contents of TA in A. acutangulus hairy root lines were analyzed by HPLC. Three independent samples extracted from the transgenic lines and the control lines were tested with SPSS software. One sample t test was used to identify the accumulations of TA which showed significant changes in different lines and control. The capacities of transgenic root lines to yield TA were shown in Figure 4. P lines showed higher level of TA content than control (0.935 mg/g dw), which ranged from 1.151 to 2.004 mg/g dw (Figure 4A, 5B and 5E). T lines also produced larger level of TA (1.682 mg/g dw) than the control (Figure 5C and 5E, Table 2). Specially, the yield of TA in line T12 reached to 2.604 mg/g dw (Figure 4B). The average content of TA in PT lines was significantly enhanced, which was much higher than the average content of the control. The levels of TA in PT lines were within the range from 0.263 to 8.104 mg/g dw. 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) (Figure 4C, 5D and 5E). The accumulations of scopolamine and anisodine were obviously improved in PT18 (P < 0.001). The yields of scopolamine and anisodine in PT18 were 49-folds and 19- folds higher than the amounts in the BC line, respectively. Moreover, the content of single alkaloid among all hairy root lines was in accordance with the content of TA (Figure 4A, B and 4C). For example, the content of TA in line PT18 was significantly higher compared with that in the control lines (P < 0.001). So the contents of four alkaloids in line PT18 were also much higher than control. The result above confirmed that biotechnological approach was a high-efficiency way to enhance the production of tropane alkaloids.


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)

TA production analyzed by HPLC. A) TA content in transgenic P lines. B) TA content in transgenic T lines. C) TA content in transgenic PT lines. BC, control hairy root cultures generate from blank-vector transformation. The values are means ± S.D of triplicate analyses. *, **, and *** Significant difference at P < 0.05, 0.01, and 0.001 respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: TA production analyzed by HPLC. A) TA content in transgenic P lines. B) TA content in transgenic T lines. C) TA content in transgenic PT lines. BC, control hairy root cultures generate from blank-vector transformation. The values are means ± S.D of triplicate analyses. *, **, and *** Significant difference at P < 0.05, 0.01, and 0.001 respectively.
Mentions: The contents of TA in A. acutangulus hairy root lines were analyzed by HPLC. Three independent samples extracted from the transgenic lines and the control lines were tested with SPSS software. One sample t test was used to identify the accumulations of TA which showed significant changes in different lines and control. The capacities of transgenic root lines to yield TA were shown in Figure 4. P lines showed higher level of TA content than control (0.935 mg/g dw), which ranged from 1.151 to 2.004 mg/g dw (Figure 4A, 5B and 5E). T lines also produced larger level of TA (1.682 mg/g dw) than the control (Figure 5C and 5E, Table 2). Specially, the yield of TA in line T12 reached to 2.604 mg/g dw (Figure 4B). The average content of TA in PT lines was significantly enhanced, which was much higher than the average content of the control. The levels of TA in PT lines were within the range from 0.263 to 8.104 mg/g dw. 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) (Figure 4C, 5D and 5E). The accumulations of scopolamine and anisodine were obviously improved in PT18 (P < 0.001). The yields of scopolamine and anisodine in PT18 were 49-folds and 19- folds higher than the amounts in the BC line, respectively. Moreover, the content of single alkaloid among all hairy root lines was in accordance with the content of TA (Figure 4A, B and 4C). For example, the content of TA in line PT18 was significantly higher compared with that in the control lines (P < 0.001). So the contents of four alkaloids in line PT18 were also much higher than control. The result above confirmed that biotechnological approach was a high-efficiency way to enhance the production of tropane alkaloids.

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