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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 construction of recombinant vectors (Kai et al. 2010). A) pCAMBIA1304+-AaPMT. B) pCAMBIA1304+-AaTRI. C) pCAMBIA1304+-AaPMT -AaTRI. The AaPMT and AaTRI cDNAs are driven by the Cauliflower mosaic virus promoter (35S). The direction of transcription is marked with arrowheads. The restriction enzyme sites BamH I, Sac I, Bgl II and BstE II were signed.
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Figure 7: The construction of recombinant vectors (Kai et al. 2010). A) pCAMBIA1304+-AaPMT. B) pCAMBIA1304+-AaTRI. C) pCAMBIA1304+-AaPMT -AaTRI. The AaPMT and AaTRI cDNAs are driven by the Cauliflower mosaic virus promoter (35S). The direction of transcription is marked with arrowheads. The restriction enzyme sites BamH I, Sac I, Bgl II and BstE II were signed.

Mentions: The vectors pBI121 (Clontech) and pCAMBIA1304 (CAMBIA) were double-digested with HindIII and EcoRI. The purified smaller DNA fragment containing a GUS expression cassette from pBI121 was cloned into the large pCAMBIA1304 fragment to generate the recombinant plasmid pCAMBIA1304+ [19]. The full-length AaPMT cDNA was inserted into pCAMBIA1304+ in place of the mGFP5 and GUSA genes to generate pCAMBIA1304+-AaPMT expression vector containing AaPMT gene under the digestion of BgLII and BstEII (Takara Biotech Co., Ltd) (Figure 7A). Similarly, the pCAMBIA1304+-AaTRI was also constructed in place of the mGFP5 and GUSA genes under the digestion of BgLII and BstEII (Figure 7B). On the basis of pCAMBIA1304+-AaTRI, the full-length AaPMT cDNA was used to replace GUS gene in pCAMBIA1304+-AaTRI under the digestion of SacI and BamHI (Takara Biotech Co., Ltd) to generate the expression vector pCAMBIA1304+-AaPMT-AaTRI containing both AaPMT and AaTRI genes (Figure 7C). The genes AaPMT or/and AaTRI were under the control of strong cauliflower mosaic virus (CaMV) 35S promoter. The blank vector pCAMBIA1304+ without exogenous gene (such as AaPMT or AaTRI) was used as the control.


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 construction of recombinant vectors (Kai et al. 2010). A) pCAMBIA1304+-AaPMT. B) pCAMBIA1304+-AaTRI. C) pCAMBIA1304+-AaPMT -AaTRI. The AaPMT and AaTRI cDNAs are driven by the Cauliflower mosaic virus promoter (35S). The direction of transcription is marked with arrowheads. The restriction enzyme sites BamH I, Sac I, Bgl II and BstE II were signed.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: The construction of recombinant vectors (Kai et al. 2010). A) pCAMBIA1304+-AaPMT. B) pCAMBIA1304+-AaTRI. C) pCAMBIA1304+-AaPMT -AaTRI. The AaPMT and AaTRI cDNAs are driven by the Cauliflower mosaic virus promoter (35S). The direction of transcription is marked with arrowheads. The restriction enzyme sites BamH I, Sac I, Bgl II and BstE II were signed.
Mentions: The vectors pBI121 (Clontech) and pCAMBIA1304 (CAMBIA) were double-digested with HindIII and EcoRI. The purified smaller DNA fragment containing a GUS expression cassette from pBI121 was cloned into the large pCAMBIA1304 fragment to generate the recombinant plasmid pCAMBIA1304+ [19]. The full-length AaPMT cDNA was inserted into pCAMBIA1304+ in place of the mGFP5 and GUSA genes to generate pCAMBIA1304+-AaPMT expression vector containing AaPMT gene under the digestion of BgLII and BstEII (Takara Biotech Co., Ltd) (Figure 7A). Similarly, the pCAMBIA1304+-AaTRI was also constructed in place of the mGFP5 and GUSA genes under the digestion of BgLII and BstEII (Figure 7B). On the basis of pCAMBIA1304+-AaTRI, the full-length AaPMT cDNA was used to replace GUS gene in pCAMBIA1304+-AaTRI under the digestion of SacI and BamHI (Takara Biotech Co., Ltd) to generate the expression vector pCAMBIA1304+-AaPMT-AaTRI containing both AaPMT and AaTRI genes (Figure 7C). The genes AaPMT or/and AaTRI were under the control of strong cauliflower mosaic virus (CaMV) 35S promoter. The blank vector pCAMBIA1304+ without exogenous gene (such as AaPMT or AaTRI) was used as the control.

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