Co-expression of AaPMT and AaTRI effectively enhances the yields of tropane alkaloids in Anisodus acutangulus hairy roots
© Kai et al; licensee BioMed Central Ltd. 2011
Received: 16 October 2010
Accepted: 28 April 2011
Published: 28 April 2011
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.
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.
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.
Tropane alkaloids (TA) including anisodamine, anisodine, hyoscyamine and scopolamine are widely used as anticholinergic agents, which act on the parasympathetic nervous system and exclusively exist in Solanaceous plants such as Anisodus, Atropa, Datura, Duboisia, Hyoscyamus, and Scopolia [1–3]. Specially, anisodamine is not as toxic as atropine and has fewer negative effects on the central nervous system than scopolamine . Furthermore, anisodamine and anisodine also showed protective effects on acute lung injury induced by oleic acid and microvascular injury of acute renal failure in rats [5, 6].
The hairy root culture system offered many advantages such as high genetic stability, rapid and hormone-free growth, which was considered as a promising way to produce bioactive components from the medicinal plants [7, 8]. It has been proven that the application of small scale jar fermenters for culturing hairy roots induced from several Solanaceous plants is a very prospective method for production of TA [9–12]. Now TA biosynthetic pathway and key enzyme gene identification have been made clear, so it is possible to enhance TA production in the hairy root culture by biotechnology methods [13–15].
A. acutangulus is a Chinese native medicinal plant with higher content of total alkaloids and it has been considered as an attractive plant resource for TA yield [13, 16]. But the natural amounts of anisodamine, anisodine and scopolamine are not very high in A. acutangulus. However, these alkaloids are all important in phyto-medicine with rapid increasing market demand. Thus, it is essential to improve their yields by biotechnological approaches. [17, 18].
Identification of transformed root lines through PCR analysis
Positive clones of transgenic hairy roots.
Number of root lines
Established root lines
P1 P2 P3 P5 P6 P7 P9 P12 P16
T3 T4 T5 T6 T12
PT2 PT3 PT4 PT5 PT6 PT7 PT10 PT12 PT13 PT15 PT17 PT18 PT19 PT20 PT21 PT22 PT24 PT25 PT26 PT27 PT28
Morphological characterization of the transgenic hairy roots
Analysis of the transcript level of AaPMT and AaTRI
Total RNA was isolated from the positive root lines and RT-PCR, real-time fluorescence quantitative analysis were used to determine the expression levels of AaPMT and AaTRI. As shown in Figure 2B and 2C, the different expression levels illustrated that total AaPMT gene (AaPMT T) and total AaTRI gene (AaTRI T) transcripts in transgenic PT, P lines and T lines exhibited higher expression than the native AaPMT gene (AaPMT E) and native AaTRI gene (AaTRI E) transcripts. Moreover, the expression patterns of AaPMT E and AaTRI E in the above transgenic lines showed similar levels with AaPMT T and AaTRI T in BC lines. On the contrary, the AaPMT T and AaTRI T transcripts in the P, T and PT lines had higher transcript levels, which demonstrated that all the expression cassettes of the cDNAs encoding AaPMT and/or AaTRI were introduced into A. acutangulus and expressed at varying degrees in corresponding transgenic lines.
Two independent transgenic lines with AaPMT and AaTRI genes, two lines with AaPMT gene and two lines with AaTRI gene were chosen (Figure 2C). The average expression levels of AaPMT and AaTRI gene in PT6 and PT18 were 13.95-fold and 29.63-fold higher when compared with control, respectively. In P3 and P9, the mean expression level of AaPMT gene was 10.93-fold higher as compared to control. In T4 and T12, the expression levels of AaTRI gene on average were 17.59-fold higher when compared with non-transgenic clones.
TA profile in transgenic hairy roots
The average productions of transgenic lines.
0.114 ± 0.001
1.300 ± 0.006
0.094 ± 0.002
0.0503 ± 0.001
1.558 ± 0.036
0.141 ± 0.010
1.376 ± 0.03
0.104 ± 0.004
0.0611 ± 0.011
1.682 ± 0.050
0.193 ± 0.01
1.914 ± 0.021
0.167 ± 0.005
0.120 ± 0.003
2.395 ± 0.016
0.0738 ± 0.0005
0.829 ± 0.047
0.0218 ± 0.0003
0.0106 ± 0.0005
0.935 ± 0.051
The difference of average yields of TA in P, T, PT, and BC lines was showed in Table 2. It illustrated that the content of TA in PT lines (2.395 mg/g dw) was the highest, when compared with T lines (1.682 mg/g dw), P lines (1.558 mg/g dw) and BC lines (0.935 mg/g dw). The average content of hyoscyamine in PT lines (1.914 mg/g dw) was much higher than that of BC lines (0.829 mg/g dw), but there is no remarkable difference between T (1.376 mg/g dw) and P lines (1.300 mg/g dw). The average content of anisodine in PT lines (0.167 mg/g dw) showed further higher than that of BC lines (0.0218 mg/g dw). The average content of anisodine was almost the same between T (0.104 mg/g dw) and P lines (0.0937 mg/g dw). To scopolamine, its average content in PT lines was 0.12 mg/g dw, which also showed much higher than the content in BC lines (0.0106 mg/g dw). It was improved by inconceivable 11.32 times compared to the BC lines.
Antioxidant activity analysis of transgenic hairy roots
TA production in A. acutangulus hairy root cultures was indeed enhanced by biotechnology approach. However, in present study, considerable variation in morphology between different lines was observed, which was similar to the result reported by Jouhikainen et al . The change of external conditions such as the nuances of ingredient in different 1/2 MS liquid nutrient media, the nuance of temperature in the gyratory shaker and the diversification of internal conditions may lead to the different root morphologies. . However, in our experiment, the yields of TA in abnormal hairy roots were higher than those of normal ones. But they would be broken down when inoculated in liquid medium for some time and they were not routinely subcultured. The results implied that root morphology could lead to the content change of secondary metabolite production .
The expression levels of the AaPMT T varied from line to line but correlated with their corresponding content of TA in P lines. The average content of TA in P lines showed higher than that of BC lines. Moreover, the contents of anisodamine, anisodine, hyoscyamine and scopolamine were all improved in contrast to those in BC lines, respectively. These results showed that AaPMT gene in P lines has a positive influence on the flow of metabolites through TA biosynthesis pathway in A. acutangulus, which suggested that AaPMT gene is a new promising target site for metabolic regulation of tropane-alkaloid pathway in A. acutangulus . However, overexpression of PMT gene in A. belladonna and H. niger hairy root cultures have no significant promotion on accumulation of TA [1, 22], which may be due to species-related or different specific post-translational regulation of the native enzyme in respect to the foreign one . Sometimes, genetic manipulation of single enzymes to increase flux through the pathway may display various and even paradox results between different species .
The yield of TA in T lines was higher than the control and even P lines, which accounted for that AaTRI gene is a more effective regulatory target than AaPMT gene for improving metabolic flux in TA biosynthetic pathway. The average content of TA in T lines (1.682 mg/g dw) were higher than that in P lines (1.558 mg/g dw) or BC lines (0.935 mg/g dw). So strong expression of AaTRI T in A. acutangulus hairy roots could up-regulate content of TA . As shown in Figure 1, two tropinone reductases (TRI and TRII) formed the branching point of tropane alkaloid biosynthesis. High expression of TRII gene would lead to increasing accumulation of calystegines in the roots; while strong expression of TRI gene was accompanied with increased level of TA and decreased level of calystegine . In a word, high TRI activity in T lines has positive effect on the conversion from pseudotropine to tropine, which would provide more key precursor for synthesis of TA.
Overexpression of single gene encoding a key enzyme may increase flux through the pathway, but its effect may be limited by other rate-committee step to some degree. Therefore, regulation of two or multiple genes would be more suitable to achieve significant gains in product accumulation. This may especially be true in branched pathways in which precursors can be channeled into a variety of metabolites away from the desired product . In our work, line PT18 was the most successful one for production of TA, which was attributed to co-overexpression of two key genes (AaPMT and AaTRI). These results showed AaPMT and AaTRI make cooperative effect than single gene in the accumulation of TA in PT lines. In the hairy root lines, overexpressing upstream key enzyme and downstream branch-controlling enzyme may act as a push-pull effect in which flux is pushed toward the branch point by AaPMT and then pulled toward the desired product by AaTRI (Figure 1).
The DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging activity, based on the reduction of the stable radical DPPH to yellow-colored diphenylpicrylhydrazine in the presence of a hydrogen donor, was widely used to evaluate the antioxidant activity due to its advantage of rapid and simple measure . TA has shown to possess the antioxidant activities . The antioxidant activity of TA from hairy roots was estimated by measuring DPPH radical scavenging in this study. All the tested samples were found to possess strong radical scavenging capacity, which suggested that there were no significant differences among them. It indicated that antioxidant activity of aliquots of TA remained stable, so transgenic hairy root lines P3, T12 and PT18 showed higher total antioxidant activity of TA due to higher total TA content they produced.
Hyoscyamine-6-hydroxylase (H6H) catalyzes the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine and is also an effective regulatory site for the synthesis and accumulation of TA [1, 20]. Therefore, in order to enhance the levels of TA and the capacity to convert hyoscyamine to much more scopolamine and anisodine, co-overexpression of multiple biosynthetic genes such as AaPMT, AaTRI and AaH6H in A. acutangulus may be a new promising strategy in the near future. In addition, anisodine is also an imperative tropane alkaloid in the market, which was first produced in hairy root cultures. This result suggested anisodine could be biosynthesized by TA biosynthesis pathway, which provided helpful information for our ensuing research.
In the present study, co-overexpression of AaPMT and AaTRI genes in A. acutangulus hairy roots led to significantly increased production of four kinds of TA and showed higher antioxidant activity than control because of higher total TA content. This is the first report on simultaneous introduction of PMT and TRI genes into TA-producing plant using biotechnological methods. Our study results showed that transgenic hairy root culture system is a promising approach for improvement and large-scale production of TA in the future.
Construction of Plant Expression Vectors
Plant Transformation and Root Cultivation
DNA extraction and PCR analysis
Primer Pairs Employed for the PCR Amplification of TA Biosynthetic Genes AaPMT and AaTRI
Primer Pairs 35S PROM 252F(Forward)and KR ofAaPMT andAaTRI(Reverse)
RNA extraction and gene expression analysis by RT-PCR and real-time fluorescence quantitative analysis
Primer Pairs Employed for the RT-PCR Amplification of Total Biosynthetic Genes AaPMT and AaTRI
Primer Pairs: KF ofAaPMT, AaTRI(Forward) and KR ofAaPMT, AaTRI(Reverse)
Primer Pairs Employed for the RT-PCR Amplification of Native Biosynthetic Genes AaPMT and AaTRI
Primer Pairs KF ofAaPMT, AaTRI (Forward) and 3'UTR ofAaPMT, AaTRI
3-UTR 156R: 5'-ATATCAGTTTATTGCATTATAC-3'
TA production in transgenic A. acutangulushairy roots by HPLC analysis
The extraction of TA was conducted using the method reported previous . After extraction, HPLC analysis was performed on a HITACHI L2000 HPLC system equipped with a photodiode array detector. The separation of TA was carried out on a reversed-phase symmetry column (250 mm × 4.6 mm; 5 μm). The mobile phase consisted of 22% acetonitrile (HPLC grade) and 78% diethylamin buffer (containing 0.7% diethylamine and adjust pH to 7.2 with orthophosphoric acid). The flow rate was 1.0 mL/min and the injection volume was 20 μL. Four TA compounds including anisodamine, hyoscyamine, anisodine and scopolamine were detected and quantified by comparison with authentic standard curves and retention times.
Measurement of DPPH free-radical scavenging activity
The free radical scavenging activity of TA from transgenic hairy roots (P3, T12, PT18 and BC) was measured by the DPPH method reported by Adeolu et al. (2008) with slight modifications . Briefly, the different concentration (0.25-4 μg/mL) of 1 mL of each sample was added to 1 mL DPPH (0.2 mM) solution in methanol. The tubes with reaction mixture were vortexed thoroughly and incubated at room temperature for 30 min. Spectrophotometer was used to detect the absorbance of the mixture at 517 nm. The DPPH solution in methanol (0.2 mM) was used as the control. Radical scavenging activity of TA was calculated according to the following formula: DPPH radical scavenging activity (%) = [1 - absorbance of the sample/absorbance of the control] ×100.
All the experiments including PCR identification, semi-quantitative RT-PCR, HPLC and measurement of antioxidant activity were repeated three times. Analysis was repeated three times. Results of TA content are presented as mean values ± S.D. The statistical significance of TA difference was analyzed by one sample t test using SPSS software (SPSS, Inc.).
This work was supported by Key Science and Technology Brainstorm Project of Yangtze River Delta (10140702018), National Transgenic Organism New Variety Culture Key Project (2009ZX08012-002B), National Natural Science Fund (30900110), Shanghai Science and Technology Committee Project (10JC1412000, 09QH1401900, 06QA14038, 08391911800, 073158202, 075405117, 065458022, 05ZR14093), Shanghai Education Committee Fund (09ZZ138, 06DZ015), Zhejiang Provincial Natural Science Fund (Y2080621), Fujian Science and Technology Committee Key Special Project (2008NZ0001-4), Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50401), Project from Shanghai Normal University (SK200830, CH030). Miss Pin Liu's (Shanghai Jiaotong University, China) kind assistance with HPLC analysis is also acknowledged.
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