ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 1, с. 148-152
КРАТКИЕ ^^^^^^^^^^^^^^^^ СООБЩЕНИЯ
УДК 581.1
ABSCISIC ACID ENHANCED AJMALICINE BIOSYNTHESIS IN HAIRY ROOTS OF Rauvolfia verticillata BY UP-REGULATING EXPRESSION
OF THE MEP PATHWAY GENES1 © 2014 K. Chang*, M. Chen**, L. Zeng*, X. Lan***, Q. Wang****, Z. Liao*
*Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), Laboratory of Natural Products and Metabolic Engineering, Chongqing Engineering & Technology Research Center for Sweetpotato, School of Life Sciences, Southwest University, Chongqing, China **School of Pharmaceutical Sciences, Southwest University, Chongqing, China ***Agricultural and Animal Husbandry College, Tibet University, Nyingchi of Tibet, China ****China Rural Technology Development Center, Ministry of Science and Technology, Beijing, China
Received September 27, 2012
Hairy root cultures of Rauvolfia verticillata were established and used to analyze the expression of the eight ajmalicine-pathway genes (the five MEP pathway genes, including DXR, MCT, MECS, HDS, and HDR; the three ajmalicine-pathway-specific genes, including TDC, STR, and SGD) and ajmalicine accumulation in the cultures treated with ABA. In the ABA treatment, the five MEP-pathway genes were simultaneously up-regulated at the transcriptional level, while the three ajmalicine-pathway-specific genes, including TDC, STR, and SGD were not up-regulated. The statistical analysis showed that the MEP pathway gene expression levels in the ABA treatment were significantly different from those in the control. TDC showed no response to ABA treatment, and SGD expression decreased slightly. Surprisingly, the well-known rate-limiting enzyme gene STR showed dramatically decreased expression in the ABA treatment. The HPLC analysis showed that the content of ajmalicine in the ABA-treated hairy roots of R. verticillata (196.9 ± 3.6 ^g/g dry wt) was significantly higher than that in the control (171.0 ± 7.6 ^g/g dry wt). This suggests that the higher expression levels of the MEP pathway genes could enhance ajmalicine biosynthesis and the MEP pathway plays an important role in ajmalicine biosynthesis in R. verticillata. Finally, STR might not be the rate-limiting enzyme involved in ajmalicine biosynthesis of R. verticillata.
Keywords: Rauvolfia — ABA - ajmalicine - gene expression - hairy root - MEP pathway
DOI: 10.7868/S0015330314010047
INTRODUCTION
Rauvolfia verticillata is a rare medicinal shrub belonging to the family Apocynaceae, which is the main source of ajmalicine [1]. Ajmalicine is a pharmacologically important monomeric indole alkaloid possess-
1 This text was submitted by the authors in English.
Abbreviations: DXR - 1-deoxy-D-xylulose-5-phosphate reduc-toisomerase; HDR - 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase; HDS - 1-hydroxy-2-methyl-2-(E)-bute-nyl-4-diphosphate synthase; MCT - 2-C-methyl-D-erythritol-4-phosphatecytidyl transferase; MECS - 2-C-methyl-D-erythri-tol-2,4-cyclodiphosphate synthase; MEP - 2-C-methyl-D-erythritol-4-phosphate; MVA - mevalonate; qPCR - quantitative PCR; SGD -strictosidine P-D-glucosidase; STR - strictosidine synthase; TDC -tryptophan decarboxylase; TIAs - terpenoid indole alkaloids. Corresponding author: Zhihua Liao. Key Laboratory of Eco-Envi-ronments in Three Gorges Reservoir Region (Ministry of Education), Laboratory of Natural Products and Metabolic Engineering, Chongqing Engineering & Technology Research Center for Sweet-potato, School of Life Sciences, Southwest University, Chongqing, 400715 China. Fax: 86-23-6836-7146; e-mail: zhliao@swu.edu.cn
ing therapeutic applications, such as treatment of high blood pressure, neurological function recovery, and so on [2]. Due to the low content of ajmalicine in plants and stable demand for it, it is eager to find alternative methods to produce ajmalicine. Metabolic engineering of the biosynthetic pathways of interest has been successfully applied to produce high-value natural products, such as scopolamine [3], artemisinin [4], and etc. Certainly, metabolic engineering is based on the knowledge of metabolic regulation at the molecular and biochemical levels. Ajmalicine contains two moieties, the terpenoid moiety and the indole moiety. The terpenoid moiety is biosynthesized by the plastid-ial MEP pathway, in which seven functional genes are involved, namely DXS, DXR, MCT, CMK, MECS, HDS, and HDR (fig. 1). Except DXS and CMK, the other five genes have been cloned from R. verticillata [1, 5, 6]. The indole moiety is given by the indole pathway, in which the formation of tryptamine is the key reaction catalyzed by tryptophan decarboxylase
ABSCISIC ACID ENHANCED AJMALICINE BIOSYNTHESIS IN HAIRY
149
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Strictosidine Ajmalicine
Fig. 1. The theoretical biosynthetic pathway of ajmalicine.
(TDC) [2]. The intermediates from the upstream pathways are condensed to produce strictosidine (the common precursor for all terpenoid indole alkaloids (TIAs)) under catalysis of strictosidine synthase (STR) [7]. Ajmalicine is produced from strictosidine after catalysis of strictosidine glucosidase (SGD) [8] and CR (fig. 1).
In the present study, hairy root cultures of R. verti-cillata were established and used to analyze the gene
expression of ajmalicine biosynthesis and the production of ajmalicine.
MATERIALS AND METHODS
The seeds of Rauvolfia verticillata were germinated on MS medium. Agrobacterium tumefaciens strain C58C1 harboring the pRiA4 plasmid was restored in this laboratory. The bacteria-free leaves isolated from
150 CHANG h gp.
The primers used to analyze expression of ajmalicine biosynthesis genes
Gene Forward primer sequence Reverse primer sequence
RvDXR 5' - AGATGCTGTCACTGTTGTTACTGG- 3' 5' - CTCTGGCAAGCCTTGGATACAC - 3'
RvMCT 5'-CGACTTTGACTCCTTCCCATTTC-3' 5'-CCAGCAAGCAGAATTACTGAGAC-3'
RvMECS 5'-CTTCGTTCTACTGCTCCACTTC -3' 5'-AGAGACTGCGACTGCCTTC-3'
RvHDS 5'-TGACCACAACAGACACCAAGGATG-3' 5'-GCAACTCGCAAAGCCACAGAAG-3'
RvHDR 5'-GAGGACGATGATGCGTAAGTATGG-3' 5'-TCTCTTGTAGATGCGAAGTGTTGC- 3'
RvTDC 5' - GAAGGACTGGAGAGGTGGAAG- 3' 5' -AGTCTCACAGTCAAGTCAACAAC - 3'
RvSTR 5'-TCTGTGGTTGGTTCTGAAGG-3' 5'-AGGTGCTAACATCGGTGAAG-3'
RvSGD 5'-ACGAATATGGTGGCTTCTTGAGTC-3' 5'-CAAATAGGGTTCTGTGGCTGGATC-3'
18S 5'-ATGATAACTCGACGGATCGC-3' 5'-CTTGGATGTGGTAGCCGTTT-3'
5-week-old in vitro seedlings of R. verticillata were used as the initial materials for genetic transformation. The explants were cut into small pieces (about 1.5 cm) followed by pre-incubation on MS medium in the dark for 2 days, and then infected with C58C1. After infection with C58C1, the explants were plated on MS medium for co-cultivation in the dark for 2 days. Thereafter, the explants were transferred onto MS medium containing 300 mg/L of cefotaxime to eliminate A. tumefaciens. Control explants were treated similarly except that they were not co-cultivated with Agrobac-terium. The apical tips of hairy roots induced from explants were excised and subcultured on MS medium with 300 mg/L of cefotaxime with 20-day intervals. Rapidly growing root clones with no bacterial contamination were used to establish hairy root lines. About 100 mg of fresh roots (2 cm in length) were inoculated into 100 mL of liquid MS medium in 250-mL conical flasks on an orbiter at 120 rpm in dark at 25°C. 35 days later, the hairy root cultures were harvested for the analysis of gene expression level and ajmalicine content. The genomic PCR analysis was performed to confirm the hairy root lines according to the previous report [9].
The hairy roots from the same clone were homogeneously divided into a great deal of copies and were respectively soaked in MS liquid medium with ABA for 24 h. The final concentration of ABA reached 50 ^M (dissolved in ethanol). Water dissolved in 1% ethanol acted as a control. These flasks containing hairy roots and liquid medium with chemicals were subcultured on a rotary shaker at 120 rpm in dark at 25 °C. After 24 h of culturing, the ABA-treated hairy root cultures were harvested for the future analysis. Total RNAs were isolated using the RNAplant reagent ("Tiangen", China) according to the manufacturer's instructions and stored in -80°C. cDNA was synthesized using AMV reverse transcriptase ("Takara", Japan) according to the manufacturer's instructions. Quantitative PCR of each gene was conducted with two primers specific to the coding sequence of eight known genes, including DXR (DQ779286), MCT (JX104650),
MECS (EU034699), HDS (HQ659759), HDR (EU034699), TDC (HM067439), STR (DQ872163), and SGD (AJ302044) using SYBR Premix ExTaq ("Takara"). Amplifications were performed under the following conditions: 94°C for 2 min, 40 cycles of de-naturation at 94 °C for 15 s, annealing at 55 °C for 15 s, and followed by extension at 68°C for 20 s. The housekeeping gene 18S rRNA was used as the reference gene. Three replicates were conducted. The primers used in the qPCR analysis were listed in the table. HPLC analysis of ajmalicine was performed according to our previous report [2]. Three replicates were performed in the present study.
RESULTS AND DISCUSSION
Hairy root cultures have several attractive advantages: high genetic stability compared to undifferenti-ated cultures, relatively fast growth rates compared to the normal roots, cultivation without plant growth regulators, and intact biosynthetic capacity [10]. These features make hair
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