Artemisinin, a sesquiterpene lactone endoperoxide, is a valuable and powerful antimalarial drug obtained from the aerial parts of a Chinese herb,
Artemisinin is synthesized by the isoprenoid pathway. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway (Fig. 1). In this pathway, farnesyl diphosphate (FDP) occupies a central position and serves as a common substrate for the first committed reactions of sterols and sesquiterpenes, such as artemisinin. Therefore, this point represents a potentially important controlling point for balancing sterol synthesis and sesquiterpenes synthesis. From metabolic engineering point of view (Fig. 1), there are two ways to increase the flux to artemisinin biosynthesis, on the one hand, we can overexpress the key genes involved in the biosynthesis of artemisinin; on the other hand, we can inhibit the genes involved in other pathways competing for its precursors.
Squalene synthase (SQS) catalyzes the condensation of two molecules of farnesyl diphosphate (FDP) to form the linear 30 carbon compound squalene, the first committed precursor for sterol biosynthesis (Goldstein & Brown, 1990). SQS is generally described as a crucial branch point enzyme for synthesizing sterol intriguing as a potential regulatory point that controls carbon flux into either sterol or into non-sterol isoprenoids (such as sesquiterpenes). So if the SQS gene expression is inhibited by genetic manipulation, the carbon flux into sterol may be diverted to sesquiterpenes, and the biosynthesis of sesquiterpenes may be increased. With the purpose to increase artemisinin production, we have cloned squalene synthase cDNA (SQS) (Liu et al., 2003). In this chapter, we report the construction of the antisense SQS plant expression vector, and its effects on inhibition of SQS gene expression on squalene and artemisinin biosynthesis.
2. Materials and methods
2.1. Plant materials
A high artemisinin producing
2.2. Construction of antisense plant gene expression vector
The original plasmid pSQF2 containing squalene synthase cDNA (SQS) gene, cloned from
Agrobacterium preparation and genetic transformation
The binary vector pBISQS was introduced into
Plant transformation was basically performed according to the method of Han et al (2005). The leaves of 2-week-old aseptic seedlings were immersed in 50 mL
2.4. PCR detection
The integration of SQS gene into
2.5. RNA isolation and RT-PCR detection
For reverse transcription-polymerase chain reactions (RT-PCR), 1 g of total RNA isolated from the leaves of the transformed and non-transformed plants was used. The RNA extraction protocol was done as described in Sambrook et al (1989). The first strand cDNA was synthesized by using a first-strand cDNA synthesis kit (TaKaRa), according to the manufacturer’s instructions. The resultant first-strand cDNA was used as a template, and the PCR primers, P1: 5´-GGA ACC ATG GGT AGT TTG AAA GCA GTA TTG-3, and P2: 5´-GCC TGG ATC CCT TGA CTC TCT CTT AAC TAT-3´, were designed according to the SQS gene sequence of
2.6. Squalene analysis
Squalene analysis was carried out according to the method described by Wentzinger et al (2002). For all the plants analyzed, young leaves was collected. Extraction and purification of the samples were in accordance with Wentzinger et al (2002), and the samples were analyzed by GC, the GC injection port was operated at 120°C. The oven temperature programmed from 120°C to 180°C at 15°C min-1 and from 180°C to 260°C at rate of 25°C min-1. The final temperature was maintained for 25 min. The results were compared to standards.
2.7. Determination of artemisinin
The detection of artemisinin was performed according to the method of Zhao & Zeng (1986). Fresh leaves of the transformed and non-transformed plants were collected and dried to constant weight in an oven at 50°C. Then the dried leaves were ground to fine powder. Exactly 0.05 g powder was added to an extraction bottle containing 40 mL petroleum ether (30-60°C) and treated in a supersonic bath for 2 min. The extraction mixture was filtered and the petroleum ether was evaporated. The residue was dissolved in 1 mL methanol and centrifuged at 12000 r/min to precipitate the undissolved components. The supernatant was used for detection of artemisinin by HPLC.
200 μL of the above prepared methanol solution was placed in a 10 mL tube and 800 μL methanol and 4 mL 0.2% sodium hydroxide were added, mixed and maintained in a 50°C water bath for 30 min, then the reaction mixture was cooled to room temperature. 0.5 mL of the reaction mixture was placed in a 1.5 mL Eppendorf tube, and 100 μL methanol and 400μL 0.05 M acetic acid were added, mixed and the sample purified by filtering on a NC filter (40 μm). The artemisinin standard (Sigma, MO) solutions with concentrations of 3, 6, 12, 24 and 48 μg/mL were prepared in the same way as the sample.
C18 reverse column was 4.6×250 mm, 5 μm. The mobile phase was 0.01 M phosphate buffer (pH 7.0) : methanol (55:45), with flow rate 1 mL/min. The wavelength of the UV detector was 288.6 nm and the injection volume was 20 μL. Artemisinin standard appeared at 4 min 30 s under the above mentioned conditions.
3.1. Regeneration of transgenic
Leaf discs, which were infected with
3.2. Molecular analysis of transgenic plants
To investigate the presence of antisense SQS gene in the putatively transformed plants, genomic DNA of 4 Kan-resistant plants regenerated from the leaves inoculated with EHA105 was isolated, and PCR analysis was performed. The antisense SQS gene was detected as 1660-bp fragments in all 4 analyzed plants (Fig. 3). The amplified fragments were of the same size as the predicted one. The fragment in the nontransformed plant was not amplified.
RT-PCR was performed using specifically designed primers according to the squalene synthase cDNA sequence in
3.3. Detection of squalene content
In order to determine the effects of inhibiting squalene synthase gene expression on sterol biosynthesis, the leaves of transgenic lines SQS3, SQS5, and these of the control 001 were selected to detect squalene content. The results of GC-MS showed that in SQS3 and SQS5 transgenic lines, squalene content is decreased by 19.4% and 21.6% respectively in comparison with the control (Fig. 5).
3.4. Determination of artemisinin
Artemisinin was detected by HPLC. The results indicated that artemisinin content of SQS3 and SQS5 transgenic lines was increased by 23.2% and 21.5%, respectively compared with that of the control (Fig. 6) and in SQS2 transgenic line, the artemisinin content manifested no obvious variation compared with the control.
The above results demonstrated a clear negative correlation between squalene content and artemisinin content, which implies that the inhibiting of squalene synthase gene expression caused part of the flux for squalene biosynthesis diverting to artemisinin biosynthesis.
Since squalene synthase is commonly depicted as the incipient and crucial branch point enzyme of the isoprenoid pathway to sterol biosynthesis, it has attracted considerable interest as a potential regulatory point that controls carbon flux into sterols. Several researchers reported the induction of sesquiterpene phytoalexins biosynthesis had been correlated with suppression of sterol biosynthesis in elicitor-treated tobacco cell cultures (Chappell et al., 1989; McGarvey & Croteau, 1995; Yin et al., 1997). The induction of one enzyme and suppression of the other are thought to be one mechanism that regulates the production of squalene and sesquiterpene (Devarenne et al., 1998). The biosynthesis of artemisinin belongs to the isoprenoid pathway, in this pathway, squalene synthase and amorpha-4,11-diene synthase are positioned at putative branch points in isoprenoid metabolism, these two enzymes catalyze the common farnesyl diphosphate to form squalene and amorph-4,11-diene, respectively. Furthermore, amorpha-4,11-diene synthase is considered as a key enzyme in artemisinin biosynthesis (Bouwmeester et al., 1999), so squalene synthase can be considered as a competitive enzyme of artemisinin biosynthesis. Therefore, the inhibiting of SQS gene expression may increase the biosynthesis of artemisinin.
In order to increase artemisinin content, we introduced antisense squalene synthase gene into
Our results as well as other related reports all indicated that it is possible to increase artemisinin content of
The antisense squalene synthase (SQS) gene was transferred into
This work was supported by the National Natural Science Foundation of China (NSFC) (No. 61173098) and the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2-EW-J-29).
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