Promotion of Artemisinin Biosynthesis in Transgenic a. Annua by Overexpressing ADS, CYP71AV1 and CPR...

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Industrial Crops and Products 49 (2013) 380–385 Contents lists available at SciVerse ScienceDirect Industrial Crops and Products journa l h om epa ge: www.elsevier.com/locate/indcrop Promotion of artemisinin biosynthesis in transgenic Artemisia annua by overexpressing ADS, CYP71AV1 and CPR genes Xu Lu a,b , Qian Shen a , Ling Zhang a , Fangyuan Zhang a , Weimin Jiang a , Zongyou Lv a , Tingxiang Yan a , Xueqing Fu a , Guofeng Wang a , Kexuan Tang a,a Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, PR China b State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China a r t i c l e i n f o Article history: Received 17 November 2012 Received in revised form 8 April 2013 Accepted 27 April 2013 Keywords: Artemisinin biosynthesis pathway Overexpression of ADS, CYP71AV1 and CPR Southern blot Real-time quantitative PCR High-performance liquid chromatography a b s t r a c t Artemisinin is an effective anti-malarial drug isolated from A. annua, which has an enormous commer- cialization demand all over the world. However, the low artemisinin content of A. annua greatly limits the commercialization of artemisinin. In this study, we report the results of our experiments, where for the first time we have achieved the overexpression of ADS, CYP71AV1 and CPR genes in A. annua. Eight transgenic A. annua plants were obtained through Agrobacterium tumefaciens-mediated transfor- mation, which was confirmed by PCR. Southern-blot analyses showed that some of the transgenic lines had low copies of the integration transgenes. The results of real time-qPCR showed that the expression levels of ADS, CYP71AV1 and CPR genes were significantly increased, too. The HPLC analyses showed that the artemisinin contents were significantly increased in these transgenic plants. One of the transgenic plants, ACR16, was found to contain 2.4-fold higher (15.1 mg/g DW) artemisinin than the control plants (pCAMBIA2300 transgenic plants). All above results showed that overexpression of ADS, CYP71AV1 and CPR genes in A. annua could promoted the metabolic flux flows toward biosynthesis of artemisinin and effectively increase the level of artemisinin content in transgenic A. annua plants. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Artemisia annua is an annual herbaceous plant in the fam- ily Asteraceae, which is capable of producing a wide range of terpenoids including artemisinin. Artemisinin, a sesquiterpene lac- tone, is currently the best therapeutic against both drug-resistant and cerebral malaria-causing strains of Plasmodium falciparum (Weathers et al., 2006). Malaria is a global health problem with more than 1 billion people living in areas with a high risk of the disease (Graham et al., 2010). So, there is an enormous demand for the supply of artemisinin all over the world. However, the artemisinin content of A. annua is very low (0.01–0.8% dry wt) and this limits the commercialization of artemisinin greatly (Lei et al., 2011). With the elucidation of the biosynthetic pathway of artemisinin, several key enzymatic genes involved in artemisinin Abbreviations: ADS, amorpha-4,11-diene synthase; CYP71AV1, cytochrome P450 dependent hydroxylase; CPR, cytochrome P450 oxidoreductase; DBR2, double bond reductase 2; ALDH1, aldehyde dehydrogenase; RT-Q-PCR, real-time quantita- tive PCR; HPLC, high-performance liquid chromatography. Corresponding author. Tel.: +86 021 34206916; fax: +86 021 34206916. E-mail addresses: [email protected], [email protected] (K. Tang). biosynthesis have been cloned and characterized from A. annua (Fig. 1). Amorpha-4,11-diene synthase (ADS) is the first committed step in artemisinin biosynthesis which produce the amorpha- 4,11-diene (Bouwmeester et al., 1999; Mercke et al., 2000). In the following step, amorpha-4,11-diene is hydroxylated to yield artemisinic alcohol. This reaction is catalyzed by a cytochrome P450 dependent hydroxylase (CYP71AV1) and NADPH: cytochrome P450 oxidoreductase (CPR) as the native redox partner (Ro et al., 2006; Teoh et al., 2006). These two enzymes can also oxidize the alcohol to artemisinic aldehyde and then further on to artemisinic acid (Ro et al., 2006). Artemisinic acid can be used as the substance to synthesis the artemisinin by semi-chemical synthesis (Dietrich et al., 2009). Then, this route is completed by the discovery of double bond reductase 2 (DBR2) and aldehyde dehydrogenase 1 (ALDH1) (Zhang et al., 2008; Teoh et al., 2009). The conversion of dihydroartemisinic acid to artemisinin has been suggested to be the enzyme-independent reactions (Sy and Brown, 2002; Brown and Sy, 2004). In a similar way, artemisinic acid is converted to arteannuin B via enzyme-independent reactions, too (Brown and Sy, 2007). All above researches provided the theoretical possibility to improve artemisinin yield in transgenic A. annua through regulating the artemisinin biosynthetic pathway. Recently, some efforts have been made to try to increase artemisinin 0926-6690/$ see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2013.04.045

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Transcript of Promotion of Artemisinin Biosynthesis in Transgenic a. Annua by Overexpressing ADS, CYP71AV1 and CPR...

  • Industrial Crops and Products 49 (2013) 380 385

    Contents lists available at SciVerse ScienceDirect

    Industrial Crops and Products

    journa l h om epa ge: www.elsev ier .com

    Promotion of artemisinin biosynthesis in transgeby overexpressing ADS, CYP71AV1 and CPR genes

    Xu Lua,b, in Tingxian ga,

    a Plant Biotech ool of University, Shab State Key Lab ina

    a r t i c

    Article history:Received 17 NReceived in reAccepted 27 A

    Keywords:Artemisinin biOverexpression of ADS, CYP71AV1 and CPRSouthern blotReal-time quantitative PCRHigh-performance liquid chromatography

    ial dr. How. In tthe oe obta. Sou

    had low copies of the integration transgenes. The results of real time-qPCR showed that the expressionlevels of ADS, CYP71AV1 and CPR genes were signicantly increased, too. The HPLC analyses showed thatthe artemisinin contents were signicantly increased in these transgenic plants. One of the transgenicplants, ACR16, was found to contain 2.4-fold higher (15.1 mg/g DW) artemisinin than the control plants(pCAMBIA2300 transgenic plants). All above results showed that overexpression of ADS, CYP71AV1 andCPR genes in A. annua could promoted the metabolic ux ows toward biosynthesis of artemisinin and

    1. Introdu

    Artemisiily Asteraceterpenoids tone, is curand cerebr(Weathers more than disease (Grfor the supartemisininthis limits t2011).

    With tartemisinin

    AbbreviatiP450 dependebond reductastive PCR; HPLC

    CorresponE-mail add

    0926-6690/$ http://dx.doi.oeffectively increase the level of artemisinin content in transgenic A. annua plants. 2013 Elsevier B.V. All rights reserved.

    ction

    a annua is an annual herbaceous plant in the fam-ae, which is capable of producing a wide range ofincluding artemisinin. Artemisinin, a sesquiterpene lac-rently the best therapeutic against both drug-resistantal malaria-causing strains of Plasmodium falciparumet al., 2006). Malaria is a global health problem with1 billion people living in areas with a high risk of theaham et al., 2010). So, there is an enormous demandply of artemisinin all over the world. However, the

    content of A. annua is very low (0.010.8% dry wt) andhe commercialization of artemisinin greatly (Lei et al.,

    he elucidation of the biosynthetic pathway of, several key enzymatic genes involved in artemisinin

    ons: ADS, amorpha-4,11-diene synthase; CYP71AV1, cytochroment hydroxylase; CPR, cytochrome P450 oxidoreductase; DBR2, doublee 2; ALDH1, aldehyde dehydrogenase; RT-Q-PCR, real-time quantita-, high-performance liquid chromatography.ding author. Tel.: +86 021 34206916; fax: +86 021 34206916.resses: [email protected], [email protected] (K. Tang).

    biosynthesis have been cloned and characterized from A. annua(Fig. 1). Amorpha-4,11-diene synthase (ADS) is the rst committedstep in artemisinin biosynthesis which produce the amorpha-4,11-diene (Bouwmeester et al., 1999; Mercke et al., 2000). Inthe following step, amorpha-4,11-diene is hydroxylated to yieldartemisinic alcohol. This reaction is catalyzed by a cytochromeP450 dependent hydroxylase (CYP71AV1) and NADPH: cytochromeP450 oxidoreductase (CPR) as the native redox partner (Ro et al.,2006; Teoh et al., 2006). These two enzymes can also oxidize thealcohol to artemisinic aldehyde and then further on to artemisinicacid (Ro et al., 2006). Artemisinic acid can be used as the substanceto synthesis the artemisinin by semi-chemical synthesis (Dietrichet al., 2009). Then, this route is completed by the discovery ofdouble bond reductase 2 (DBR2) and aldehyde dehydrogenase 1(ALDH1) (Zhang et al., 2008; Teoh et al., 2009). The conversion ofdihydroartemisinic acid to artemisinin has been suggested to bethe enzyme-independent reactions (Sy and Brown, 2002; Brownand Sy, 2004). In a similar way, artemisinic acid is converted toarteannuin B via enzyme-independent reactions, too (Brown andSy, 2007).

    All above researches provided the theoretical possibilityto improve artemisinin yield in transgenic A. annua throughregulating the artemisinin biosynthetic pathway. Recently,some efforts have been made to try to increase artemisinin

    see front matter 2013 Elsevier B.V. All rights reserved.rg/10.1016/j.indcrop.2013.04.045 Qian Shena, Ling Zhanga, Fangyuan Zhanga, Weimg Yana, Xueqing Fua, Guofeng Wanga, Kexuan Tannology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Schnghai 200240, PR Chinaoratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR Ch

    l e i n f o

    ovember 2012vised form 8 April 2013pril 2013

    osynthesis pathway

    a b s t r a c t

    Artemisinin is an effective anti-malarcialization demand all over the worldthe commercialization of artemisininfor the rst time we have achieved Eight transgenic A. annua plants wermation, which was conrmed by PCR/ locate / indcrop

    nic Artemisia annua

    Jianga, Zongyou Lva,

    Agriculture and Biology, Shanghai Jiao Tong

    ug isolated from A. annua, which has an enormous commer-ever, the low artemisinin content of A. annua greatly limitshis study, we report the results of our experiments, whereverexpression of ADS, CYP71AV1 and CPR genes in A. annua.ined through Agrobacterium tumefaciens-mediated transfor-

    thern-blot analyses showed that some of the transgenic lines

  • X. Lu et al. / Industrial Crops and Products 49 (2013) 380 385 381

    Fig. 1. The bithase; CYP71Aoxidoreductas

    production.(FPS) gene yield, of 2.5type plantsmulti-geneof multi-geimprove th(Zhang et agenic lines amorpha-4higher (1.7annua plant

    Table 1Primers used in this study.

    Primers Purpose Primer sequence (53)

    P P

    P P

    P P2 P

    R R

    R R

    R RF RR R1304F1304RP35S ADSR2CYPR2CPRR2NPTIIRADSF4ADSR4CYPF4CYPR4CPRF4CPRR4Actin-Actin-osynthesis pathway of artemisinin. ADS, amorpha-4,11-diene syn-V1, cytochrome P450 dependent hydroxylase; CPR, cytochrome P450e; DBR2, double bond reductase 2; ALDH1, aldehyde dehydrogenase.

    Overexpression of farnesyl pyrophosphate synthaseexhibited higher artemisinin content (13.3 mg/g) and- and 3.6-fold, respectively, than that detected in wild-

    (Banyai et al., 2010). Recent research indicated thats overexpression could regulate the expression levelsnes simultaneously, and was the optimum strategy toe biosynthetic ability of plant secondary metabolitesl., 2004; Alam and Abdin, 2011). One of the trans-by over-expressing of HMG-CoA reductase gene and,11-diene synthase gene was found to contain 7.65-fold3 mg/gDW) artemisinin than the non-transgenic in A.s (Alam and Abdin, 2011).

    In this sthe high artiment and hHere, we retime we haCPR genes tartemisinin

    2. Method

    2.1. Plant m

    The seedences, SoutA. annua plaisolation remanufactur

    2.2. Vector

    The ORF(DQ318192ing site attvectors. TheBstEII and lThe expressminator we(with SwaI)pMD18-T vvectors weCYP71AV1-nand ligatioligated intosites). The genes wereplementarywas introduand the pos

    Supplemonline vers

    2.3. Plant t

    Seeds of1 min and trite) for 10distilled wamedium (SCR ATTTAAATCAGGAGTCAAAGATCAAACR GCTGCAGCCCCGGGGCCCGATCTAGTAACATAGATCR TTCGTCAACATGGTGGAGCACR AGTGCCCGTTGTATTTCGCR GGAAGGCTTTTTTTGGTGGATTTGCR CTTCCGTCCGTCATCAACCTCACR CCCTGATGCTCTTCGTCCAT-Q-PCR AATGGGCAAATGAGGGACACT-Q-PCR TTTCAAGGCTCGATGAACTATGT-Q-PCR, Probe CACCCTCCACTACCCTTGT-Q-PCR, Probe GACACATCCTTCTCCCAGCT-Q-PCR AGCCTCTTTGCCACCTCCTT-Q-PCR GAACAGACTCCCTTGTGAACGT-Q-PCR CCAGGCTGTTCAGTCTCTGTATT-Q-PCR CGCTCGGTAAGGATCTTCATCA

    tudy, we choose wild-type A. annua plants, which haveemisinin content, to do the heredity transformed exper-ope to get the high content transgenic A. annua plants.port the results of our experiments, where for the rstve achieved the overexpression of ADS, CYP71AV1 andogether in A. annua and obviously increased the level of

    content.

    s and materials

    aterials

    s of A. annua were obtained from the School of Life Sci-hwest University in Chongqing, PR China. Leaves fromnts were collected for RNA extraction using plant RNAagent (Tiangen Biotech, Beijing, China) following theers instructions.

    construction

    of ADS (AF138959), CYP71AV1 (DQ268763) and CPR) genes, with the SpeI digesting site and the BstEII digest-ached, were respectively cloned in pMD18-T simple

    ORF of the three genes were digested with SpeI and theigated into the pCAMBIA1304 to replace the GUS gene.ion cassettes containing the 35S promoter and nos ter-re respectively amplied with the primers of p1304-F

    and p1304-R (with SmaI and PstI) and ligated into theectors. All the primers used were in Table 1. The middlere named as pMD18T-p35S-ADS-nos, pMD18T-p35S-os and pMD18T-p35S-CPR-nos, respectively. By cuttingn, the three expression cassettes were respectively

    the pCAMBIA2300 (with Swa1 and Pst1 restrictionexpression vector containing ADS, CYP71AV1 and CPR named pCAMBIA2300-ADS-CYP71AV1-CPR(ACR) (sup- Fig. 1). After conrmation by sequencing, the plasmidced into the Agrobacterium tumefaciens strain EHA105,itive clone was used for plant transformation.entary material related to this article found, in theion, at http://dx.doi.org/10.1016/j.indcrop.2013.04.045.

    ransformation

    A. annua were surfaced sterilized in 70% ethanol forhen treated with 10% (v/v) NaOCl (sodium hypochlo-

    min. After rinsing 5 times thoroughly with sterileter, the seeds were placed on Murashige and Skoog (MS)igmaAldrich, MO, USA) with 88 mM sucrose and 0.7%

  • 382 X. Lu et al. / Industrial Crops and Products 49 (2013) 380 385

    agar (pH5.8) (Murashige and Skoog, 1962). Cultured in a chamber,the temperature was at 25 2 with 16 h light/8 h dark and light at8000 lx photoperiod. After 2 weeks, the leaves of the seedings wereused as transformation. The leaves of seedings were cut into 0.5-cm-diameteleaf disk traA. tumefacieThen the lemedium Mnaphthalencarbenicilli(every 10 dregeneratedinto rootingcillin). Aftewere transftransplante

    2.4. PCR an

    The gento 40 mg frgreenhousebromide) mintroduced with ADS, Cby PCR metKANR2. Thevolume conTaq Versiomal cyclingamplicatioadditional 1in 1.0% agarfor 10 min Shanghai, Cwas used foand then co

    2.5. Southe

    The genlated and p60 g of eafor 16 h andthen transfebrane (GE Hpartial cDNusing AmerEngland) action of the phybrid-oveDetection Mfacturers inexposure to

    2.6. Real-ti

    All RNA to use. Aliqtranscriptasthesis of r

    The ampan iCycler iwith gene-sthe SYBR E

    conrm changes in gene expression. For each reaction, 2 l ofdiluted cDNA (corresponding to 0.8 ng of total RNA), 2.5 l of EASYdilution, 12.5 l of 2 SYBR Premix ExTaqTM, 0.25 M of forwardprimer, 0.25 M of reverse primer, and nuclease-free water were

    to aere t 95

    agaed e exand St leain froft Eed thfore

    alystogr

    mowereamp

    Instthe centries. Tous

    sam couions methSD coa (50ere

    weree was ched th

    ults

    btain

    you straCAMtivellectioon mycin

    (Figransolog

    tranhe grd ints mA10

    R an

    omid NPorwaductr discs used as the explants in A. tumefaciens-mediatednsformation (McCormick et al., 1986). The leaves andns strain EHA105 were co-cultivated at 25 for 3 days.aves discs were transferred to kanamycin selectionS1 (MS + 2.5 mg/L 6-benzoyladenine (6-BA) + 0.3 mg/Le-1-acetic acid (NAA) + 50 mg/L kanamycin + 250 mg/Ln). After subcultured 30 days of the kanamycin selectionays for 3 times) some kanamycin-resistant buds were. Then the kanamycin-resistant buds were transferred

    medium MS2 (half-strength MS + 250 mg/L carbeni-r roots were formed (about 1215 days), the plantletserred into soil. After 12 months of growth they wered into the greenhouse for further growth.

    alysis in transgenic A. annua

    omic DNA for PCR detective was isolated from 30esh leaves from 4 months old plantlet grown in the

    by using the modied CTAB (cetylmethylammoniumethod (Stewart and Via, 1993). For the presence ofCYP71AV1 gene (the CPR were in the same T-DNA borderYP71AV1, CPR and KAN gene were respectively detectedhod) by using primers P35S, ADSR2, CYPR2, CPRR2 and

    PCR reaction was carried out in a 20 l nal reactiontaining approximate 50 ng DNA template using Premixn2.0 (TaKaRa, Dalian) followed its instruction. The ther-

    conditions was 94 for 4 min, followed by 30 cycles ofn (94 for 30 s, 56 for 30 s and 72 for 1 min) and by 72 for0 min. A 10 l aliquot PCR solution was electrophoresedose gel (with 0.25 g ml1 ethidium bromide) at 140 Vthen visualized under Gel Image System (Tanon 3500,hina). The PCR analysis with right length DNA fragmentsr rapid screening of putative transformanted A. annuanrmed as transgenic regenerates by Southern blotting.

    rn blot analysis

    omic DNA of transgenic and wild type plants was iso-uried by the modied CTAB method. Approximatech sample genomic DNA was digested by EcoRI at 37

    separated by electrophoresis in 0.8% agarose gel andrred on to a positively charged Hybond-N + nylon mem-ealthcare, England). An alkaline-phosphatase-labeledA sequence of CYP71AV1 as the probe was prepared bysham AlkPhos Direct Labeling Reagents (GE Healthcare,cording to the manufacturers instructions. Hybridiza-robe with the membrane was carried out overnight in

    n at 55. Hybridization signals were detected by CDP-Starodule (GE Healthcare, England) following the manu-structions. The hybridized signals were visualized by

    Fuji X-ray lm at room temperature for 5 h.

    me quantitative PCR analysis

    samples were digested with DNase I (RNase-free) prioruots of 0.4 g total RNA were employed in the reversee reaction using random hexamer primers for the syn-st strand cDNA.lication reactions of RT-Q-PCR were performed onQTM Real-Time PCR Machines (Bio-Rad, Watford, UK)pecic primers, Actin primers Actin-F and Actin-R, andxScript RT-PCR kit (Takara, Shiga, Japan) protocol to

    added used w(15 s asis andperformget gen(Livak from afor actMicrosindicat(0 h be

    2.7. Anchroma

    Fiveannua (0.1 g/sZhisununder were cparticlSartori

    Thesystemconditwater/The EL345 kPgains wSigma volum(Waterrepeat

    3. Res

    3.1. O

    ThefaciensCPR (prespecday seselectikanameratedwere tmorphrootedAfter tplanteobviouand EH

    3.2. PC

    GenCPR anUsing fbp pro nal volume of 25 l. The thermal cycle conditions1 min at 95 C followed by 40 cycles of amplicationC, 30 s at 56 C, and 30 s at 72 C). Melt curve analy-rose gel electrophoresis following each RT-Q-PCR wereto assess product specicity. Quantication of the tar-pression was carried out with comparative CT methodchmittgen, 2001). Average CT values for the target genest three PCRs were normalized to average CT valuesom the same cDNA preparations and analyzed usingxcel. The relative expression of ADS, CYP71AV1 and CPRe increasing fold of the gene expression over the controltreatment).

    is of artemisinin contents by high-performance liquidaphy (HPLC)

    nths after transplanted to soil, the leaves from A. dried at 45 C and grounded. The dried-leaf powderle) was extracted with methanol (1 ml) using a Shanghairument Co. Ltd. model JYD-650 ultrasonic processorondition of 37 C 50 W for 30 min. Then the samplesfuged for 10 min at 3910 g to remove the suspendedhe nal supernatant was ltrated through a 0.45 mmembrane.ples were analyzed using a Waters Alliance 2695 HPLCpled with a Waters 2420 ELSD detector. The HPLCwere as follows: column, Waters C18; mobile phase,anol (40:60, v/v) for artemisinin; owrate, 1 ml/min.nditions were optimized at a nebulizer-gas pressure of

    lbf/in.2) and drift-tube temperature of 45 C, and the set at 7 for artemisinin. Authentic artemisinin from

    used as the standard. For each sample, the injections 20 l, and the results were analyzed using Empowerromatography data software). The measurement wasree times.

    and discussion

    ment of transgenic A. annua plants

    ng leaves of A. annua were transformed with A. tume-in EH105 harboring pCAMBIA2300-ADS-CYP71AV1-BIA2300-ACR) plasmid and EHA105 (pCAMBIA2300)y by A. tumefaciens-mediated transformation. After a 30-n, the untransgenic tissues could not survive on theedium containing 50 mg/l kanamycin (Fig. 2A). After the

    selection, some kanamycin-resistant buds were regen-. 2B). Two to three centimeter long transgenic budsferred into the rooting medium and grown to normaly after approximately 15-day cultivation (Fig. 2C). Well-sgenic plants were transplanted to the soil (Fig. 2D).owth of 12 months, the transgenic plants were trans-o the greenhouse for further growth. There were noorphological differences between pCAMBIA2300-ACR5 (pCAMBIA2300) transgenic plants.

    alysis of target genes in transgenic plants

    c PCR was used to detect if exogenous ADS, CYP71AV1,TII genes have integrated into the genome of A. annua.rd primer F35S and ADS-specic primer ADSR2, a 1027-

    was amplied with pMD18T-p35S-ADS-nos as positive

  • X. Lu et al. / Industrial Crops and Products 49 (2013) 380 385 383

    Fig. 2. Obtainmof transgenic A

    template. U1, ACR-8, Acould generdid not shotransgenic CYP71AV1-s580-bp andaimed prodgenic lines from all thewhich couldsame amplithe existencCPR genes i

    3.3. Southe

    In orderplants, Sougency condsequence otransgenic lconrmed t(Fig. 4). ThACR52 had ent of transgenic A. annua plants. (A) Obtainment of kanamycin-resistant plants through selecting on MS medium with 50 mg/L kanamycin. (B) Regeneration. annua plants. (C) Rooting of transgenic A. annua plants. (D) Transplantation of partial transgenic A. annua plants.

    nder the same condition, eight transgenic lines ACR-CR-10, ACR-16, ACR-30, ACR-31, ACR-52 and ACR-53ate the 1027-bp products after PCR amplication, whichw up with transgenic plants of empty vector and non-plants as template (Fig. 3A). Likewise, using F35S andpecic primer CYPR2, CPR-specic primer CPRR2, the

    729-bp products were amplied, respectively. Theucts were amplied respectively in all the eight trans-(Fig. 3B and C). A 635-bp product could be amplied

    transgenic A. annua with F35S and NPTIIR2 as primers, not be amplied from non-transgenic plants under thecation condition (Fig. 3D). The above results validatede of NPTII gene and 35S-promoted ADS, CYP71AV1 andn transgenic A. annua.

    rn blot analysis

    to investigate the inserted copies in the transgenicthern blot analysis was performed under high strin-ition. The alkaline- phosphatase-labeled partial cDNAf CYP71AV1 was used as the probe. The examinedines showed different sizes of hybridized bands whichhat they were from independent transformation eventse results also showed that ACR1, ACR10, ACR31 andlow inserted copies in the genomes (Fig. 4).

    Fig. 3. Polymerase chain reaction detection of ADS, CYP71AV1, CPR and NPTII genein transgenic A. annua plants. Primers used for detecting were F35S, ADSR2, CYPR2,CPRR2 and NPTIIR2. Total genomic DNA was isolated from young leaves by CTABmethod. Plasmid DNA from the Agrobacterium carrying pCAMBIA2300 + ACR wasused as a positive control. M, DNA size marker DL2000; V, A. annua plant trans-formed with blank pCAMBIA2300; C, wild-type A. annua; , water control; +, positivecontrol. Numbers indicate the lines of transgenic A. annua (e.g. 1 indicates ACR1).

  • 384 X. Lu et al. / Industrial Crops and Products 49 (2013) 380 385

    Fig. 4. Southegenomic DNA the AgrobacteGenomic DNAACR10, ACR3137 C with Eco

    3.4. Express

    The eightion. The mof these traindicated thdifferent trvidual genehigh expression of ADSplants, respsix overexpthan that inwas increastively (Fig. 1.7-fold in t

    The mRNent transgewhich may Krol et al., 1and CPR weannua plantrn blot analysis of CYP71AV1 in transgenic A. annua plants. Totalwas isolated from young leaves by CTAB method. Plasmid DNA fromrium carrying pCAMBIA2300 + ACR was used as a positive control.

    samples (60 mg per sample) of four transgenic A. annua plants (ACR1, and ACR52) were used for Southern blot were digested overnight atRI.

    ion of ADS, CYP71AV1 and CPR in transgenic A. annua

    t transgenic lines were subjected to further examina-RNA expressions of ADS, CYP71AV1 and CPR in leavesnsgenic lines were analyzed by RT-Q-PCR. The resultat expression prole of three target genes varied fromansgenic lines. Compared to control lines, except indi--silenced lines, most of the transgenic lines exhibitedsion level (Fig. 5). The results showed that the expres-

    was increased from 1.1- to 2.5-fold in the transgenicectively (Fig. 5A). The expression levels of ADS in theseressed lines, especially ACR52, were signicantly higher

    the control lines (Fig. 5A). The expression of CYP71AV1ed from 1.1- to 2.2-fold in the transgenic plants, respec-5B). The expression of CPR was increased from 1.1- tohe transgenic plants, respectively (Fig. 5C).A expression of each target gene was varied in differ-

    nic lines. There are some individual gene-silenced linesbe due to DNA methylation or co-suppression (Van der990; Tang et al., 2007). The expression of ADS, CYP71AV1re increased from 1.1- to 2.5-fold in the transgenic A.s, which could be due to their positional effect (Meyer,

    Fig. 5. Expresgenic lines: ACblank pCAMBIas internal con

    1995; SpikeCYP71AV1 aA. annua linin the ACR5much lowealso not thecontent of functions o

    3.5. Artemi

    Above aof this stuACR and pCmonths of gartemisininDW which pared to co(Fig. 6). Artsion prole analysis of ADS, CYP71AV1 and CPR by RT-Q-PCR. Trans-R1, ACR8, ACR10, ACR16, ACR30, ACR31, ACR51 and ACR52; vector,A2300 transgenic plants. RT-Q-PCR analysis was performed with Actintrol.

    r and Thompson, 1996). The mRNA expression of ADS,nd CPR were also difference in the same transgenice. The expression of ADS and CYP71AV1 were highest2 line, however, the expression of CPR in ACR52 was

    r than other lines. The artemisinin content of ACR52 was highest in all the transgenic lines. So, the artemisininthe transgenic lines should be affected by the complexf ADS, CYP71AV1 and CPR genes.

    sinin contents in transgenic A. annua

    ll, the content of artemisinin was the central focusdy. Artemisinin contents in all the pCAMBIA2300-AMBIA2300 transgenic plants were analyzed after 5rowth in the greenhouse by HPLCELSD. The content of

    were improved varying from 9.4 mg/g DW to 15.1 mg/gwere 1.5- to 2.4-fold in all eight transgenic lines com-ntrol plants (artemisinin content is about 6.4 mg/g DW)emisinin accumulation was highest in transgenic line

  • X. Lu et al. / Industrial Crops and Products 49 (2013) 380 385 385

    Fig. 6. Artemisinin content in different lines. Artemisinin content analysis was per-formed by HPLC. Transgenic lines: ACR1, ACR8, ACR10, ACR16, ACR30, ACR31, ACR51and ACR52; vector, blank pCAMBIA2300 transgenic plants. Data are the means ofdeterminations of the three copies of each transgenic line. Vertical bars representstandard deviation.

    ACR16 at 15.1 mg/g DW, which was 2.4-fold than that of controlplants (pCA

    4. Conclus

    In the sexcess of 50were achievline ACR16 artemisininaccumulatiooverexpresplants shouthe overexpbreak the limway, and pof artemisin

    Acknowled

    This wo2011AA1002011ZX080

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    Promotion of artemisinin biosynthesis in transgenic Artemisia annua by overexpressing ADS, CYP71AV1 and CPR genes1 Introduction2 Methods and materials2.1 Plant materials2.2 Vector construction2.3 Plant transformation2.4 PCR analysis in transgenic A. annua2.5 Southern blot analysis2.6 Real-time quantitative PCR analysis2.7 Analysis of artemisinin contents by high-performance liquid chromatography (HPLC)

    3 Results and discussion3.1 Obtainment of transgenic A. annua plants3.2 PCR analysis of target genes in transgenic plants3.3 Southern blot analysis3.4 Expression of ADS, CYP71AV1 and CPR in transgenic A. annua3.5 Artemisinin contents in transgenic A. annua

    4 ConclusionsAcknowledgmentsReferences