Limits...
Enhanced artemisinin yield by expression of rol genes in Artemisia annua.

Dilshad E, Cusido RM, Palazon J, Estrada KR, Bonfill M, Mirza B - Malar. J. (2015)

Bottom Line: Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis.TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. erum.dilshad@gmail.com.

ABSTRACT

Background: Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug.

Methods: In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC-MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined.

Results: Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4-12-fold increase in artesunate and 1.2-3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.

Conclusion: Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.

No MeSH data available.


Related in: MedlinePlus

Statistical and quantitative analysis of artemisinin and derivatives: comparative and statistical analysis of artemisinin content and its derivatives in wild type A. annua and transgenics of rol B and rol C gen. Letters on the bars show LSD ranking
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4625606&req=5

Fig4: Statistical and quantitative analysis of artemisinin and derivatives: comparative and statistical analysis of artemisinin content and its derivatives in wild type A. annua and transgenics of rol B and rol C gen. Letters on the bars show LSD ranking

Mentions: Differences were observed in the content of artemisinin and derivatives in transformed and untransformed plants. All of the transgenic lines of rolB and rol C gene showed enhancement of artemisinin and derivatives (Fig. 4). Transgenics of rolB gene showed 2.7–9.2-fold increase in artemisinin, 4–12.6-fold increase in artesunate and 1.2–3-fold increase in D.H.A. Whereas in case of rol C gene transformants 4–4.6-fold increase in artemisinin, 4.4–9.1-fold increase in artesunate and 1.5–2-fold increase in D.H.A concentration was observed. Previously it is reported that rol genes (rol A, rol B and rol C) when expressed individually or combined result in increased plant secondary metabolism by transcriptional activation of defense genes through an unknown mechanism [38]. The effects of individual rol genes of TL-DNA of Agrobacterium rhizogenes, A4 strain on production of ginsenoside of Panax ginseng cell cultures has been reported [17]. In this report, rolC cultures accumulated 1.8–3 times more ginsenoside than control plant [17]. Rol B transgenics of Rubia cordifolia showed enhanced production of anthraquinones [38]. Further, rolB gene from A. rhizogenes when expressed in tomato improved foliar tolerance against fungal pathogens [39]. However, in the current study differences in artemisinin content of rol B and rol C transgenics were observed. Rol B gene was found to be more active in the enhancement of artemisinin content than rol C gene. As previously reported, high expression of the rol B gene dramatically increased the biosynthesis of secondary metabolites in transformed plant cells [38]. Compared to the rol B gene, the rol C gene activated the biosynthesis of secondary metabolites to a lesser extent. Evidence indicates that each of the rol genes has its own role in plant metabolic processes [14]. Transgenic plants showing increased artemisinin concentration and altered morphology were analysed by semi quantitative RT-PCR to check that whether the results correlate with rol gene expression. Results confirmed the findings to be the result of presence of rol gene transcripts. Also it was observed that the lines TB3 and TC1 had higher artemisinin content and more transcripts of rol B and rol C gene, respectively (Fig. 2c, d). As these genes are under the control of CaMV35S promoter, their effect on the induction of secondary metabolism become more clear due to systemic high level of expression [40].Fig. 4


Enhanced artemisinin yield by expression of rol genes in Artemisia annua.

Dilshad E, Cusido RM, Palazon J, Estrada KR, Bonfill M, Mirza B - Malar. J. (2015)

Statistical and quantitative analysis of artemisinin and derivatives: comparative and statistical analysis of artemisinin content and its derivatives in wild type A. annua and transgenics of rol B and rol C gen. Letters on the bars show LSD ranking
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4625606&req=5

Fig4: Statistical and quantitative analysis of artemisinin and derivatives: comparative and statistical analysis of artemisinin content and its derivatives in wild type A. annua and transgenics of rol B and rol C gen. Letters on the bars show LSD ranking
Mentions: Differences were observed in the content of artemisinin and derivatives in transformed and untransformed plants. All of the transgenic lines of rolB and rol C gene showed enhancement of artemisinin and derivatives (Fig. 4). Transgenics of rolB gene showed 2.7–9.2-fold increase in artemisinin, 4–12.6-fold increase in artesunate and 1.2–3-fold increase in D.H.A. Whereas in case of rol C gene transformants 4–4.6-fold increase in artemisinin, 4.4–9.1-fold increase in artesunate and 1.5–2-fold increase in D.H.A concentration was observed. Previously it is reported that rol genes (rol A, rol B and rol C) when expressed individually or combined result in increased plant secondary metabolism by transcriptional activation of defense genes through an unknown mechanism [38]. The effects of individual rol genes of TL-DNA of Agrobacterium rhizogenes, A4 strain on production of ginsenoside of Panax ginseng cell cultures has been reported [17]. In this report, rolC cultures accumulated 1.8–3 times more ginsenoside than control plant [17]. Rol B transgenics of Rubia cordifolia showed enhanced production of anthraquinones [38]. Further, rolB gene from A. rhizogenes when expressed in tomato improved foliar tolerance against fungal pathogens [39]. However, in the current study differences in artemisinin content of rol B and rol C transgenics were observed. Rol B gene was found to be more active in the enhancement of artemisinin content than rol C gene. As previously reported, high expression of the rol B gene dramatically increased the biosynthesis of secondary metabolites in transformed plant cells [38]. Compared to the rol B gene, the rol C gene activated the biosynthesis of secondary metabolites to a lesser extent. Evidence indicates that each of the rol genes has its own role in plant metabolic processes [14]. Transgenic plants showing increased artemisinin concentration and altered morphology were analysed by semi quantitative RT-PCR to check that whether the results correlate with rol gene expression. Results confirmed the findings to be the result of presence of rol gene transcripts. Also it was observed that the lines TB3 and TC1 had higher artemisinin content and more transcripts of rol B and rol C gene, respectively (Fig. 2c, d). As these genes are under the control of CaMV35S promoter, their effect on the induction of secondary metabolism become more clear due to systemic high level of expression [40].Fig. 4

Bottom Line: Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis.TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. erum.dilshad@gmail.com.

ABSTRACT

Background: Despite of many advances in the treatment of malaria, it is still the fifth most prevalent disease worldwide and is one of the major causes of death in the developing countries which accounted for 584,000 deaths in 2013, as estimated by World Health Organization. Artemisinin from Artemisia annua is still one of the most effective treatments for malaria. Increasing the artemisinin content of A. annua plants by genetic engineering would improve the availability of this much-needed drug.

Methods: In this regard, a high artemisinin-yielding hybrid of A. annua produced by the centre for novel agricultural products of the University of York, UK, was selected (artemisinin maximally 1.4 %). As rol genes are potential candidates of biochemical engineering, genetic transformation of A. annua with Agrobacterium tumefaciens GV3101 harbouring vectors with rol B and rol C genes was carried out with the objective of enhancement of artemisinin content. Transgenic lines produced were analysed by the LC-MS for quantitative analysis of artemisinin and analogues. These high artemisinin yielding transgenics were also analysed by real time quantitative PCR to find the molecular dynamics of artemisinin enhancement. Genes of artemisinin biosynthetic pathway were studied including amorphadiene synthase (ADS), cytochrome P450, (CYP71AV1) and aldehyde dehydrogenase 1 (ALDH1). Trichome-specific fatty acyl-CoA reductase 1(TAFR1) is an enzyme involved in both trichome development and sesquiterpenoid biosynthesis and both processes are important for artemisinin biosynthesis. Thus, real time qPCR analysis of the TAFR1 gene was carried out, and trichome density was determined.

Results: Transgenics of rol B gene showed two- to ninefold (the decimal adds nothing in the abstract, please simplify to two- to ninefold) increase in artemisinin, 4-12-fold increase in artesunate and 1.2-3-fold increase in dihydroartemisinin. Whereas in the case of rol C gene transformants, a fourfold increase in artemisinin, four to ninefold increase in artesunate and one- to twofold increase in dihydroartemisinin concentration was observed. Transformants with the rol B gene had higher expression of these genes than rol C transformants. TAFR1 was also found to be more expressed in rol gene transgenics than wild type A. annua, which was also in accordance with the trichome density of the respective plant.

Conclusion: Thus it was proved that rol B and rol C genes are effective in the enhancement of artemisinin content of A. annua, rol B gene being more active to play part in this enhancement than rol C gene.

No MeSH data available.


Related in: MedlinePlus