RNAi-mediated modulation of squalene synthase gene expression in Artemisia annua L. and its impact on artemisinin biosynthesis
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Artemisinin, an endoperoxide sesquiterpene lactone, is an antimalarial phytoconstituent isolated from Artemisia annua L. plants. Artemisinin-based combination therapies (ACTs) are recommended by WHO for treating malaria caused by multidrug-resistant Plasmodium falciparum sp. The main cause of instability in supply and cost escalation of ACTs in countries endemic to malaria is low content (0.01–1.1%) of artemisinin in these plants. In this study, the expression of SQS gene encoding squalene synthase (SQS), a key enzyme of the sterol biosynthetic pathway that competes for carbon precursor with that of artemisinin biosynthetic pathway was suppressed by hpRNAi-technology in A. annua to study its impact on artemisinin biosynthesis. Southern blot analyses of transgenic lines obtained have shown single (TR2, TR4 and TR10) and multiple copies (TR1, TR3, TR7, TR8 and TR9) of transgenes in their genomes. The GC/MS analysis showed decreased in squalene content by 65.7%, while HPLC result showed an increase in artemisinin content by 40.2% in transgenic lines, TR3. The plants from TR3 were also grown in environmentally controlled polyhouse and yielded 28.3% higher artemisinin as compared to the non-transgenic plants. The increased artemisinin levels coupled with reduced squalene contents in the transgenic lines of A. annua were well correlated with the up-regulation of artemisinin biosynthetic pathway genes and suppression of SQS in the qPCR analyses.
KeywordsArtemisia annua L. Asteraceae Artemisinin Malaria and RNAi-technology
Artemisinin-based combination therapies
Squalene synthase enzyme
Squalene synthase gene
Gas chromatography–mass spectrometry
High performance liquid chromatography
Murashige and Skoog
Naphthalene acetic acid
National Institute Standard and Technology
Octopine synthase gene
Shoot-induction selection medium
Neomycin phosphatransferase gene
Authors are thankful to University Grants Commission (UGC), Government of India, for financial support to Department of Biotechnology under the scheme of UGC-SAP (DRS-1) to develop research advanced facilities used in this study. A. Ali and Khan S are thankful to UGC for providing Research Fellowship under UGC-SAP (BSR) scheme. The pHANNIBAL RNAi vector was gifted by CSIRO Plant Industry, PO Box 1600 Canberra ACT 2601, Australia. Technical help provided by Dr. Ajay Kumar, AIRF, School of Physical Sciences, JNU, New Delhi, India, for squalene analysis is greatly acknowledged.
MZA and AA developed the idea and critically editing the MS. AA conducted the experiments, wrote/finalized the MS. MMA helped in writing and data analysis. MAK helped in writing MS and interpretations of data. SK helped in experiments, writing and data analysis. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that he/she have no conflict of interest.
- Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
- Looa CSN, Lama NSK, Yua D, Sub X, Lud F (2017) Artemisinin and its derivatives in treating protozoan infections beyond malaria. Pharm Res 117:192–217Google Scholar
- Luximon AB, Jhurry D (2017) Artemisinin and its derivatives in cancer therapy: status of progress, mechanism of action, and future perspectives. Cancer Chemother Pharmacol 79(3):451–466Google Scholar
- Wang H, Song Y, Shen H, Liu Y, Li Z, Wang H (2012) Effect of antisense squalene synthase gene expression on the increase of artemisinin content in Artemisia anuua. In: Çiftçi YO (ed) Transgenic plants—advances and limitations. InTech, Rijeka. ISBN 978-953-51-0181-9Google Scholar
- World Health Organization (2005) World Malaria Report 2005. WHO, GenevaGoogle Scholar
- World Health Organization (2016) World Malaria Report 2016. WHO, Geneva.Google Scholar
- Yan L, He-Chun Y, Hong W, Guo-Feng L (2003) Molecular cloning, Escherichia coli expression and genomic organization of squalene synthase gene from Artemisia annua. L. Acta Bot Sin 45:608–613Google Scholar
- Zhang L, Jing F, Li F, Li M, Wang Y, Wang G, Sun X, Tang K (2009) Development of transgenic Artemisia annua (Chinese wormwood) plants with an enhanced content of artemisinin, an effective anti-malarial drug, by hairpin-RNA mediated gene silencing. Biotechnol Appl Biochem 52:199–207CrossRefGoogle Scholar
- Zhao SS, Zeng MY (1986) Determination of qinghaosu in Artemisia annua L. by high performance liquid chromatography. Chin J Pharm Anal 6:3–5Google Scholar