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.
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