RNAi-mediated modulation of squalene synthase gene expression in Artemisia annua L. and its impact on artemisinin biosynthesis

Abstract

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.