Isolation and Identification of Novel Genes Involved in Artemisinin Production from Flowers ofArtemisia annuaUsing Suppression Subtractive Hybridization and Metabolite Analysis

Abstract

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Artemisinin is highly effective against multidrug-resistant Plasmodium falciparum and it has been widely used as part of the artemisinin-based combination therapies against malaria. To elucidate the biosynthetic pathway of artemisinin and to clone related genes in Artemisia annua, differentially expressed genes between blooming flowers and flower buds were isolated and characterized by a combined approach of suppression subtractive hybridization (SSH) and metabolite analysis. A total of 350 cDNA clones from a subtractive cDNA library were randomly picked, sequenced and analyzed and 253 high-quality sequences were obtained. BLASTX comparisons indicated that about 9.9 % of the clones encoded enzymes involved in isoprenoid (including artemisinin) biosynthesis. The expression of 4 gene transcripts involved in artemisinin biosynthesis was examined by RT-PCR and the results confirmed the higher expression of these transcripts in blooming flowers than in flower buds. In addition, 2 putative transcript factors transparenta testa glabra 1 (TTG1) and ENHANCER OF GLABRA3 (GL3), which promote trichome initiation, were presented in the library. Finally, this study demonstrated that the increase of expression level of the putative TTG1 gene correlated with the improvement of glandular trichome density and artemisinin production in A. annua leaves. The subtractive cDNA library described in the present study provides important candidate genes for future research in order to increase the artemisinin content in A. annua.

Reproductive Development Modulates Gene Expression and Metabolite Levels with Possible Feedback Inhibition of Artemisinin in Artemisia annua

“…Although trichome counts have been reported for A. annua, data were obtained only from the adaxial surface of leaves and apparently only from plants in the vegetative phase of growth (Lommen et al, 2006;Liu et al, 2009). Likewise, instead of correlating trichome numbers to their neighboring epidermal cell population, counts have been generally reported as trichomes per unit leaf area and range from 20 to 80 trichomes mm 22 , the number usually declining with leaf maturity (Lommen et al, 2006) although increasing with plant age (Lommen et al, 2006;Liu et al, 2009). Additionally, studies have typically focused on only the adaxial surfaces and the youngest leaves on a given plant, which have the highest density of trichomes.…”

“…A recent gene expression study in A. annua by Liu et al (2009) used the A. annua 'H050139'. Their work showed little to no change in FPS or CYP levels, whereas our study found large decreases in both transcript levels accompanying the transition from floral bud to full flower.…”

Chitosan oligosaccharide and salicylic acid up-regulate gene expression differently in relation to the biosynthesis of artemisinin in Artemisia annua L.

“…These transcription factor genes do not directly take part in the artemisinin synthesis pathway but indirectly regulate the artemisinin production. WRKY1 was suggested to regulate the expression of the ADS gene [8] and TTG1 was reported to promote glandular trichome initiation [9]. The expression of TTG1 was up-regulated by COS treatment revealing the possibility of a positive effect of COS on glandular trichome formation.…”

“…AaWRKY1: A. annua WRKY transcription factor 1; ADS: amorpha-4,11-diene synthase; CPR: cytochrome P450 reductase; CYP: cytochrome P450 monoxygenase; DBR: artemisinic aldehyde 11 (13) reductase. The genes selected for analysis in this paper were underlined in figure. ENHANCER OF GLABRA3 (GL3), which promote trichome initiation were isolated from an A. annua subtractive cDNA library [9].…”

Trichome Specific Expression: Promoters and Their Applications

“…AaAS was initially identified from a leaf cDNA library by similarity to known sesquiterpene synthases from plants (Mercke et al, 2000;Wallaart et al, 2001) and its specific pattern of expression in trichomes was later confirmed (Bertea et al, 2005;Olofsson et al, 2011). A succession of oxidation steps requiring a P450 mono-oxygenase (CYP71AV1) and an aldehyde dehydrogenase leads to artemisinic acid, while the synthesis of de-hydro-artemisinic acid requires the intervention of reductase (DBR2) (Zhang et al, 2008;Liu et al, 2009;Teoh et al, 2009;Wang et al, 2009;Zhang et al, 2009;Weathers et al, 2011). Although much progress has been achieved in the elucidation of the artemisinin pathway, relatively little was done with regards to promoter identification.…”