A 1 539 by squalene synthase (AaSQS) cDNA was cloned from a high-yield Artemisia annua L. strain 001 by reverse transcription-polymerise chain reaction (RT-PCR). The amino acid sequence of AaSQS is 70%, 77%, 44% and 39%a identical to that of squalene synthases from Arabidopsis thaliana, tobacco, human and yeast, respectively. The AaSQS genomic DNA has a complex organization containing 14 exons and 13 introns. Full-length or C-terminal truncated cDNA was subcloned into prokaryotic expression vector pET30a and the constructed plasmid was introduced to Escherichia coli strain BL21 (DE3) for induced overexpression. No squalene synthase protein with expected molecular mass was observed in E. cola containing the putative full-length squalene synthase cDNA, however, overexpression in E. coli was achieved by truncating 30 amino acids of hydrophobic region at the carboxy terminus.
New advances in molecular biology of plant secondary metabolic pathway were summarized. The biosynthesis of artemisinin was used as an example, combined with the research work in the authors’ laboratory. Some new ways for producing plant secondary metabolites were proposed.
A 1 886 bp full-length sesquiterpene synthase (AaSES) cDNA was cloned front a high-yield Artemisia annua L. strain 001 by a rapid amplification of cDNA end (RACE) strategy. AaSES is 59% identical to Artemisia cyclase cDNA clone cASC125, 50% identical to epi-cedrol synthase from A. annua , 48% identical to amorpha-4, 11-diene synthase from A. annua, 39% identical to the 5-epi-aristolechene synthase from tabacco, 38 % identical to vetispiradiene synthase front H. muticus, 41 % identical to the, delta-cadinene synthase from cotton. The coding region of the cDNA was inserted into a procaryotic expression vector pET-30a and overexpressed in E. coli BL21 ( DE3). The cyclase proteins extracted front bacterial culture were found largely in an insoluble protein fraction. AaSES expresses in leaves, stems a-rid flowers, not in roots as indicated by Northern blotting analysis.
