Cloning and molecular analysis of two new sesquiterpene cyclases from Artemisia annua L.

Artemisia annua L. is the only source of artemisinin, a new promising antimalarial drug (Qinghaosu Antimalarial Coordinating Research Group, Chin. Med. J. 92 (1979) 811). Our efforts are focused on the overproduction of this valuable medicine by genetic engineered A. annua plants. Therefore, we decided to isolate the gene(s) encoding sesquiterpene cyclase(s) in A. annua as a first step in improving artemisinin yield. Four partial genomic clones, gASC21, gASC22, gASC23 and gASC24, were isolated through polymerase chain reaction (PCR) with degenerated primers based on homologous boxes present in sesquiterpene cyclases from divergent sources. Intron-exon organisation of those partial genomic clones was analysed and it was shown that A. annua contains a gene family for sesquiterpene cyclases. Based on gASC21, gASC22, gASC23 and gASC24 sequences, the full-length cDNA clones cASC34 and cASC125 were subsequently isolated by rapid amplification of cDNA ends PCR. The derived amino acid sequences of both full-length clones show high homology with sesquiterpene cyclases from plants. Reverse transcription-PCR analysis revealed transient and tissue specific expression patterns for cASC34 and cASC125, in contrast to the constitutively expressed 8-epicedrol synthase, a previously reported sesquiterpene cyclase from A. annua. Both cASC34 and cASC125 could only be detected in flowering plants when artemisinin concentration is at highest.