Cloning of artemisinin biosynthetic cDNAs and novel ESTs and quantification of low temperature-induced gene overexpression

Abstract

To isolate and verify novel genes from qinghao (Artemisia annua) based on the development-specific and environment-induced transcriptomics, leaves have been harvested from the flowering A. annua plants and exposed to low temperature for isolation of total RNAs and cloning of full-length cDNAs and cDNA fragments, or expressed sequence tags (ESTs). After being sequenced and browsed for homology, these sequences have been submitted to GenBank. Among the accessed 75 sequences, 4 full-length cDNAs are highly homologous to the known A. annua genes, but 71 ESTs are absent in the sequence records of A. annua genes, in which 34 sequences are homologous to other plant genes, including 24 identified protein-coding sequences and 10 unidentified protein-coding sequences, while other 37 sequences are not present in the sequence records of any plant genes, representing the first cloned plant genes. In order to investigate the responsive patterns of A. annua genes to extreme environmental stresses, especially low temperature, the expression levels of 3 critical qinhaosu (artemisinin) biosynthetic genes, ADS, CYP71AV1 and CPR, have been measured in pre-and post-chilling A. annua seedlings cultured in vitro by semi-quantitative PCR (SQ-PCR). Consequently, ADS and CYP71AV1 genes are strongly induced by chilling, but CPR gene is not significantly affected by such treatment. Furthermore, induction of these genes by chilling can be potently suppressed by Ca2+ channel inhibitor LaCl3 or Ca2+ chelator EGTA, suggesting a putative involvement of Ca2+-CaM signal transduction pathway in chilling-induced overexpression of ADS and CYP71AV1 genes. The real-time fluorescent quantitative PCR (RFQ-PCR) assay of A. annua seedlings exposed to chilling has shown that the expression level of CaM gene is up-regulated for more than 2.5 folds, thereby confirming our above inference on the relevance of Ca2+-CaM-mediated signal transduction to chilling-induced gene overexpression. Finally, 7 newly isolated A. annua ESTs have been functionally annotated by RFQ-PCR, which indicates that the chilling stress-induced overexpression levels of D/LTSRP, UBE, AR/DAP and POD1 genes are augmented approximately for 8, 5, 2.3, and 1.5 folds, respectively, but those of CHI and RGP genes are not predominantly up-or down-regulated. The present study has preliminarily explored the chilling-induced overexpression patterns of 3 artemisinin biosynthetic genes and 7 novel A. annua ESTs at the transcriptional level, which should further facilitate our understanding of the intrinsic rule and mechanism underlying the coordinative regulation manner of artemisinin biosynthesis and accumulation, and yield substantial insight into improvement of A. annua by the metabolic engineering-guided breeding strategy.