The jasmonate-responsive transcription factor CbWRKY24 regulates terpenoid biosynthetic genes to promote saponin biosynthesis in Conyza blinii H. Lév.

Conyza blinii H. Lév., the most effective component is saponin, is a biennial medicinal material that needs to be overwintered. WRKY transcription factors family is a large protein superfamily that plays a predominant role in plant secondary metabolism, but their characteristics and functions have not been identified in C. blinii. The CbWRKY24 sequence was selectedfrom the transcriptome database of the C. blinii leaves constructed in our laboratory. Phylogenetic tree analysis revealed that it was associated with AaWRKY1 which can regulate artemisinin synthesis in Artemisia annua. Expression analysis in C. blinii revealed that CbWRKY24 was mainly induced by methyl jasmonate (MeJA) and cold treatments. Transcriptional activity assay showed that it had an independent biological activity. Overexpression of CbWRKY24 in transient transformed C. blinii resulted in improved totalsaponins content, which was attributed to upregulate the expression level of keys genes from mevalonate (MVA) pathway in transient transformed plants compared to wild type (WT) plants. Meanwhile, overexpression the CbWRKY24 in transient transformed tomato fruits showed that the transcript level of related genes in lycopene pathway decreased significantly when compared to WT tomatofruits. Additionally, the MeJA-response-element was found in the promoter regions of CbWRKY24 and the histochemical staining experiments showed that promoter had GUS activity in transiently transformed tobacco leaves. In summary, our results indicated that we may have found a transcription factor that can regulate the biosynthesis of terpenoids in C. blinii.

ß-Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae.

Conyza blinii H.Lév. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane-type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from ß-amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a ß-amyrin synthase, which mediates cyclization of 2,3-oxidosqualene to yield ß-amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative ß-amyrin synthase, named CbßAS. The open reading frame of CbßAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of ß-amyrin synthases. Transcription of CbßAS was upregulated 4-24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbßAS in Saccharomyces cerevisiae successfully yielded ß-amyrin. The chemical structures and concentrations of ß-amyrin were confirmed by GC-MS/MS. The target yeast ultimately produced 4.432 mg·L-1 ß-amyrin. Thus, CbßAS is an OSC involved in conyzasaponin biosynthesis.