Transcriptome-based Discovery of AP2/ERF Transcription Factors Related to Terpene Trilactones Synthesis in Ginkgo biloba

Ginkgo biloba is a unique tree in China with medicinally and phylogenetically important characteristics. Terpene trilactones (TTL) is a key active pharmaceutical ingredient in Ginkgo, so the content of TTL in Ginkgo has become one of the important indices for evaluating quality of the medicinal materials. By transcriptome sequencing on samples treated by chlormequat, ultraviolet (UV) and drought, totally 59820 contigs and 37564 unigenes were obtained. Furthermore, 18234 unigenes were annotated through COG, KEGG and GO analysis. There were 78 AP2/ERF transcription factors, 23 factors of up-regulation and 66 factors of down-regulation that were related with synthetic pathway of TTL in Ginkgo. Phylogenetic tree clustering analysis indicated that there were 42 AP2s could be clustered into ERF, DREB and RVA subfamilies. EMSA analysis demonstrated that GbERF13, GbERF25 and GbERF27 could bind with regulatory elements, such as E-box, in the upstream of GbMECPs promoter. Expression analysis showed that the expression level of GbERF25 was the highest in root, and GbERF25 and GbERF27 were expressed in relatively high transcription levels in leaf and other tissues. The results of qRT-PCR indicated that CCC treatment could significantly improve expression levels of ERF25 and ERF27, and UV and drought could induce transcription levels of ERF13 and ERF25, respectively. The results implied that ERF25 and ERF27 might involve in the induction and regulation of CCC treatment on synthesis of bilobalide in G. biloba. ERF13 might participate in the regulation of bilobalide synthesis induced by UV, and EFR25 might involve in the regulation of the synthesis induced by drought. During annual cycle of expression, the transcription levels of ERF13, ERF25 and ERF27 had significantly positive correlation with diterpene level with correlation coefficient 0.975. It implied that these transcription factors mainly acted on the MEP pathway that regulated synthesis of bilobalide. The aim of the research was to indicate the mechanism of environment or cultivation measure regulating target gene of TTL metabolic pathway by AP2/ERF, and establish metabolic network of AP2/ERF regulating TTL synthesis.