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Basic helix–loop–helix (bHLH) proteins, one of the most important and largest transcription factor family in plants, play important roles in regulating growth and development, stress response. In recent years, many bHLH family genes have been identified and characterized in woody plants. However, a systematic analysis of the bHLH gene family has not been reported in Ginkgo biloba , the oldest relic plant species. In this study, we identifed a total of 85 GbbHLH genes from the genomic and transcriptomic databases of G. biloba , which were classified into 17 subfamilies based on the phylogenetic analysis. Gene structures analysis indicated that the number of exon–intron range in GbbHLHs from 0 to 12. The MEME analysis showed that two conserved motifs, motif 1 and motif 2, distributed in most GbbHLH protein. Subcellular localization analysis exhibited that most GbbHLHs located in nucleus and a few GbbHLHs were distributed in chloroplast, plasma membrane and peroxisome. Promoter cis-element analysis revealed that most of the GbbHLH genes contained abundant cis-elements that involved in plant growth and development, secondary metabolism biosynthesis, various abiotic stresses response. In addition, correlation analysis between gene expression and flavonoid content screened seven candidate GbbHLH genes involved in flavonoid biosynthesis, providing the targeted gene encoding transcript factor for increase the flavonoid production through genetic engineering in G. biloba .

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Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in southern China. The biosynthetic and metabolic pathways for a core secondary metabolite in L. macranthoides, chlorogenic acid (CGA), have been elucidated in many species. However, the mechanisms of CGA biosynthesis and the related gene regulatory network in L. macranthoides are still not well understood. In this study, CGA content was quantified by high performance liquid chromatography (HPLC), and CGA levels differed significantly among three tissues; specifically, the CGA content in young leaves (YL) was greater than that in young stems (YS), which was greater than that in mature flowers (MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads (average length 90 bp) and 76,453 unigenes (average length 703 bp). A total of 3,767 unigenes were involved in biosynthesis pathways of secondary metabolites. Of these unigenes, 80 were possibly related to CGA biosynthesis. Furthermore, differentially expressed genes (DEGs) were screened in different tissues including YL, MF and YS. In these tissues, 24 DEGs were found to be associated with CGA biosynthesis, including six phenylalanine ammonia lyase (PAL) genes, six 4-coumarate coenzyme A ligase (4CL) genes, four cinnamate 4-Hydroxylase (C4H) genes, seven hydroxycinnamoyl transferase/hydroxycinnamoyl-CoA quinate transferase HCT/HQT genes and one coumarate 3-hydroxylase (C3H) gene.These results further the understanding of CGA biosynthesis and the related regulatory network in L. macranthoides.

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