| Dragon’s Blood is a traditional Chinese medicine with anti-inflammatory,analgesic,hemostasis,antibacterial and anti-tumor pharmacological activities.At present,there are many studies on the chemical composition,pharmacological activity and clinical application of Dragon’s Blood,and the mechanism of its formation is still unclear.Dracaena camhodiana(Dracaena cambodiana Pierre ex Gagn.)is the main source of Chinese Dragon’s Blood,and its main active ingredient in the Dragon’s Blood is mainly flavonoids.The biosynthesis of plant fiavonoid compounds is controlled by structural genes(key enzyme genes for the synthesis of flavonoid compounds)and regulatory genes(transcription factors).bHLH transcription factors are widely present in plants and play an important regulatory role in the biosynthesis of flavonoids.In this study,the Dracaena cambodiana bHLH genes was recognized at the transcriptome level,and a bHLH gene DcbHLH5 related to flavonoid accumulation was identified,and the mechanism of its regulation of flavonoid synthesis was studied.This study laid the foundation for revealing the flavonoid biosynthesis of Dracaena cambodiana and the formation mechanism of Dragon’s Blood.Based on transcriptome data and genomic mapping sequences,45 bHLH genes were identified in Dracaena cambodiana with a complete open reading frame.The protein encoded by DcbHLHs contains 143-762 amino acids with a molecular weight between 12.75 and 83.85 kDa.There are 28 isoelectric points(PI)less than 7,and 17 of them greater than 7.Both bHLH protein families contain a highly conserved bHLH domain(helix-loop-helix).There are differences in the number of exons in DcbHLHs,and most of them are multi-exon structures.Phylogenetic tree analysis indicated that DcbHLHs were distributed in five of the 12 categories,of which DbbHLH5 was clustered with other flavonoid-related bHLHs in the Illf subclass.The bHLH domain of the Illf subclass contains H5一E9-R13 active sites and is involved in the regulation of flavonoid biosynthesis.The results of gene expression analysis based on transcriptome data showed that most of the DcbHLHs gene expression was up-regulated or down-regulated after induction by a blood-inducing agent.Real-time PCR showed that DcbHLH5 gene expression was induced by four hormones,JA,ABA,6-BA and BR.DcbHLH5 had the highest expression after ABA treatment and the most positive response to 6-BA and MeJA.The results of stress treatment showed that the expression of DcbHLH5 gene was induced by UV-B and NaCI,but not by PEG and Sucrose stress.The DcbHLH5 gene expression was highest after UV-B treatment and the response was most positive.Subcellular localization analysis indicated that DcbHLH5 was localized in the nucleus.It was shown by yeast one-hybrid assay that DcbHLH5 was able to bind to the promoters of DcCHS1,DcCHS2 and DcCHI1.Dual luciferase transient expression analysis indicated that DcbHLH5 was able to enhance the promoter activity of DcCHSl,DcCHS2 and DcCHI1 The yeast two-hybrid assay showed that DcbHLH5 could interact with DcWD40-1.It is speculated that DcbHLH5 and DcWD40-1 may synergistically regulate the expression of multiple flavonoid structural genes involved in the biosynthesis of flavonoids. |
