ABSTRACT
Paclitaxel is an important anticancer drug. The phytohormone jasmonic acid can significantly induce the biosynthesis of paclitaxel in Taxus, but the molecular mechanism has not yet been resolved. To establish the jasmonic acid signalling pathway of Taxus media, based on the gene of the jasmonic acid signalling pathway of Arabidopsis thaliana, sequence analysis was performed to isolate the jasmonic acid signal from the transcriptome, a transcriptional cluster of pathway gene homologs and the full length of 22 genes were obtained by RACE PCR at 5' and 3': two EI ubiquitin ligase genes, COI1-1 and COI1-2;7 MYC bHLH type transcription factor (MYC2, MYC3, MYC4, JAM1, JAM2, EGL3, TT8); 12 JAZ genes containing the ZIM domain; and MED25, one of the components of the transcriptional complex. The protein interaction between each were confirmed by yeast two hybridization and bimolecular fluorescence complementation based on similar genes interaction in Arabidopsis. A similar jasmonate signaling pathway was illustrated in T. media. All known paclitaxel biosynthesis genes promoters were isolated by genome walker PCR. To investigate the jasmonate signaling effect on these genes' expression, the transcription activity of MYC2, MYC3 and MYC4 on these promoters were examined. There are 12, 10 and 11 paclitaxel biosynthesis genes promoters that could be activated by MYC2, MYC3 and MYC4.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cyclopentanes/metabolism , Oxylipins/metabolism , Paclitaxel/biosynthesis , Plant Proteins/metabolism , Taxus/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/classification , Basic Helix-Loop-Helix Transcription Factors/genetics , Phylogeny , Plant Proteins/classification , Plant Proteins/genetics , Promoter Regions, Genetic , Signal Transduction , Two-Hybrid System TechniquesABSTRACT
Jasmonate, an effective elicitor, can induce the biosynthesis of paclitaxel, a well-known anticancer drug, in Taxus cell culture. The jasmonate signaling pathway has been well studied in Arabidopsis, and many early jasmonate-responsive genes have been found to be involved in signaling pathway. In Taxus, only a few late jasmonate-responsive genes related to paclitaxel biosynthesis were identified. So, identification of early responsive genes and knowledge of the jasmonate signaling pathway are essential for understanding the effects of jasmonate on paclitaxel biosynthesis and for improving paclitaxel production in Taxus cells. In this study, total RNA of Taxus × media cells cultured in liquid medium was extracted after 0, 0.5, 3, and 24 h of methyl jasmonate treatment. Three biological independent repetitions were performed. The 12 extracted RNA samples were integrated and sequenced on an Illumina HiSeq 2500 platform using the paired-end method. A total of 45,583 transcript clusters were obtained by de novo assembly of the sequenced reads. Based on the transcriptome data, the digital gene expressions of each RNA sample were investigated. We found that after 0.5, 3, and 24 h of methyl jasmonate treatment; 134, 1008, and 987 unigenes were differentially expressed. For the secondary metabolism pathways, phenylalanine pathway unigenes were responsive to jasmonate after 3 h of treatment, while genes related to paclitaxel biosynthesis were induced after 0.5 h of treatment. The digital gene expression levels of candidate genes related to paclitaxel biosynthesis were confirmed by qRT-PCR. Transcriptome sequencing and digital gene expression profiling identified early jasmonate-responsive genes in cultured Taxus × media cells. The comprehensive time series jasmonate-responsive gene expression data have provided transcriptome-wide information about the mechanism of paclitaxel biosynthesis regulation by jasmonate signaling.