| Objective:Mining the key proteins of the JAZMONATE ZIM-domain protein family in patchouli based on the transcriptome for preliminary bioinformatics analysis,cloning and characterization;Identifying systematically patchouli PcJAZ6 and analyzed its exogenous methyl jasmonate(MeJA)induced expression pattern;Detecting the protein interaction between downstream transcription factors and PcJAZ6,and conducting a preliminary analysis of the effect of patchoulol biosynthesis with the help of transient overexpression;Constructing Virus-induced Gene Silencing(VIGS)technology in patchouli to verify the function of PcJAZ6 gene to regulate PcPTS expression and patchouli alcohol synthesis.Methods:1.Spraying patchouli seedlings with 300 μM MeJA for 8 hours,extracting RNA for transcriptome sequencing,and mining key proteins of patchouli JAZ family for cloning and characterization.2.Bioinformatics analysis was performed on PcJAZ6 using MEGA and DNAMAN software,and Real-time q-PCR was used to detect the expression of PcJAZ6 after MeJA treated plants to analyze the response changes at different treatment times,and the protoplast system was used to study its subcellular localization.3.Yeast two-hybrid(Y2H)and Firefly Luciferase Complementation Imaging Assay(LCI)were used to verify the transcription factors interacting with PcJAZ6.The original plant transient overexpression technology was used to detect and analyze the effect of PcJAZ6 on the regulation of patchouli alcohol synthase gene expression,and the change in patchouli alcohol content was detected by gas chromatography-mass spectrometry(GC-MS)for preliminary functional analysis.4.Using the TRV virus and PcPDS gene,construct the patchouli VIGS technology,through which the PcJAZ6 gene can be quickly silenced,and its participation in gene expression and patchouli alcohol synthesis function verification.Results:In this study,we identified six JAZ proteins,PcJAZ3,PcJAZ4,PcJAZ6,PcJAZ7,PcJAZ10,and PcJAZ11 through transcriptome data,and obtained gene cloning and protein characterization.The PcJAZ6 protein of patchouli was systematically cloned and identified,and it was found that PcJAZ6 was localized in the nucleus,and the gene expression level was up-regulated by induction of exogenous MeJA.PcJAZ6 interacts with the transcription factors PcMYC2bl and PcMYC2b2.Overexpression of PcJAZ6 reduces the biosynthesis of patchouli alcohol.A virus-induced gene silencing system in patchouli was established and evaluated.Using the TRV virus vector and the endogenous PcPDS gene of patchouli as an indicator,a decrease in gene expression was detected after 42 days of silencing,and a leaf albino phenotype was observed.Based on virus-induced PcJAZ6 silencing,the relative expression level of PcJAZ6 was significantly reduced compared to the control,while the expression of PcPTS was up-regulated by about 80%.Moreover,the relative expression of the transcription factors PcMYC2b1 and PcMYC2b2 interacting with PcJAZ6 increased,resulting in a significant increase in the synthesis and accumulation of patchouli alcohol.Based on this,we have drawn a mechanism model of patchouli PcJAZ6 participating in JA signal-mediated regulation of patchouli alcohol synthesis.Conclusion:For the first time,we have isolated and identified one of the JAZ proteins in the patchouli:PcJAZ6.PcJAZ6 was strongly induced by MeJA and localized in the nucleus.PcJAZ6 has significant interactions with PcMYC2bl and PcMYC2b2 in yeast systems and plants.Transient overexpression of PcJAZ6 in patchouli original plants resulted in varying levels of PcPTS,PcMYC2b1 and PcMYC2b2 reductions,which reduced the accumulation of patchouli alcohol.The patchouli VIGS technology system was successfully established and evaluated and applied to the gene silencing of PcJAZ6,resulting in a significant increase in patchouli alcohol content.In summary,PcJAZ6 acts as a repressor to inhibit the activity of transcription factors PcMYC2bl/PcMYC2b2,regulate down stream PcPTS gene expression,and then affect JA signal-mediated patchouli alcohol synthesis. |
