Transcriptional Regulation Mechanism Of EGCG Biosynthetic Genes CsLAR And CsUGT84A In Camellia Sinensis(L.)O.Ktze

EGCG is not only an important secondary metabolite of tea plants,but also a major component in determining the quality and physiological activity of tea leaves,but its biosynthesis and transcriptional regulation mechanism are not yet clear.However,the mechanisms of EGCG biosynthesis and transcriptional regulation are still unclear.Therefore,it is important to study the regulatory mechanism of EGCG biosynthesis in Camellia sinensis(L.)O.Ktze.Based on our previous study,transcriptomes of tea leaves were sequenced after sprayed with nano-selenium fertilizers.The profilings of differential expression genes were obtained from transcriptome data.Two transcriptional factors(TFs)were selected and identified which might regulate EGCG biosynthesis.The characteristics of these two TFs were analyzed using bioinformatics.In addition,the molecular mechanisms of these two TFs were further investigated and regulated the expression of the biosynthesis gene of EGCG.The major results are as followed:1.Based on the transcriptome data,five TFs Csb HLH46,CsWRKY70,CsMYB106-like,CsMYB88,and CsMYC2-like were selected.The full-length CDS regions of CsWRKY70 and CsMYB88 were cloned successfully.Amino acid sequence alignment and phylogenetic analysis showed that CsWRKY70 has a typical WRKYGQK heptapeptide sequence followed by a C2 HC zinc finger structure,which belongs to class III of the WRKY family.Further,CsMYB88 has an R2R3 domain and it belongs to the R2R3 subfamily of MYB.Subcellular localization showed that both CsWRKY70 and CsMYB88 were localized in the nucleus and belonged to nuclear proteins.Transcriptional activity analysis showed that CsWRKY70 was a transcriptional activator with transcriptional activation activity,while CsMYB88 has no self-activating activity.2.Real-time quantitative polymerase chain reaction(q RT-PCR)showed that the expression levels of CsWRKY70 and CsMYB88 were reduced compared to the control group(CK)after sprayed with nano-selenium fertilizers in tea leaves.Meanwhile,the expression levels of CsLAR and CsSCPL1 A were decreased and the expression level of CsUGT84 A was increased.These results indicated that CsWRKY70 and CsMYB88 might influence the EGCG biosynthesis in tea leaves via regulating the gene expression of CsLAR,CsSCPL1 A and CsUGT84 A.3.The upstream promoter sequences of the CsLAR and CsUGT84 A were cloned.Cis-acting elements analysis showed that both CsLAR and CsUGT84 A promoters contained W-box(5’-(C/T)TGAC(T/C)-3’)and MYBRs(5’-TAACCA-3’)elements that could directly bind to both WRKY and MYB families TFs.4.The conserved domains of CsWRKY70(129-188aa)and CsMYB88(1-100aa)proteins were induced and purified using prokaryotic expression and protein purification methods.The molecular weights of the two recombinant proteins were 33.4 KDa and 37.6 KDa,respectively.Electrophoretic mobility shift assay(EMSA)revealed that CsWRKY70 and CsMYB88 proteins could specifically bind to the cis-acting elements in the promoters of CsLAR and CsUGT84 A genes.Dual-luciferase assay showed that CsWRKY70 could activate the transcriptional activity of the CsLAR promoter,while CsMYB88 could repress the transcriptional activity of the CsUGT84 A promoter.