The Molecular Mechanism Of Transcriptional Regulation Of Key Genes PnSS And PnSE In The Synthesis Of Panax Notoginseng Saponins

Panax notoginseng(Burk.)F.H.Chen is a perennial herb of the Araliaceae.It has been cultivated in China for more than 400 years.Saponins are the main active components of P.notoginseng,which have various effects such as stanching bleeding,anti-thrombosis,anti-inflammatory,and anti-tumor.The synthetic pathway of saponins had been elucidated,among which PnSS and PnSE were the key enzyme genes in the saponin synthesis pathway,but the transcriptional regulation mechanism in saponin biosynthesis was still unknown.In this study,the upstream promoters of the PnSS and PnSE genes of P.notoginseng were cloned based on previous research.The promoter sequence was used as the target sequence to screen the transcription factor that specifically regulated the biosynthesis of triterpene saponins in the c DNA library of P.notoginseng.The transcription mechanism has been preliminarily discussed in this study.The main research conclusions were as follows:1.The promoter sequence of 1582 bp upstream of the PnSS gene and the promoter sequence of 938 bp upstream of the PnSE gene were cloned.The promoter structure was analyzed by online software.It was found that it not only included the basic structural elements TATA-box,CAAT-box,etc.,but also included a variety of cis-acting elements,such as growth regulation elements,light response elements,and hormone responsive elements such as abscisic acid(ABA)and gibberellin(GA)response,MYB binding components and anaerobic components(ARE),etc.The promoter sequence was constructed on the plant expression vector fused with the GUS gene,and the tobacco transient expression system was used,and the corresponding hormones were sprayed exogenously and respectively.It was found that the PnSS and PnSE gene promoters can respond to exogenous ABA,GA,Eth and SA signals.The existence of corresponding hormone response elements on the promoter was verified.2.The PnSS and PnSE promoter fragments were constructed into the bait vector of yeast one-hybrid,and transformed into Y1 H yeast,which was screened for the lowest aureobasidin A(Ab A)inhibitory concentration.The results showed that the bait strain SS1262 containing a 231 bp PnSS promoter fragment can be inhibited by 100 ng/m L Ab A,and the bait strain SE697 containing a 297 bp PnSE promoter fragment can be inhibited by 400 ng/m L Ab A,and other bait strains cannot be inhibited by 1000 ng/m L Ab A.These two yeast strains were named YS1262 and YE697 respectively,and used for screening of P.notoginseng c DNA library.Finally,two MYB transcription factors Pn MYB2 and MYB-like were screened,and one WRKY transcription factor Pn WRKY1 was screened.The genes encoding ORF107 A,DNK,TP,COX11,GYRA,23 S r RNA and DCD proteins were also screened.3.The full length of ORF regions of 8 genes ORF107 A,MYB2,TP,WRKY1,DNK,23 S r RNA,COX11,DCD were cloned,and they were transferred into yeast and X-gal reaction was used for mutual verification.The results showed that ORF107 A,TP,DNK and COX11 proteins interacted with PnSS promoter fragments,23 S r RNA and DCD proteins interacted with PnSE promoter fragments,MYB2 and WRKY1 interacted with PnSS and PnSE.4.The prokaryotic expression vector containing DNK,COX11,DCD,MYB2 gene was constructed and expressed in BL21(DE3)Escherichia coli strain,37 KD COX11,50 KD DCD and 45 KD MYB2 His tag fusion proteins were obtained,all of which belong to inclusion body proteins.The Pn MYB2 protein was purified and probes were designed for EMSA verification experiments.The results of EMSA showed that Pn MYB2 protein can bind to the CAACTG motif present on the PnSS promoter and the ACCAAC motif on the PnSE promoter,which proved that Pn MYB2 belonged to MYB transcription factor and can co-regulate PnSS and PnSE genes.5.The GFP fusion vector containing DNK,COX11,DCD and MYB2 genes was constructed and transfected into Agrobacterium tumefacien to infect tobacoo.The subcellular localization was observed by laser confocal microscopy.The subcellular localization results indicated that DNK and COX11 were located in the chloroplast,DCD was located in the nucleus and cytoplasm,and MYB2 was located in the nucleus.