Study On The Transcriptional Regulation Of SmCPS1 And SmKSL1 Are Involved In Tanshinone Biosynthesis

Salvia miltiorrhiza,is a traditional Chinese herbal.Tanshinone is one of the main medicinal ingredients in S.miltiorrhiza,and is widely used in modern medicine.At present,the tanshinone synthesis pathway has been in-depth research and improvement.SmCPS1 and SmKSL1 are two key gene in downstream of the tanshinone biosynthesis.While,the tanshinones biosynthesis specific transcriptional regulation is unclear.This study uses transcriptional regulation theory to finding the transcription factors which specific regulate tanshinone biosynthesis.To further explore the regulation of tanshinone biosynthesis pathway,this research take promoter SmCPS1 and SmKSL1 gene as target to selecting transcription factors which specific regulate tanshinones biosynthesis from S.miltiorrhiza cDNA Library.Then,the functional researches of the screened transcription factors were carried on.The main conclusions as following:(1)Seven S.miltiorrhiza landscares which had different tanshinone contents were selected to analysis the association between their gene expression level of SmCPS1 and SmKSL1 and their tanshinone contents.The results showed that SmCPS1 and SmKSL1 gene expression had significant positive correlation with tanshinone IIA and tanshinone I.The subcellular localization of two key genes showed that they were located in the plastid.(2)In this study,the promoter region upstream of the SmCPS1 and SmKSL1(KY937191 and KY937192)were cloned,1057 bp and 876 bp respectively.The online prediction and analysis results of their features showed that two promoters region contained the basic structural elements(TATA-box,GC island,CAAT),growth and development necessary elements,some specific response to hormonal signals elements(GARE,GCC-box,P-box),MYB transcription factor binding sites(MBS)and anaerobic response element(ARE)etc.The PGL3 dual luciferase system was used to test activities of two promoters.The results showed that their activity enhanced with their length increases.Then,we used tobacco transient expression system to verify the SmCPS1 and SmKSL1 promoter were sensitive to exogenous GA and Eth signal(p<0.01).(3)The SmCPS1(826 bp)and SmKSL1(649 bp)promoter were cloned into yeast one hybrid bait plasmid.Then,the constructed plasmid transformed into yeast and named PYC826 and PYK649 respectively.The minimum AbA concentration(200 ng/mL and 700 ng/m L respectively)was selected in yeast deficiency culture medium(SD/-Ura).Then,the yeast Y1 Hgolden system was used to screen transcription factors from S.miltiorrhiza cDNA library.The results showed that a total of 10 transcription factors were screened out,and they are belongs to the AP2/ERF gene family.Their annotation respectively as following:KY988300,MH006593,MH006594,MH006595,MH006596,MH006597,MH006598,MH006599,MH006600,MH006601.(4)In this research,two full-length AP2/ERF family transcription factor genes were cloned.According to the similarity of the nucleotide sequence with arabidopsis ERF genes,they were named as SmERF6(KY988300)and SmERF8(MH006600).The ORF regions are 561 bp and 654 bp respectively.Their structure contain an AP2 domain.Phylogenetic analysis of SmERF6 and SmERF8 showed that they were classified to IXb and VIIa ERF subfamily respectively.And the subcellular localization results of SmERF6 and SmERF8 showed they were expressed in the nucleus.And two genes were responsive to exogenous Eth and GA treatments.Tissue specific expression of SmERF6 and SmERF8 showed their highest expressed in root and root head respectively.(5)The interaction between SmERF6,SmERF8 and the promoter region of SmCPS1 and SmKSL1 were positive which verified by the yeast one hybrid in vivo.By the 0.3 mM or 0.4 mM IPTG induced the expression of SmERF6 and SmERF8 in Escherichia coli Rossetta strains.The purification protein of SmERF6 and SmERF8 with MBP tag were obtained.SmERF6 and SmERF8 proteins are respectively 62 KD and 66 KD.And their MBP tag fusion protein verified for Werstern blotting.In vitro,the interaction between GCC-box and SmERF6 and SmERF8 protein verified by EMSA.(6)Using the genetic transformation system of S.miltiorrhiza hairy root,SmERF6 and SmERF8 were overexpressed in S.miltiorrhiza hairy roots.By PCR identification,we have successfully obtained positive SmERF6 and SmERF8 over-expression hairy root lines.In transgenic lines,the expression level of SmERF6 and SmERF8 significantly higher than control(p<0.05).The SmCPS1 and SmKSL1 gene expression also significantly increased(p<0.05).The tanshinone content(dihydrotanshinone,cryptotanshinone,tanshinone I and tanshinone IIA)significantly increased in SmERF6 and SmERF8 overexpression hairy root lines(p<0.05).The total phenolic acid and total flavonoids content decreased,at the same time,the growth of hairy roots was significantly inhibited(p<0.05).(7)In order to further study the function of SmERF6 and SmERF8,we used artificial target microRNA silencing experiments(RNAi)to interfering the expression of SmERF6 and Sm ERF8 in the S.miltiorrhiza hairy roots system.The results showed that amiRSmERF6 and amiRSmERF8 were the successful overexpreessed in S.miltiorrhiza hairy roots,and the expression of amiRSmERF8 and amiRSmERF6 were significantly increased(p<0.05),the expression level of SmERF6 and SmERF8 were significantly decreased(p<0.05).Then,the expression level of SmCPS1 and SmKSL1 had also been down-regulation(p<0.05).The HPLC results showed that the tanshinone contents were significantly decreased in the ami RSmERF6 and amiRSmERF8 overexpression lines(p<0.05).However,there was a homeostasis between tanshinone and total phenolic acid and total flavonoids content.The growth of hairy roots were normal.(8)In addition,the cis-elements analysis of SmCPS1 and SmKSL1 promoter showed that they contained the GRAS transcription factor binding element which is specific response to GA.Therefore,we retrieved 28 EST sequence which contained the GRAS domain in S.miltiorrhiza transcriptome database.We obtained 5 full-length SmGRAS genes by RT-PCR,and they were named Sm GRAS1~5(KY435886,KY435887,KY435888,KY435889,KY435890).The expression of five genes exogenous could induced by gibberellin(GA)and ethephon(Eth)treatmhent(p<0.05,p<0.01).SmCPS1 and SmKSL1 were also sensitive to these two exogenous signals(p<0.05,p<0.01).At the same time,the accumulation of tanshinone contents were also induced in S.miltiorrhiza hairy root.In summary,this study was aim to regulate tanshinone biosynthesis by the transcriptional regulation of SmCPS1 and SmKSL1.We used the SmCPS1 and SmKSL1 promoter as the target to screening out 10 transcription factors.Further research cleared up that SmERF6 and SmERF8 were involved in tanshinone biosynthesis by specific regulated gene expression of SmCPS1 and SmKSL1,and their function verified by RNAi.Meanwhile,we made a preliminary exploration on SmGRAS gene which were candidate transcription factors in tanshinone biosynthesis.This study provides a reference for tanshinone biosynthesis and regulation mechanism,and provides a reliable reference for obtain of high tanshinone production and excellent germplasm resources breeding by biotechnology in S.miltiorrhiza.