Functional Identification Of AP2/ERF Transcription Factor Involved In The Regulation Of Bioactive Compound Biosynthesis In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge.is one of the important medicinal plants and used widely as the traditional Chinese medicine(TCM).Tanshinone and salvianolic acid are the major bioactive ingredients,possessing various pharmacological activities.The medicine"Danshen dripping pills" which contains S.miltiorrhiza as the major medicinal material has a remarkable curative effect on cardiovascular and cerebrovascular diseases.The biosynthetic pathways of tanshinone and phenolic acid compounds in S.miltiorrhiza have been studied extensively,but the mechanism of regulation is still largely unknown.AP2/ERF transcription factors have been verified to be involved in the regulation of the bioactive ingredients in various medicinal plants,including sesquiterpenes artemisinin,diterpene paclitaxel,etc.However,the AP2/ERF playing roles in regulation of bioactive compound biosynthesis in S.miltiorrhiza has not been identified.The main results of this research are as below:1.The genome of S.miltiorrhiza has been completely sequenced in the previous study,and 170 AP2/ERF transcription factors have been identifed,named Sm001-Sm170.Based on the transcriptome data of different organs(root,stem,leaf and flower)and root tissues(pericarp,phloem and xylem)of S.miltiorrhiza,two AP2/ERF genes were selected.The expression profile of Sm008 was consistent with that of SmRAS1 gene,which participates in the biosynthesis of phenolic acid compounds;while Sm082 was highly expressed in the root.The expression profiles of two AP2/ERF genes were also verified by qRT-PCR method.The nucleus-subcellular localization of these two putative AP2/ERF transcription factors were detected by the transient transformation of tobacco.2.In this study,the genetic transformation system of S.miltiorrhiza has been constructed,and the transgenic hairy roots of S.miltiorrhiza have also been obtained.To analyze the efficiency of these two genes transformed into hairy roots of S.miltiorrhiza,the expression level of Sm008 was detected in the transgenic hairy roots of the over-expression lines and RNAi lines.Totally,three RNAi lines(8i-1/3/6)and over-expression lines(8oe-1/8/9)of Sm008 were selected.Compared with the control line,the content of salvianolic acid B was decreased in the three RNAi lines and was increased in the three over-expression lines of Sm008.In addition,the color of Sm008 transgenic root hairy and the liquid culture medium looks like the color of tanshinone compounds.Therefore,the content variation of tanshinone in the hairy roots and liquid culture medium were tested for the RNAi transgenetic lines of Sm008.The results showed that the contents of dihydrotanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA were also decreased in both hairy roots and culture medium of two RNA lines(8i-1/3),compared with the control line.The results indicated that Sm008 gene could regulate the biosynthesis of tanshinone and salvianolic acid compounds.The expression level of genes in the biosynthesis of tanshinone and salvianolic acid in transgenic hairy roots were analyzed in order to predict the target genes of transcription factor.The results showed that Sm008 may regulate 4CL2 in the biosynthesis of the salvianolic acid,or regulate CPS1,KSL1 and CYP76AH1 in the biosynthesis of tanshinone.3.Four RNAi lines(82i-1/5/7/19)and one ove-expression line(82oe-9)of Sm082 were selected.The contents of dihydrotanshinone I,cryptotanshinone,tanshinone I and tanshinone IIA were decreased in the four RNAi lines of Sm082 and the contents of dihydrotanshinone I and cryptotanshinone were increased in the overexpression line of Sm082.Meanwhile,the content of salvianolic acid B have not been effected by the expression level of Sm082 in transgenic hairy roots.The results indicated that Sm082 gene regulate the biosynthesis of tanshinones.The analysis of expression level of genes in the biosynthesis of tanshinone showed that Sm082 may regulate CPS1 and KSL1.In this study,the transgenic plantlets corresponding to the Sm082 transgenic hairy roots were also obtained.The fluorescence of the roots and leaves of the transgenic plantlets and the gene expression were detected.The results showed that the transgenic plantlets still carry the GFP reporter gene and maintained the inhibition/over-expression of Sm082 gene.4.The heterologous expression of Sm008 and Sm082 in the E.coli system was carried out.Only Sm008 can be detected as soluble protein.Sm082 was not induced in the supernatant and precipitation.Furthermore,the GCC box was identified from the promoter of the key genes encoding enzymes involved in the biosynthetic pathway of tanshinone and phenolic acid compounds.The promoter of SmRAS1 and SmTATl,both of that are involved in phenolic acid compound biosynthesis,and the promoter of SmCPSl,that participates in tanshinone biosynthesis,were cloned to be used for the target gene identification for these two transcription factors.The results showed that Sm008 and Sm082 transcription factor played an important roles in regulation of salvianolic acid and tanshinone biosynthesis.The molecular mechanism of the function for these two genes need to be explored and confirmed in the following work.This study provides the basic data for illustration on the regulation mechanism for bioactive compound biosynthesis and accumulation in S.miltiorrhiza.