Functional Characterization Of SmAP2/ERF82 In Regulating The Biosynthesis Of Tanshinones And The Growth And Development Of Salvia Miltiorrhiza

Salvia miltiorrhiza is a traditional Chinese herbal medicine,and its active ingredient tanshinone is widely used to treat cardiovascular and cerebrovascular diseases.Therefore,the research on tanshinone biosynthesis and regulation has been a hot research area.In this study,a transcription factor named SmAP2/ERF82 which was highly expressed at the root was selected from the transcriptome data of S.miltiorrhiza based on the previous study in our lab.The expression profile of this gene detected by real-time quantitative PCR confirmed that SmAP2/ERF82 was highly expressed in the root and the phloem of S.miltiorrhiza.Protein subcellular localization experiments showed that SmAP2/ERF82 was localized in the nucleus,and it does not have self-activating activity.The recombinant plasmids of SmAP2/ERF82-RNAi and over-expressed(SmAP2/ERF82-oe)were constructed and transformed into S.miltiorrhiza induced by Agrobacterium tumefaciens ACCC10060,and the transgenic hairy roots of S.miltiorrhiza were obtained.By examining the efficiency of SmAP2/ERF82 gene suppression and overexpression in transgenic hair roots,a total of three RNAi lines(82i-1/5/9)and three overexpression lines(82oe-3/5/7)were screened.We used UPLC to detect the content of tanshinone compounds in SmAP2/ERF82-RNAi and overexpression transgenic hairy roots;and salvianolic acid compounds in RNAi lines.The results showed that the content of dihydrotanshinone Ⅰ and tanshinone Ⅰ was decreased in the SmAP2/ERF82-RNAi lines,while the content of these two compounds was increased significantly in the SmAP2/ERF82-overexpression lines,in comparison to the control lines,indicating that SmAP2/ERF82 positively regulated the biosynthesis of tanshinones in S.miltiorrhiza.However,the content of salvianolic acids in SmAP2/ERF82-RNAi lines was not different with that in control lines,suggesting that SmAP2/ERF82 had no effect on salvianolic acid biosynthesis and metabolism.To further analyze the regulatory mechanism of SmAP2/ERF82 in tanshinone biosynthesis,we analyzed the expression levels of key genes involved in the tanshinone biosynthetic pathway in the transgenic hairy root lines.The results showed that the expression levels of key enzyme-coding genes CPS1 and CYP76AH3 in SmAP2/ERF82-RNAi strains were significantly reduced,while the expression levels of IDI1 and CPS1 were increased in SmAP2/ERF82-overexpression lines(except 82oe-7 line).We found SmAP2/ERF82 binded to the promoter regions of IDI1,CPS1 and CYP76AH based on yeast one-hybrid analysis,indicating that SmAP2/ERF82 regulates tanshinone biosynthesis by regulating the expression of key enzyme genes in tanshinone biosynthetic pathway.The recombinant plasmids of SmAP2/ERF82-RNAi and SmAP2/ERF82-oe were transformed into agrobacterium EH105,and then infected leaves of S.miltiorrhiza to obtain transgenic tissue culture seedlings.One RNAi lines(82i-6)and three overexpression lines(82oe-1/15/16)were obtained.Compared with the control line(pki),the growth of SmAP2/ERF82-RNAi lines was normal.However,compared with the control line(pkoe),the SmAP2/ERF82-overexpression lines grew dwarfly,with the small and shriveled leaves and the relatively sparse roots,that indicated SmAP2/ERF82 might play essential role in regulation of the morphological development of S.miltiorrhiza.To further verify the role of SmAP2/ERF82 in regulation of the growth and development of S.miltiorrhiza,the SmAP2/ERF82 overexpressed recombinant plasmid was transformed into Arabidopsis thaliana induced by agrobacterium GV3101.The phenotype of T1 generation of the control lines was normally,while the SmAP2/ERF82 overexpressed lines grew short and were less bolting,indicating that the overexpression of SmAP2/ERF82 gene in A.thaliana affected the bolting process and the development of A.thaliana.The seedling of salvia miltiorrhiza grown from transgenic hairy roots were used to detect the changes in gene expression between the transgenic lines and control lines based on the Illumina deep RNA sequencing(RNA-seq)strategy.The results showed that the expression levels of GA20ox and GA3ox,the key enzyme genes in the gibberellin biosynthesis pathway,were up-regulated in the RNAi lines,while the expression level of GA20ox and GA2ox were down-regulated in the overexpressed lines,suggesting that SmAP2/ERF82 may be involved in gibberellin biosynthetic pathway.In summary,SmAP2/ERF82 plays a positive role in regulating the expression levels of key enzyme genes IDI1,CPS1 and CYP76AH3 to further regulate the biosynthesis of tanshinons in S.miltiorrhizae.In addition,SmAP2/ERF82 may regulate plant growth and development by negatively regulating the biosynthesis of gibberellin.This study identified the function of SmAP2/ERF82 transcription factor and clarified its role in the regulation of tanshinone biosynthesis and the plant development,which will facilitate to explore the produce of tanshinones by genetic engineering and the cultivation of S.miltiorrhiza in future.