High-throughput Sequencing Of Salvia Miltiorrhiza Transcriptome And Functional Study Of Some Genes Related To Tanshinone Synthesis

Salvia miltiorrhiza Bunge is a well-known medicinal plant in the Labiatae family.Its dry roots or rhizomes has such effects as removing blood stasis and odynolysis,activating blood to promote menstruation,cooling the blood,treating boils,etc.And it is used in the treatment of cardiovascular,cerebrovascular,hyperlipidemia,and acute ischemic stroke diseases.Up to date,more than 70 components have been isolated and structurally identified from Danshen.These active ingredients can be divided into two groups:one is lipid-soluble tanshinones,such as tanshinone ?,tanshinone?A,tanshinone ?B,and cryptotanshinone;and the other is water-soluble phenolic acids,including rosmarinic and salvianolic acids,etc.Because reproduction and genetic manipulation is simple and secondary metabolites are abundant for S.miltiorrhiza,it has become a model organism for study of medicinal plants now.Many studies have attempted to elucidate the biosynthesis of salvianolic acids and tanshinones in S.miltiorrhiza by cloning and characterizing the genes that encode for key enzymes.However,the genetic background and its regulators of biosynthesis pathways of these two group compounds,especially in their late steps,remain unclear.In addition,the molecules regulate transcription of key enzymes in pathways related to active ingredients are-also still unknown.Based on these problems,we performed our work as followings.(1)To obtain detailed and general data,we employed Solexa deep RNA sequencing(RNA-seq)to evaluate the transcriptome of S.miltiorrhiza throughout its life cycle.About 1.2Gb nucleotides were obtained and were assembled into 56,774 unigenes(GenBank accession No.SRA020132).In total,more than 200 unigenes(more than 60 novel genes)were identified which are related in the terpenoid backbone biosynthesis pathway and in diterpenoid biosynthesis.In all,523 unigenes related to 47 enzymes for the phenylpropanoid pathway and 156 unigenes for 17 enzymes in the tyrosine-derived pathway were obtained in this transcriptome database.Therefore,this broad coverage by these secondary metabolic genes provides more abundant information for examining the biosynthesis process of secondary metabolites in S.miltiorrhiza.(2)Digital expression profiles of MeJA and fungal elicitor treatments of S.miltiorrhiza were studied,and 2,131 and 1,652 different expression genes(DGE)were selected out respectively.13 and 4 DGEs were involved in the biosynthesis pathways of active ingredients.Among these DGEs,54 and 44 transcripts factor were also selected,which include many transcription factor families'members of MYB,AP2-EREBP,bHLH and WRKY.These families has associated with the regulation of secondary metabolism in other species,the biological function of specific TFs in S.miltiorrhiza need further research to determine.(3)Based on the analysis of Solexa high-throughput sequencing data,SmFPPS1,SmIPI1 and SmGGPPS3,were cloned and characterized using BD-walking PCR and RT-PCR.And then their characterizations,expression patterns were studied.?Length of SmFPPS1 is 1,320 by and consists of a 1,050 bp of the ORF encoding a polypeptide of 349 amino acid residues.SmFPPS1 composed with ?-helix is located in the cytoplasm and does not have the GGPP synthase activity.SmFPPS1 mainly are expressed in flowers and can be induced by MeJA and pathogens.? The full-length of SmIPI1 is 1,243 bp encoding a polypeptide of 226 amino acid residues.Structural analysis showed that SmIPI1 belonging to ?/? protein contains an IPP isomerase domain and shares high similar in their primary structure and active sites,etc.SmIPI1 was constitutively expressed throughout its life cycle and can be the induced by MeJA and pathogens.?The full-length of SmGGPPS3 is 2,908 bp,containing a 960 bp of ORF encoding 319 amino acids.SmGGPPS3 also including transfer isopentenyl transferase domain shares high identity with the GGPPS sequences reported in other plants.Furthermore,the result of genetic complementation expression of SmGGPPS3 in Escherichia coli revealed that SmGGPPS3 encoded a functional GGPP synthase.SmIPI1 was constitutively expressed in whole plant and can also be the induced by MeJA and pathogens.(4)38 transgenic plants of over-expression SmGG/PPS1 in S.miltiorrhiza were got using a procedure for Agrobacterium tumefaciens-mediated genetic transformation.Content of tanshinone IIA in the transgenic lines reached more than 2 folds comparing with control plants.By using real-time PCR,SmHMGR1,SmDXS1,SmDXS2,SmCPS2,SmGGPPS2 and SmGGPPS3 were up regulated in transgenic lines,revealing mechanisms of its high tanshinone ?A content.Compounds such as chlorophyll and carotenoids derived from GGPP also had high contents in transgenic lines.(5)Total tanshinone content and tanshinone ?A content reached 1.35?1.51 folds and 1.57?1.85 folds respectively in the S.miltiorrhiza transgenic lines of over-expression AtMYB40 comparing with control,and contents of water-soluble phenolic compounds had no significant change in strains.Expression of SmHMGR1,SmDXS2 and SmCPS1 were activated by AtMYB40,however,the expression of SmGGPPSl was suppressed.These preliminarily reveal the regulatory mechanism of tanshinone biosynthesis by AtMYB40 in transgenic lines.And AtMYB40 also activated expression of SmPYS3,promoting the synthesis of carotenoids.