Salvia Miltiorrhiza Hairy Root Genes Induced By Expression Analysis Of Its Active Ingredients Biosynthesis Gene Cloning Research

Salvia miltiorrhiza Bunge is a famous traditional Chinese medicine, recorded very early in "Shen Nong's herbal classic ". It has such effecs as removing blood stasis and odynolysis, activating blood to promote menstruation, cooling the blood and treating boils etc. It is often applied to the treatment of cardiovascular system and hematological system diseases. The main components of S.miltiorrhiza are lipid soluble diterpene quinone compounds and hydrosoluble phenolic acid ones. The lipid soluble ingredient of diterpene quinone compounds mainly refer to tanshinones. Their metabolites often participate in many of intracorporal biochemical reaction and act as coenzyme to promote or interfer some of these bioreactions. These functions are often shown as bioactivities of antibiosis, bacteriostasis and antiinflammatory. At the same time, tanshinones also showe bioactivities in inhibiting platelet aggregation, elevating ablity of bearing hypoxia and ameliorating blood supply of coronary artery. So to speak, tanshinone is one of importmant dugs for the treatment of cardiovascular system diseases. Besides, the hydrosoluble compounds, salvianolic acid, have potential pharmacological effects on hepatoma, diabetes caused nephronia, depression and AIDS. Our experiments mainly focus on the study of tanshinones.Problems of different qualities in medical materia of danshen roots from different culture region plus longtime growth cycle and low content of active components often lead to uncertain clinical effects. Plant cell suspension culture has been considered an alternative to traditional agricultural processes for producing valuable secondary metabolites. Hairy roots, the result of genetic transformation by Agrobacterium rhizogenes, have attractive properties for secondary metabolite production.They often grow as fast as or faster than plant cell cultures and do not require hormones in the medium. The greatest advantage is that hairy root cultures often exhibit about the same or greater biosynthetic capacity for secondary metabolite production compared to their mother plants. (Banerjee et al., 1998; Kittipongpatana et al., 1998). Many valuable secondary metabolites are synthesized in roots in vivo, and often synthesis is linked to root differentiation (Robins et al., 1991; Flores et al., 1999). Even in cases where secondary metabolites accumulate only in the aerial part of an intact plant, hairy root cultures have been shown to accumulate the metabolites. Salvia miltiorrhiza, with advantages in few cromosomes(2n=14), rapid growth and easy breed, is increasingly believed as a model plant for the study of secondary metabolic pathway and gene control research. Recentely it is rising in study on tissue and cells culture from Salvia miltiorrhiza, especially for the Agrobacterium rhiyogenes induced hairy roots culture system.As to the study of hairy roots culture of Salvia miltiorrhiza, elevating the active ingredient accumulation has been always thought to the goal of most of such culture studyies. Using elicitors to improve the accumulation of secondary metabolites is believe to be one of the most promising way. There were a lot of reports on optimizing cuture condition, screening of elicitors and contents assaying for active ingredients etc. But the were few of reports about mechanism of secondary metabolism and reasons of elicitors work.It is very important to make it clear that how tanshinone produce and which pathway it comes from. Structural analysis showed that tanshinones are belong to diterpene quinines. Theoretically, tanshinones biosynthesis maninly operate via none-MVA pathway, the 1-deoxy-D-xylulose 5-phosphate(DXP) pathway, since mevalonicacid (MVA) pathway in the cytosol is responsible for the synthesis of Sesquiterprenes and triterprenes, and the mevalonic acid-independent pathway (or none-MVA Pathway) in the plastids is responsible for the synthesis of monoterpenes, diterpenes and tetraterpenes. It was reported that YE-induced tanshinone accumulation in the Salvia miltiorrhiza hairy roots appeared to be mainly derived from a none-MVA pathway by assaying the enzyme activity of HMGR and 1-deoxy-D-xylulose-5-phosphate synthase(DXS)(Ge et al., 2005).As the IPP or DMAPP was the result the terminal product of MVA or DXP pathway, IPP isomerase, a reversible enzyme which interconverts isopentenyl-diphosphate (IPP) and DMAPP, is the first enzyme in common between the mevalonic acid and DXP pathways. To continue, the prenyltransferase reaction involves the condensation of an acceptor isoprenoid or non-isoprenoid molecule to an allylic diphosphate. The reaction involves a head-to-tail of IPP and its isomer, DMAPP to form 5 carbon compound, GPP. Then FPPS catalize GPP to generate FPP. The Condensation reaction of FPP and IPP produces GGPP catalyzed by prenyltransferases.In the down-stream of isoprenoids pathway, the plastid is the main if not the unique site for the synthesis of GGPP that constitutes the backbone for the synthesis of diverse diterpenes. All diterpenes are derived from geranylgeranyl diphosphate (GGPP) and are prevalent throughout the plant. It worthes noting that diterpene skeleton of tanshinone and gibberellic acid (GA) are similar. This observation led to the prediction that the first steps leading to tanshinone from GGPP are identical to those described for GA biosynthesis: GGPP is first converted by protonation-initiated cyclization to copalyl diphosphate (CDP) by CDP synthase (CPS) . So it would be a problem of whether the CPS/KS involved in GA or in diterpenes biosynthesis has to wait to be made clear. Some reports demonstrated that transcription of CPS that can only be involved in defensive secondary metabolism is up-regulated in response to MJ . While transcription of the GA specific CPS gene (OsCPS1ent) is not significantly altered.This provided us a good method to get the evidence for function stdudy of the two cyclization synthases. And so the diterpene cyclases is focal point that we try to make clear.The molecular study of secondary metabolism on Salvia miltiorrhiza confronted a great difficulty as few gene sequence information especially for the functional genes was available. Only 15 protein sequence and even fewer of protein sequences related to secondary metabolism could be found in GenBank database. Besides, the downstream of GGPP metabolism in diterpene metabolic pathway is now still unknow. So correct and feasible experimental design and proper technology would be the key factor to the success for the experiment.After well known about the background of study on Salvia miltiorrhiza mentioned above, we put forward a new concept of branch of a phenotypic cloning method for unknown functional gene cloning, the"Ingredient difference phenotypic cloning". The so-called "Iingredient difference phenotypic cloning" is a kind of method that is applied to clone unknown genes whose encoding enzymes are closely related to the secondary metabolism to the plants by phenotype characteristic differences of secondary metabolites(including the different content of ingredients and containing or not containing of one or more kinds of compounds). Based on the instruction of this method, we succeeded in cloning 6 genes which were closely related to tanshinones and salvianolic acid secondary metabolism. Whin the 6 genes, two of them encoding diterpene cyclase was found, which improved the study on tanshinoens metabolic pathway from Salvia miltiorrhiza. This work mianly acquired following achievements:1)The effects of elicitors, YE+ Ag⁺ and MJ, on tanshinones accumulation of Salvia miltiorrhiza were studied in this experiment. Result showed YE+ Ag⁺ could effectively improve tanshinone accumulation, which provide the experimental evidences for choosing samples treatment time and samples selection for microarray hybridization. The result of MJ treatment of hairy roots showed that MJ could effectively enhanced the accumulation of tanshinones in hairy roots and released them into culture medium. This result validated the hybridization of cDNA microarray and at the same time help to analyized the likely functions of CPS and KS.2) This work succeeded in screening useful clones of differential expressed genes by Cluster analysis and t test according to ratio values from cDNA microarray hybridization results. The YE+Ag⁺ induced differential genetic transcription information were acquired through electronic splicing , BLASTX annotation, KOBAS and KEGG metablism pathway analysis. Five genes related to tanshinone metabolism and one gene related phenolic acid metabolism were found according to the genetic transcription information. This would do a great help to elucidate mechanlism of elicitor effects and get the full length of cDNA by RACE technique and analyze tanshinones secondary metabolic pathways.3) In this work, 6 full length cDNA were cloned and sequenced from Salvia miltiorrhiza through SMART^TM RACE method based on cDNA fragments acquired from cDNA microarray. These 6 genes encoding enzymes distributed at different branches or points of secondary metabolic pathways and engaged in regulating productions of diterpene metabolites like tanshinones and salvianolic acids from hairy roots of Salvia miltiorrhiza.4) According to the full length of cDNA, this work analyzed the characteristic of 5 translated proteins inculding analysis of ORF, functional prosite, hydrophobicity, secondary structure prediction, three dimensional structure prediction, homology alignment, Phylogenetic tree and metabolic pathway etc. KEGG analysis results showed that SmAACT initiate the condensation reaction of two Acetyl COA to genenrate one AACT in MVA pathway. SmCMK is the only kinase in DXP pathway. SmIPPI, a reversible enzyme which interconverts IPP and DMAPP, is the first enzyme in common between the mevalonic acid and DXP pathways. SmFPPS, at downstream of IPP metabolic pathway, catalizes GPP to generate FPP. The Condensation reaction of FPP and IPP produces GGPP catalyzed by prenyltransferases.In the down-stream of isoprenoids pathway, the plastid is the main if not the unique site for the synthesis of GGPP that constitutes the backbone for the synthesis of diverse diterpenes. All diterpenes are derived from geranylgeranyl diphosphate (GGPP) and are prevalent throughout the plant. We had succeeded in cloning two diterpene cyclase, CPS and KS. Amino acide sequence analysis showed that CPS and KS belong to B type and A type cyclase respectively containing "DDXXD" and "DXDD" motif respcetively. In A type cyclase, a C-terminal conserved aspartate DDXXD motif is involved in the diphosphate ionization-initiated cyclization of the substrate. In type B, a N-terminal conserved aspartate motif E/DxD(D,N) is involved in the protonationinitiated cyclization of GGPP. According to the anlysis of proein characteristic above, it will proveide important basic theory in instructing further studies on gene function and the predict tanshinone metabolism pathwy as well.5) This work succeeded in establishing method for dectecting of mRNA expression levels by SYBGREEN quantitive real-time PCR and analyzed the mRNA expression levels of Sm AACT, SmHMGR, SmCMK, SmIPPI, SmFPPS, SmCPS and SmKS in hairy roots of Salvia miltiorrhiza at different time points after treated by elicitors of YE+Ag⁺ and MJ. The detection result proved the validity of hybridization of cDNA microarray and showed dependablity of mRNA expression levels with accumulation of tanshinones. Resuts in detail was shown as follows: A) Succeeded in proving the correctness and reliability of results of cDNA microarray hybridization by analyzing mRNA expression levels at different time after treatment of YE+Ag⁺. B)Primarily proved in mRNA level by analyzing genge expression level of SmHMGR and SmCMK that the secondary metabolism of tanshinones mainly operated via DXP pathway, but existed "crosstalk" in MVA. C) Initially validated the functions of SmCPS and SmKS by analysis of mRNA expression from hairy roots after treated with elicitiors of MJ. Two diterpene cyclases in downstream of diterpene metabolic pathway, SmCPS and SmKS, were successfully cloned and analyzed by this experiment. Result of KEGG analysis showed that their catalyzed reaction in GA pathway is initiated the cyclization of the substrate of GGPP, and then derived diverse diterpenes. As is branch point of secondary metabolism of diterpene and GA pathway, the elucidating of functions of two cyclases of SmCPS and SmKS showed very important in this study. Quantitive real time PCR result showed that mRNA expressions levels of SmCPS and SmKS from hairy roots enhanced soon after treated with MJ. It was suggested that SmCPS and SmKS had the immediate relation with tanshinone metabolism and at the same time indicated that SmCPS was independent in GA pathway. So to speak, by analyzing all the genes cloned from hairy roots of S.miltiorrhiza, which regulated tanshinones metabolism would help to predict tanshinones metabolic pathway.6) Through cloning and analysis of expressions of genes encoding key enzymes in diterpene metabolic pathway, we firstly predicted tanshinones metabolic pathway by experiment, which provided molecular evidence for biosynthesizing and regulating of active ingredients in S.miltiorrhiza through genentic enginerring .In summary, we had cloned several genes wihich was closely related to secondary metablism of S.miltiorrhiza through "Ingredient difference phenotypic cloning" method based on cDNA microarray techniques. From analysis results of secondary metabolic pathway and mRNA expression , we firstly and seccussfully predicted the primary tanshinone metabolic pathways, which would facilitate the developments of studies on active ingredients related genes regulation, functional genonmics and mechanism of formation of standar medical material of danshen roots.