Clone And Analysis Of Secondary Metabolism Related Genes: SmKSL3 And SmSPLs In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge, belonging to labiatae(Lamiaceae) sage(Salvia Linn), is an important medicinal plant. The earliest record of Salvia miltiorrhiza can be found in more than two thousand years ago in the Sheng Nong’s Herbal Classic in which S.Miltiorrhiza was listed as one of the top grade drugs. Salvia miltiorrhiza has a long history in treatment of many cardiovascular and cerebrovascular diseases. The active components of Salvia miltiorrhiza are divided into two categories: water-soluble phenolic acids and lipid-soluble tanshinones. Tanshinones belong to diterpene which were nature products with various structure synthesized via isoprene metabolism pathways. In order to find out further steps of the bio-synthesis process of tanshinone, in this research, using molecular clone, gene structure analysis, gene expression partern analysis and genetic transformation technology, we studied secondary metabolism related gene SmKSL3 and transcript factor SmSPLs in Salvia miltiorrhiza; and we established T-DNA insertional activation-tagging mutant library and intended to find more new genes related of effective ingredients synthesis in Salvia miltiorrhiza. Followings are the main work and results:1.clone and analysis of SmKSL3Based on the data of Salvia miltiorrhiza genome and previous reports, using 5′RACE and 3′ RACE, from a 285 bp contig we cloned a new KSL homologous fragments,and using RT-PCR for the total length, we firstly cloned another important new KSL gene in Salvia miltiorrhiza, SmKSL3.SmKSL3 contains a 5′ untranslated region(5′ UTR) with length of 208 bp, harboring apparent polyA structure at the 3′ end, showing complete genetic structure, its coding region is 1245 bp, protein molecular formula C2123H3321N565O638S24, containing 414 amino acids to form a hydrophobic and unstable protein. By bioinformatics analysis onconserve domain,transmembrane area, protein subcellular localization, secondary structure and the 3-Dimentional structure of raw sequence of SmKSL3, these results revealed that SmKSL3 belongs to class I terpene synthase, particularly tansinone synthase,it involves in catalyze GPP, FPP or GGPP cyclization to produce monoterpene,sesquiterpenes or diterpene. Analysis of phylogenetic tree of genes share high sequence similarity with SmKSL in other nine planta, the result showed that the genetic distance between Salvia miltiorrhiza and other species is far: no matter dicotyledonous,monocotyledon, woody or herb, they all have a certain genetic distance. Those species are not used as medicinal plants, it suggested that: as a medicinal plant, Salvia miltiorrhiza may have different downstream biosynthesis process of its active ingredients.In order to investigate the subcellular localization of SmKSL, we constructed SmKSL1 – GFP and SmKSL3- GFP fusion expression vectors and observed transient expression in Nicotiana benthamiana. The result showed SmKSL3- GFP located in cytoplasm, and SmKSL1- GFP located in chloroplast. Gene expression pattern analysis showed SmKSL1 and SmKSL3 transcript expression level was high in root; while treated with methyl jasmonic acid(JA), SmKSL1 highly expressed at the 24 th hour, significantly increase of SmKSL3 were obseved at the 36 th hour. Thus presumably SmKSL1 and SmKSL3 are involved in Salvia miltiorrhiza secondary metabolism process; in time and space they were relatively independent. SmKSL1- GFP located in plastid, mainly involved in plastid terpenoid synthesis, while SmKSL3 lacking the organelles positioning signal, mainly correlated with cytoplasm terpenoid synthesis. The corresponding at different time points to outer stimulating(JA) of SmKSL1 and SmKSL3, plays an important physiological role in Salvia miltiorrhiza.For further research on function of SmKSL1 and SmKSL3,we constructed the prokaryotic expression vector, and successfully expressed and purified the proteins( protein purity > 90%). We also constructed an over-expression vector for SmKSL3 with double-CaMV35 S promoter in PBI121 plasmid. In addition, using artificial micro RNA technology we constructed SmKSL3- artificial miRNA knock-down vectors. Both kinds of vectors were transfered into Salvia miltiorrhiza viaagrobacterium-mediated genetic transformation method, preliminary identification of genetic Salvia miltiorrhiza plants were carried out,3 strains for SmKSL3 over expression and 6 strains for SmKSL3 knock-down were obtained. These works lay solid foundation for further in-depth study on SmKSL3 function and the function division between SmKSL1 and SmKSL3.2. clone and analysis of SmSPLsSQUAMOSA promoter binding protein-likes(SPLs) are plant-specific transcription factors playing vital regulatory roles in plant growth and development. There is no information about SPLs in Salvia miltiorrhiza(Danshen). Through genome-wide identification and subsequent molecular cloning, we identified total 15 SmSPLs with divergent sequence features, gene structures, and motifs. Comparative analysis showed sequence conservation between SmSPLs and their Arabidopsis counterparts. A phylogenetic tree clusters SmSPLs into six groups. Many of the motifs identified commonly exist in a group/subgroup, implying their functional redundancy. Eight SmSPLs were predicted and experimentally validated to be targets of miR156/157.SmSPLs were differentially expressed in various tissues of S. milltiorrhiza. The expression of miR156/157-targeted SmSPLs was increased with the maturation of S.miltiorrhiza, whereas the expression of miR156/157 was decreased, confirming the regulatory roles of miR156/157 in SmSPLs and suggesting the functions of SmSPLs in S.miltiorrhiza development. The expression of miR156/157 was negatively correlated with miR172 during the maturation of S. miltiorrhiza. MiR156/157,miR172 and SPLs regulate the transit time of plant vegetative growth to reproductive growth.The change of different growing phase has great impact on plant secondary metabolism. These results indicate the significance and complexity of SmSPL-, miR156-, and miR172-mediated regulation of developmental timing in S. Miltiorrhiza.3. construct of T-DNA insertional activation-tagging mutantsIn order to find more genes involving in secondary metabolism pathways in Salvia miltiorrhiza, we constructed a T- DNA insertional vector exhibited both the function of enhancer activation tag and gene trap; based on the formal reported Agrobacterium tumefaciens-mediated genetic transformation method, we triedestablishing Salvia miltiorrhiza mutant library. In the process, we optimized an easy repeatable screening method with streptomycin in Salvia miltiorrhiza genetic transformation, and had got a mutant plantlet with obvious phenotype.These works lay foundation of discovery and identification of more new secondary metabolism related genes in Salvia miltiorrhiza.