Characterization Of SmGGPPS Gene Family In Medicinal Plant Salvia Miltiorrhiza Bge.(labiatae)

The traditional Chinese medicine Danshen is the dried roots and rhizomes of Salvia miltiorrhiza Bunge in the family Lamiaceae.Tanshinones are a group of lipid-soluble bioactive components in Danshen,which are mostly abietane diterpenes.Geranylgeranyl diphosphate?GGPP?is a versatile precursor of various primary metabolites and secondary metabolites in plants,providing biosynthetic backbones for terpenoids,gibberellins,carotenoids,chlorophyll,etc.Geranylgeranyl diphosphate synthase?GGPPS?catalyzes the condensation of the isoprenyl pyrophosphat?IPP?with the farnesyl pyrophosphate?FPP?to produce GGPP,this is a critical step in the metabolic pathway of terpenoids.Therefore,GGPPS has been considered as an important regulatory target in the terpenoids biosynthetic pathway engineering.So,this study takes SmGGPPS gene family as the research object,by analyzing the co-expression genes,the genetic variation of the SmGGPPS gene family and its correlation with the content of tanshinones in the roots,and explored the relationship between the different members of the SmGGPPS gene family and the different metabolic branches of diterpenoid biosynthetic pathway in Danshen,and explored the regulatory mechanism of SmGGPPS gene family on the biosynthesis of tanshinones at the same time.The main results are as follows:?1?The members of the SmGGPPS gene family is co-expressed with six genes of the diterpenoid metabolic pathway,it suggesting that they may be involved in different metabolic branches of diterpenoid biosynthetic pathway in Danshen.Based on the analysis of co-expression of AtGGPPS gene family in Arabidopsis thaliana?L.?Heynh,and the phylogenetic relationship between A.thaliana and Danshen,we predicted some candidate genes co-expressed with the SmGGPPS.Then,after experimental verification we found that SmGGPPS1 gene is co-expressed with SmCPS1,SmKAO1 and SmKAO2 genes;SmGGPPS2 gene is co-expressed with SmKAO2 gene;and SmGGPPS3 gene is co-expressed with SmKSL1,Sm CPS1,SmCPS_ent,SmKO and SmKAO1 genes.It is known that Sm CPS1and SmKSL1 are key enzymes in the tanshinone biosynthesis pathway,and Sm CPS_ent,SmKO,SmKAO1,and SmKAO2 are key enzymes in the gibberellin biosynthesis pathway.Since the co-expressed genes have similar biological functions,it is speculated that the SmGGPPS1 and SmGGPPS3 genes are mainly related to the biosynthesis of tanshinones and gibberellins.The SmGGPPS2 gene is mainly involved in the biosynthesis of gibberellin,but not related to the biosynthesis of tanshinones.?2?The genetic variation at different levels was revealed by SRAP molecular markers and SmGGPPS functional gene coding region and promoter region sequences in Danshen.The SRAP molecular markers was used to analyse the genetic variation of Danshen sampling from different origins,and the results show that the genetic variation of Danshen genome based on SRAP molecular markers was relatively large.Analysis of the genetic variation of the SmGGPPS gene family in the DNA sequence,amino acid sequence,promoter sequence and transcription factor binding site show that the genetic variation of SmGGPPS1 and SmGGPPS2 genes mainly existed in the promoter region.The mutation of the promoter sequence resulted in some transcription factor binding sites?TFBSs?are mutated,while the variation of coding region sequence and amino acid sequence of the SmGGPPS1 and SmGGPPS2 genes are small.However,the genetic variation of the SmGGPPS3 gene mainly exists in the intron region and the promoter,and the sequence variation of intron 1 is the largest.In addition,the mutation of the SmGGPPS3 gene promoter sequence causes partial TFBSs to mutate,whereas the variation of the DNA sequence of the SmGGPPS3 gene didn't cause variation in the amino acid sequence of the SmGGPPS3 gene,and its amino acid sequence was highly conserved.?3?The genetic variation of the proximal promoter of SmGGPPS genes is correlation with the content of tanshinones in Danshen.The content of tanshinones in Danshen samples with different genetic backgrounds was determined by ultraviolet spectrophotometry.And the correlation analysis was carried out by Mantel test.The results show that there was a significant correlation between the promoter sequence polymorphisms at-50bp and-100bp of Sm GGPPS1 gene and the content of tanshinones,and the closer to the transcription start site,the greater the correlation between the promoter sequence polymorphisms and the content of the tanshinones component.At the same time,there was a significant correlation between the variation of AP2,GRAS TFBSs in the promoter region of-50bp,-100bp,-200bp of SmGGPPS1 gene and the content of tanshinones.In addition,there was a significant correlation between the variation of WRKY TFBSs in the promoter region of-118bp of the SmGGPPS3 gene and the content of tanshinones.However,there was no significant correlation between the genetic variation of Danshen genome based on SRAP molecular markers and the content of tanshinones.Similarly between DNA sequence polymorphism,amino acid sequence polymorphism of SmGGPPS1,SmGGPPS2,SmGGPPS3 gene and the content of tanshinones.Also between the promoter sequence polymorphisms of SmGGPPS2 and SmGGPPS3 gene and the content of tanshinones,and between the variation of TFBSs in the promoter of SmGGPPS2 gene and the content of tanshinones.In summary,the members of the SmGGPPS gene family may be involved in different metabolic branches of diterpenoid biosynthetic pathway in Danshen.Thus,the SmGGPPS gene family may play an important role in the regulation of metabolic fluxes in the downstream pathway of terpenoids biosynthetic pathway in Danshen.At the same time,our study reveals to that the AP2,GRAS,WRKY transcription factors participate in the regulation of the biosynthesis of tanshinones by acting on the promoter of different members of the SmGGPPS gene family.That is great significant to further understanding the regulation mechanism of tanshinones biosynthesis pathway,and it also lays a foundation for the development of molecular markers related to the quality of Danshen and for the further exploration of the regulatory mechanisms of terpenoids biosynthesis pathway.