Multi-omics Analysis Of Salvia Miltiorrhiza Root Color Formation And Tanshinone Biosynthesis Regulation

Salvia miltiorrhiza Bunge(S.miltiorrhiza)is a perennial herb of the Lamiaceae family.Its roots are generally red.As a traditional Chinese medicinal material,the dried root is known as Danshen.It has a very good curative effect in the treatment of cardiovascular and cerebrovascular diseases.However,due to the depletion of wild resources,the deterioration of the quality of cultivated S.miltiorrhiza,and the impediments to continuous cropping,the supply of S.miltiorrhiza is insufficient to meet market demand.In this study,a natural mutant of S.miltiorrhiza and its inbreding progeny were used as research materials,combined with research methods such as genome resequencing,transcriptomics,and metabolomics,to investigate the causes of white root mutant formation.Moreover,further explored the downstream pathways of tanshinone biosynthesis and identified its regulatory mechanisms,as well as candidate CYP450 genes and MYB transcription factors that may be related to tanshinone synthesis,in order to provide new ideas for the study of tanshinone biosynthesis pathways and lay the groundwork for subsequent breeding of high-quality S.miltiorrhiza and industrial tanshinone production.Simultaneously,it serves as a resource for secondary metabolism research on other medicinal plants.The following are the study's main findings:1.Mutant phenotypic analysis and comparison of physiological and biochemical indicatorsThe self-bred progeny of S.miltiorrhiza mutants showed trait segregation,with a 3:1 segregation ratio,indicating that the mutants' root color traits were controlled by a single dominant gene.Phenotypic statistics revealed that there was no significant difference in the root traits of white root and red root S.miltiorrhiza during the middle growth stage(120 d);however,in the later stage of development(270 d),the underground part of white root S.miltiorrhiza developed more vigorously,with multiple lateral roots,and its aerial part plant height is short.Only a trace amount of tanshinone IIA was detected in the pericarp of white root S.miltiorrhiza,but no tanshinone I and cryptotanshinone were detected,and the content of phenolic acid salvianolic acid B was not significantly different from that of red root.It is speculated that the phenotype of white root in the mutant is caused by the lack of tanshinones.2.Genome resequencing and variant analysis of mutantsBased on next-generation sequencing,a resequencing library of S.miltiorrhiza mutant was created.Following data filtering,a total of 43.9 Gb clean data was generated.Compared with the reference genome of S miltiorrhiza,the comparison rate of samples was 89.06%,and the contents of GC was 38.79%,and the average coverage depth was 28.7×.A total of 4,739,028 SNPs and 1,264,831 Indel loci were discovered through variation analysis.Sequence analysis of known key enzyme genes in tanshinone synthesis pathway found that there were multiple base insertions and mutations in the coding sequence of SmCYP71D375,which may affect its normal transcription and translation.In addition,more SNPs were found in SmCYP71D373,and most of them were heterozygous mutations.The possible mutation sites of mutants were explored at the gene level.3.Transcriptomic and metabolomic analysis of different root tissue parts from red root and white root S.miltiorrhizaA total of 190.58 Gb of clean data was obtained,the GC content distribution ranged from 47.74%to 49.75%,and the Q30 was greater than 91.76%.A total of 1,587 differentially expressed genes were discovered after comparing the pericarps of white root and red root.They were annotated to diterpene and quinone biosynthesis pathway,phenylpropane biosynthesis pathway and flavone biosynthesis pathway by KEGG analysis,respectively.The results of qRT-PCR were highly correlated with the RNA-Seq data.Furthermore,by the WGCNA analysis,the turquoise module was screened,which contains multiple known key enzyme genes in the biosynthetic pathway of tanshinone and potential unknown genes in the downstream biosynthesis pathway of tanshinone.In the metabolomics of white root and red root,a total of 507 metabolites were detected,and 112 differential metabolites were screened using a comparative analysis of the pericarp of white root and red root.Most flavonoids and quinones have a low content in BG-Z,but a high content in HG-Z and CK-Z,whereas the contents of amino acids and lipids were more likely to be in BG-Z.In addition,we also screened 15 metabolites as metabolic markers,including 8 phenanthraquinones,which can distinguish white root and red root Danshen from metabolites.4.Reasons for the formation of root color in white root S.miltiorrhiza mutantsThe expression parrern of several key enzyme genes in tanshinone biosynthesis pathway in white root S.miltiorrhiza,such as SmGGPPS1,SmCPS1,SmCPS5,SmKSL2,SmKSL3,SmKSL7,SmKSL8,and SmCYP76AK1,was significantly lower than that in red root S.miltiorrhiza,which might affect the metabolic synthesis of some intermediate products of tanshinone.For example,the contents of intermediate products ferruginol and sugiol decreased sharply,which may lead to the decrease of the contents of several main tanshinones,including tanshinone ?,tanshinone ? a,cryptotanshinone,dihydrotanshinone ? and tanshinone? B.The absence of these pigment components might make the root epidermis of S.miltiorrhiza white.5.Prediction and identification of genes and transcription factors related to tanshinone biosynthesisThrough the combined analysis of transcriptomics and metabolomics,17 CYP450 genes and 5 MYB transcription factors were screened out,which may be highly related to tanshinone metabolites.Two CYP450 genes named P4 and P5 and one MYB transcription factor named SmMYB71 were cloned.P4 and P5 were expressed in the roots of S.miltiorrhiza,especially in the peripheral cortex,which was consistent with the synthesis and accumulation mode of tanshinone.Phylogenetic tree analysis showed that the two new CYP450s genes were closely related to the known CYP450s genes involved in terpene synthesis,especially P5,which belonged to the same subfamily as the SmCYP71D373 and SmCYP71D375.SmMYB71 was mainly expressed in the periderm and phloem of the root,and significantly induced by MeJA,indicating that it might be related to the regulation of tanshinone synthesis.