| Salvia miltiorhiza Bunge,a plant in the genus Salvia of the Lamiaceae family,has a common red root.Its roots and rhizomes are traditional Chinese medicine named Danshen,with a long history of use and a huge market demand,making it of great economic value.It is also considered to be a suitable medicinal model plant due to its biological properties and has high academic value.Tanshinones are the main active ingredients in S.miltiorrhiza and exhibit significant pharmacological activities,such as antioxidant activity,antiinflammatory activity,cardiovascular effects,and antitumor activity.The biosynthesis of tanshinones has been studied for more than two decades but remains to be completely elucidated.Tanshinones are mostly yellow to red in colour,and their enrichment in the root bark results in a red colour in the root tissue of S.miltiorrhiza.The wild orange-rooted S.miltiorrhiza(shh)is very closely related to the red-rooted S.miltiorrhiza and is a valuable research material.In this study,the comparative analysis of orange and red roots of S.miltiorrhiza was used as an entry point to further analyse the tanshinone biosynthesis pathway and to investigate the causes of the formation of orange roots of S.miltiorrhiza.We uncovered a gene involved in an important step in tanshinones biosynthesis by comparing the metabolites content and genes expression level of orange roots and red roots.We verified its function through in vivo and in vitro experiments.And the gene was found to have a large deletion in the shh genome and may be the main reason for the variation in root colour in S.miltiorrhiza.The results of the study provide a basis for research on the quality improvement of S.miltiorrhiza and the synthetic biology of tanshinones.This study also published the chromosomal-level genome sequence of S.miltiorrhiza with orange roots,which will provide valuable biological information and genetic resources for further understanding of tanshinones biosynthesis and regulatory mechanisms as well as other biological properties.The main findings are as follows:(1)A high-quality genome sequence of S.miltiorrhiza with orange roots was obtained and genome annotation was completed.In this study,whole genome sequencing and preliminary assembly of shh genome was performed using Illumina and PacBio sequencing technologies.The 10× Genomics data were used to assist the assembly,resulting in a genome size of 530.97 Mb,Scaffold N50 of 2.01 Mb and Contig N50 of 1.05 Mb.Further Hi-C data were used to mount 93.51%of the sequences to eight chromosomes.The CEGMA assessments identified 89.52%of the conserved eukaryotic genes,indicating a more complete assembly.The genome was annotated to 32,191 protein-coding genes,with approximately 56.65%of the repetitive sequences.(2)Phylogenomic analysis revealed the evolutionary history of the orange root line shh.A phylogenetic tree was constructed for shh together with three S.miltiorrhiza lines with red-rooted,three other Salvia plants(S.bowleyana,S.hispanica,S.splendens)and 12 other plants.The orange-rooted shh clustered with the red-rooted S.miltiorrhiza as well as with the S.bowleyana.This implies that the common ancestor of shh,S.miltiorrhiza with red roots and S.bowleyana may have been a red-rooted species.Certain changes accumulated over millions of years of evolution led to the formation of the orange roots.Analysis of collinearity suggests that shh has not undergone a recent WGD and S.splendens has twice WGD.Analysis of collinearity between the shh genome and the DSS3 genome showed that the encoding genes in shh assembly and DSS3 assembly have a high degree of genome-wide collinearity,but differ significantly in some regions.(3)A gene involved in an important step in tanshinone biosynthesis was identified and found to be mutated in shh.Through genomic and transcriptomic comparison,in vitro protein assay and in vivo complementation assay,we found that Sm20GD3 responsible for 15,16-dehydrogenation of tanshinones.In addition,several homologous proteins of Sm2OGD3 from other S.miltiorrhiza lines and S.bowleyana also could convert CT,15,16-DHT and THT into TAII,TAI and 1,2-DHT,respectively.The results confirmed the catalytic roles of Sm2OGD3 in 15,16-dehydrogenation,a key reaction in the tanshinone biosynthetic network.Moreover,Sm2OGD3 in shh lost the second exon and may be the main reason for the variation in root colour in S.miltiorrhiza.(4)Genome-wide identification and chromosomal localization of the full-length 20GD and CYP gene families in shh genome.Based on the annotation results of the shh genome and manual correction of the gene structure,92 full-length 2OGD DOXC gene family members and 273 full-length CYP gene family members were identified.Based on the genomic localization of these genes with known tanshinones biosynthesis related genes,five potential secondary metabolism gene clusters were initially screened,of which two tanshinones synthesis gene clusters have been reported in previous studies.Sm2OGD3m is located on chr7 of the shh genome,with several 2OGDs clustered nearby in a DNA sequence approximately 50 kb.Of these,Sm2OGD17 is approximately 1.4 kb from Sm2OGD3m and is the gene with the highest identity to Sm2OGD3 in the 2OGD gene family.In vitro enzymatic assays demonstrated that Sm2OGD17 does not possess Sm2OGD3 activity.In summary,a high-quality chromosomal-level genome of S.miltiorrhiza with orange roots was obtained.The results revealed that the common ancestor of S.miltiorrhiza with orange/red roots and S.bowleyana is a red-rooted species.We found that Sm2OGD3 is responsible for introducing a double bond at C-15,16 of tanshinones,and it has a large deletion mutation in shh.The above results provide a theoretical basis for the improvement of the traits ofS.miltiorrhiza and the synthetic biology of tanshinones,and provide genetic information resources for further research on the biological properties of S.miltiorrhiza. |
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