Screening And Functional Identification Of CYP450 Genes Related To Tanshinone Biosynthesis In Salvia Miltiorrhiza

DanShen is the dried root and rhizome of Salvia miltiorrhiza Bunge,which is a common and important traditional medicine material and has significant curative effect on cardiovascular and cerebrovascular diseases.Diterpenoid tanshinones are the mainly active compounds of S.miltiorrhiza.S.miltiorrhiza is an important medicinal model plant.The biosynthetic pathway and synthetic biology research of tanshinone have attracted much attention in the world.However,due to the high oxidation characteristics of tanshinone,its biosynthetic pathway remains incomplete elucidating,including the catalysis of multi-step oxidase,such as CYP450 s.Our group successfully analyzed the genome and the full-length transcriptome of S.miltiorrhiza based on hybrid-seq(sencond generation and third generation sequencing).Here,we screened and investigated the key enzyme genes in tanshinone biosynthesis based on theories and approaches of genomics,transcriptomics,metabolomics,bioinformatics,etc.1.In total,161 CYP71 clan was classified into 16 families in combination with the phylogenetic analysis of CYP450 s based on the genome and full-length transcriptome of S.miltiorrhiza.Among them,CYP71 family(64)is the most abundant,followed by CYP76 family(28).Gene expression analysis revealed that 36.00% of CYP71 clan genes was highly expressed with FPKM > 10 in all tested samples.The full-length of SmCYP71AU66 and SmCYP76AK5 genes were 1503 base pair(bp),encoding 500 amino acids.The results of transcriptome and realtime quantitative PCR showed that SmCYP71AU66 and SmCYP76AK5 were highly expressed in the peridermis of root,in accordance with the accumulation of tanshinones,suggesting the potential oxidative modification in tanshinone biosynthesis.2.The expression levels of RNA interference(RNAi)lines were detected by real-time quantitative PCR.Compared with the control(empty vector),the expression of RNAi in SmCYP76AK5-1 and SmCYP76AK5-2 decreased by 35.00% and 63.00%,respectively.The content of tanshinones in transgenic hairy roots was further measured by ultra-high performance liquid chromatography(UPLC).In comparison with the control,the contents of tanshinones in the four hairy root lines of RNAi genes were decreased.Finally,we detected 24 tanshinone compounds in transgenic hairy roots by UPLC-MS/MS.By comparison,only 2 tanshinones compounds increased,and the other 22 decreased,in which 6 compounds decreased significantly(fold change < 0.5).The most declining was Miltirone,which was 0.2 times that of the control group.3.This study explored the application of CRISPR/Cas9 genome editing technology via knocking out the SmCYP76AK5 gene in S.miltiorrhiza.From the results of DNA detection,the transgenic hairy root strain lacks three bases of "GCT" at 29 bp – 31 bp of SmCYP76AK5 gene,which cause the frameshift mutation of the protein.At the same time,the efficiency of gene editing was calculated,and 40 of the 67 clones lacked "GCT" at 29 bp to 31 bp,and the sequences of 2 clones did not change.The remaining 25 clones were detected different INDELs at the sgRNA1 target sequence.These results indicate that the SmCYP76AK5 gene was successfully knocked out using CRISPR/Cas9 technology.This study provides the basis for functional identification and molecular mechanism of CYP76AK5 in tanshinone biosynthesis.