| Salvia miltiorrhiza Bunge(Labiatae)is a perennial herb,mainly used as medicine for the dry rhizomes and roots of plants,which have the functions of removing blood stasis and soothing the nerves.Tanshinone is one of the most important active components in S.miltiorrhiza.Cytochrome P450(CYP450)is a large enzyme protein superfamily widely existing in plants and plays an important role in plant metabolism.In recent years,CYP450 gene superfamily has become a research hotspot in the downstream pathways of tanshinone synthesis.Many studies have focused on how SmCYP450 work in tanshinone biosynthesis in vivo.Based on the whole genome database of S.miltiorrhiza and combined with the published research,116 full-length CYP450s in S.miltiorrhiza were identified and characterized.Through bioinformatics analysis of the physicochemical properties and phylogenetic relationship of the members of SmCYP450s,all of SmCYP450s were divided into two types.When comparing the conserved motifs and gene structure,we found that SmCYP76 gene family had highly conservative.Two SmCYP450s,SmCYP76AK2 and SmCYP76AK3,were screened out by analysis of gene expression patterns.It is speculated that they may be involved in the synthesis of tanshinones.Subsequently,we constructed overexpression and knockout vectors of these two genes and obtained transgenic lines.At the same time,prokaryotic expression vectors of the target genes were constructed,and the target protein was obtained.Above all,the effect of SmCYP76AK2 and SmCYP76AK3 on the synthesis of tanshinones was verified.And we also provide a reference for further exploration of the two genes in vitro.The main research methods and results are as follows:1.Screening out genes involved in tanshinone synthesisBased on literature review and bio informatics analysis,116 full-length SmCYP450s were obtained.Phylogenetic analyses showed that SmCYP450s was divided into A-type and non-A-type,respectively.There were 67 A-type mCYP450s(57.8%),belonging to 17 families and 49 non-A-type SmCYP450s(42.2%),belonging to 31 families.Using bioinformatics software to analyze the physicochemical properties and conserved motifs of all members,we found the conserved motifs of SmCYP76s were highly conserved and had an uinique motif 15.Among them,3 genes of the SmCYP76 family had been confirmed to be involved in the synthesis of tanshinones,and all SmCYP76s sequences are highly conserved,and there is a motif 15 unique to the motif.It is speculated that there may be other functional genes in SmCYP76.Based on the tissue-specific expression pattern and abiotic stresses responses of 9 genes in SmCYP76,we screened out SmCYP76AK2 and SmCYP76AK3 that may be involved in the synthesis and accumulation of tanshinone.These two genes were mainly expressed in the root,and responded to methyl jasmonate and salicylic acid significantly.2.Obtaining SmCYP76AK2 and SmCYP76AK3 transgenic linesSmCYP76AK2 and SmCYP76AK3(SmCYP76AK2/3)were cloned.The full-length cDNAs of the two genes were 1548 bp(SmCYP76AK2)and 1638 bp(SmCYP76AK3),encoding 495 amino acids and 491 amino acids,respectively.SmCYP76AK2/3 overexpression and knockout vectors were constructed by Gateway and CRISPR/Cas9 method.Then,an Agrobacterium-mediated gene transfer method was performed to generate transgenic plants with leaf explants from sterile S.miltiorrhiza.The constructed vector was successfully inserted into the genome of S.miltiorrhiza,including 3 SmCYP76AK2 overexpression strains(OE-AK2-1,OE-AK2-2,OE-AK2-4),7 SmCYP76AK3 overexpression lines(OE-AK3-1,OE-AK3-2～OE-AK3-7),8 knockout lines of SmCYP76AK2(CH-AK2-1,CH-AK2-2,CH-AK2-4,CH-AK2-6～CH-AK2-10)and 14 knockout lines of SmCYP76AK3(CH-AK2-1 AK3-2～CH-AK3-4,CH-AK3-6～CH-AK3-13,CH-AK3-16,CH-AK3-18,CH-AK3-20).3.Effects of SmCYP76AK2 and SmCYP76AK3 in tanshinone synthesisThe contents of tanshinone Ⅱ A,tanshinone Ⅰ,cryptotanshinone,dihydrotanshinone,and tanshinone ⅡB in the overexpressed and knockout plants was determined by LC-MS.Compared with the control group,the content of tanshinone Ⅰin SmCYP76AK2 overexpressed lines increased by 5.49,4.91,and 4.28 times;the content of cryptotanshinone increased by 27.76,16.67,and 25.88 times;the content of tanshinone ⅡB increased by 23.21,4.07,and 4.96 times;the content of dihydrotanshinone increased by 36.85,28.74,and 10.50 times;the content of tanshinone ⅡA Increased by 44.58,16.67 and 25.88 times.The contents of tanshinones in the knockout lines of SmCYP76AK2 and SmCYP76AK3 were reduced by more than 90%,indicating that SmCYP76AK2 and SmCYP76AK3 did participate in the synthesis of tanshinones.4.Purification of SmCYP76AK2 and SmCYP76AK3 proteins in vitroIn this part,the prokaryotic expression vectors of SmCYP76AK2 and SmCYP76AK3 proteins were constructed,and the target proteins were induced and purified successfully.The inducible expression of prokaryotic expression vectors and the purification of the proteins proved that SmCYP76AK2 and SmCYP76AK3 proteins were soluble proteins.Both of the two proteins were assembled in large quantities at 16℃.In summary,based on the exploration of the evolution and classification of the CYP450 superfamily system of S.miltiorrhiza,combining phylogenetic evolution and classification of CYP450 superfamily in S.miltiorrhiza,tissue-specific expression patterns and response analysis of different abiotic stresses revealed that SmCYP76AK2 and SmCYP76AK3 may participate in the synthesis of tanshinones.Further studies on transgenic lines confirmed our speculation.And our research can provid evidence for further exploration in molecular mechanism of tanshinones synthesis. |
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