| Saposhnikovia divaricata(Trucz.)Schischk is a perennial herb in the umbellaceae family.The unbolted dried roots are used as medicine,which has the effects of dispelling wind and relieving exterior,eliminating dampness and relieving pain,and relieving spasm.The chromones in S.divaricata are the main material basis of its efficacy and the main secondary metabolites.At present,limited natural resources are difficult to meet the needs of medicine and research and development.Long-term exploitation of wild resources will inevitably lead to the depletion of natural resources.Hairy root transformation culture techniques have the advantage of fast propagation speed and high growth efficiency.With its unique advantages,it has been applied to the protection of medicinal plant resources.It is an important way to obtain secondary metabolites of medicinal plants and one of the important means to explore their specific gene function.In this study,the hairy roots of S.divaricata were successfully induced by Agrobacterium tumefaciens K599,and the culture system of hairy roots for S.divaricata was established.The quality of hairy roots(the total content of four chromones: prim-O-glucosylcimifugin,cimifugin,4’-O-beta-Glucopyranosyl-5-O-methylvisamminol and sec-O-glucosylhamaudol)was evaluated.The effects of elicitors(polyethylene glycol,salicylic acid,methyl jasmonate)on the biomass and quality of hairy roots of S.divaricata were investigated.The transcriptome of the hairy roots treated with methyl jasmonate was sequenced.The expression of differential genes was used to explore the effect of methyl jasmonate on the key genes for chromone synthesis in S.divaricata.hairy roots It provides a theoretical basis for the large-scale production of secondary metabolites in S.divaricata hairy roots.The main results are as follows:1.The young leaves of aseptic seedlings of S.divaricata were selected as explants,and the hairy roots of S.divaricata were obtained by Agrobacterium K599 infection.Three excellent monoclonal strains were identified by PCR molecular identification,and the biomass and quality of the different strains were investigated.The results show that the growth of hairy roots was divided into three stages.The growth is slow at 0～14d,the logarithmic growth period was 14～28 d,and the growth rate slowed again after 28 d into the plateau period.The biomass of three hairy roots from large to small was line C(ZC)> line B(ZB)> line A(ZA),and ZC reached 6.70 g.The total content of four chromones from high to low was ZB > ZA > ZC,and ZB reach 5.49 mg/g;The biomass of the hairy roots grown in 1/2MS for 35 days reached 18.6 g,which is 1.25 times that of MS and 2.24 times that of B5,respectively.The chromone content was 2.70 mg / g,which is 2.05 times that of MS and 1.66 times that of B5.2.(1)The hairy roots of S.divaricata were induced and regulated by polyethylene glycol(PEG-6000).The hairy roots of S.divaricata were treated with different concentrations of polyethylene glycol for 7 d,14 d,21 d,28 d and 35 d,respectively.Compared with the control group,the biomass and chromone content of hairy roots of S.divaricata decreased with the increase of polyethylene glycol concentration.The higher the concentration of polyethylene glycol,the lower the biomass and chromone content.Polyethylene glycol inhibited the growth of hairy roots of S.divaricata and the accumulation of chromone content.(2)Salicylic acid was used to induce hairy roots in S.divaricata for 7 days.The hairy roots showed obvious browning and the biomass gradually decreases with increasing salicylic acid concentration.When the salicylic acid concentration reached200 μmol/L,the hairy root biomass was the lowest,5.40 g,which was 0.51 times that of the control group,but the effect of salicylic acid on the content of chromones in hairy roots was not significant.(3)The hairy roots of S.divaricata were induced by methyl jasmonate for 7 days.The content of chromone in hairy roots treated with 150 μmol/L methyl jasmonate decreased significantly to 4.92 mg/g compared with the control group,while the content of chromone in hairy roots treated with 100 μmol/L methyl jasmonate was the highest,reaching 6.45 mg/g.Methyl jasmonate affected the accumulation of chromone in hairy roots of S.divaricata,but has no significant effect on its biomass.The hairy roots were treated with 100 μmol/L methyl jasmonate for 0 h,3 h,6 h,12 h and 24 h,respectively.The content of chromones in hairy roots increased to 9.49 mg/g after 3 h of treatment,and decreased to 7.20 mg/g after 24 h of treatment.Within 24 hours,the content of chromone in hairy roots of S.divaricata increase and then slowly decrease.3.The hairy roots treated with 100 μmol/L methyl jasmonate for 0 h and 3 h were selected for transcriptome sequencing.A total of 10,380 differentially expressed genes were identified compared to the control group,including 4,750 down-regulated genes and 5,630 up-regulated genes.There are 115 differentially expressed genes in the phenylpropanoid biosynthesis pathway,of which 88 genes were up-regulated and 27 genes were down-regulated.Differentially expressed genes in this pathway are involved in encoding 11 enzymes.Among them,PAL and 4CL are the key enzymes for the synthesis of chromones,and 11 differential genes encoding these two key enzymes may be involved in chromone synthesis. |
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