Improvement Of Triterpenoid Content In Psammosilence Tunicoides Hairy Roots By Exogenous Salicylic Acid

Psammosilene tunicoides is a rare and endangered single species of medicinal plant in China.Its main active ingredient is triterpenoid saponin.the use of hairy root scale culture technology can solve the shortage of market supply of wild P.tunicoides resources.However,there is a problem of low content of active ingredients in the process of hairy roots culture,which limits the commercialization of hairy roots culture.In this study,salicylic acid(SA)was selected as an elicitor to increase the content of triterpenoid saponins in P.tunicoides hairy roots,and then analyze its molecular regulation mechanism.The main results are as below:(1)5 mg/L SA is most useful for the growth of hairy roots and triterpenoid saponin synthesis.With reference to the unsettled samples,the total production of triterpenoid saponins increased by 13 % after 5 days of treatment with 5 mg/L SA.(2)Based on Illumina Xten transcriptome sequencing technology,it was identified that the transcriptome level changes after 0,8 and 24 h of hairy roots treated with 5 mg/L SA.A total of 23.7 Gb transcriptome data were obtained,and 158045136 high quality sequences were obtained by filtration,and Q30 was more than 90.8 %.Trinity assembled and spliced182277 unigenes with the average length of 1457 bp and the N50 of 2277 bp.119960(65.81 %)unigenes can obtain annotation information in public protein databases such as Nr,Nt,KO,SwissProt,PFAM,GO and KOG.44844 unigenes can be enriched in the "metabolic process" pathway in the GO database;2207 unigenes are enriched in the "secondary metabolism" pathway in the KEGG database,of which 689(31.2 %)unigenes are associated with steroid metabolism.A total of 55 unigenes in the transcriptome data were associated with the MVA pathway,101 unigenes were associated with the MEP pathway,and 59 unigenes were associated with the triterpene skeleton synthesis pathway,with 7,13,and 7 unigenes significantly up-regulated during SA induction.In the transcriptome data:20,17,10,7 and 2unigenes in the unigenes of the WRKY,MYB,bZIP,AP2/ERF and bHLH transcription factor families were up-regulated in the whole process of SA induction.The above differentially expressed key enzyme genes and transcription factors are highly likely to be involved in the induction of SA-induced triterpenoid saponin synthesis.(3)A transcription factor of WRKY gene was cloned in P.tunicoides hairy roots and named PtWRKY1.PtWRKY1 is a member of the WRKY family GII class,and its open reading frame(ORF)is 963 bp in length;PtWRKY1 is a hydrophilic protein localized in the nucleus,and has the highest homology with Arabidopsis AtWRKY40.The expression pattern of PtWRKY1 under different elicitors was analyzed by real-time PCR.The expression of PtWRKY1 was up-regulated after SA treatment.The expression of PtWRKY1 was up-regulated after NaCl treatment for 24 h,while the expression of PtWRKY1 was significantly decreased under MeJA treatment,indicating that PtWRKY1 might be that has different functions in different saponin synthesis regulatory pathways.The results showed that the key genes related to the synthesis of triterpenoid saponins induced by SA in P.tunicoides hairy roots,which provided a basis for the subsequent improvement of the synthesis of saponins by the genetic engineering methods,which was beneficial to reduce the cultivation cost of P.tunicoides hairy roots and promoted the practical application of this technology.