Effects Of Different Growth Patterns On The Accumulation Of Isoflavones In Astragalus Mongholicus And Its Mechanism

Rationales: Astragalus membranaceus(Fisch.)Bge.var.Monghlicus(Bge.)Hsiao has been used as a traditional Chinese medicine for thousands of years.It has two origins including A.membranaceus(Fisch.)Bge.and A.membranaceus(Fisch.)Bge.var.Monghlicus(Bge.)Hsiao.At present,A.mongholicusis the mainstream species in the market,which is planted in Hunyuan(Shanxi),Dingxi(Gansu),Wuchuan(Inner Mongolia)and the surrounding areas.For the quality of medicinal materials,it is generally believed that A.mongholicus is better than A.membranaceus.Our research group is located in the authentic producing areas of A.mongholicus in Shanxi Province.The roots of A.mongholicus grow vertically downward,its length could reach about 1 m,and its growth period could be up to 6 years or more.In the 1980 s,with the increasing demand for A.mongholicus become limited,and then transplanted A.monghlicus began to emerged in the medicinal market.Transplanted A.monghlicus was planted mainly by seedling cultivation for one year and transplanting for one year,and its main root lies in the ditch.With the advantages of less growth years and low cost,transplanted A.monghlicus has gradually become a new mainstream medicinal material.Nowadays,the difference between the two growth patterns of A.mongholicus has gradually been concerned.Previous studies have shown that there is a significant difference in the appearance,the intensity of beany smell and sweetness between wild and transplanted A.monghlicus.Some pharmacodynamic experiments about immunology and anti-fatiguealso showed that the effect of wild A.monghlicus was stronger than that of transplanted A.monghlicus.As is known to all,its efficacy is closely related to the content of compounds for Chinese herbal medicine.There are researchers found that calycosin-7-O-β-D-glucoside,formononetin and astragaloside II in wild A.mongholicus were significantly higher than those in the transplanted A.monghlicus,while the content of astragaloside I in the transplanted A.monghlicus was significantly higher than that in the wild A.monghlicus.Moreover,the content of flavonoids increased gradually with the increase of growth years in A.mongholicus.According to the genetic central principle,the organisms produce proteins(enzymes)with different activities through gene regulation,and the various metabolites were produced the catalysis of enzymes.For Chinese herbal medicine,the research of plant proteomics is now mainly concentrated in a few species that complete genome sequencing,such as rice and Arabidopsis thaliana,while transcriptomics and metabonomics technology are more widely used.Many research groups at home and abroad have found genes related to the secondary metabolic pathway of A.mongholicus through Iso-seq and RNA-seq.At present,the biosynthetic pathways of astragaloside and calycosin-7-O-β-D-glucoside have been basically clarified.Based on this,the study focused on the wild and transplanted A.monghlicus.The next+third generation high-throughput transcriptome sequencing and metabonomics(UHPLC-ESI-Q-TOF-MS/MS)were used to analyze the profile of A.mongholicus with two different growth patterns from the transcriptome and metabolite levels.At the same time,the transcriptome database of A.mongholicus was constructed through Iso-seq and RNA-seq,and the differenceson the transcriptome level between wild and transplanted A.monghlicus was carried out.On this basis,the content of four differential metabolites were determined including formononetin,onion,calycosin and calycosin-7-O-β-D-glucoside,and the key enzyme genes were identified which play an important role in isoflavone biosynthesis pathway by q RT-PCR and correlation analysis.Finally,the effects of different growth patterns on the accumulation of isoflavones in A.mongholicus and its mechanism were revealed from the levels of metabolites and transcriptome.Objective: 1.To revealed the differences of A.mongholicus under different growth patterns from transcriptome and metabolite levels by the profile analysis.2.To explore the effects of different growth patterns on isoflavones in A.mongholicus.3.To elucidate the molecular mechanism of isoflavones accumulation in A.mongholicus with different growth patterns.Methods: 1.Three biological repetitions were designed for field sampling of A.mongholicus.As for fresh samples,the RNA was extracted by Trizol method.The diameter of dried samples was measured,and the diameter range of A.mongholicus in each age was divided.2.Based on UHPLC-ESI-Q-TOF-MS/MS,the metabonomics of 6-year-old wild A.mongholicus and 2-year-old transplanted A.mongholicus were analyzed.The metabolites with VIP >1 and P < 0.05 were screened by multivariate statistical analysis(PLS-DA and OPLS-DA).3.According to the Pharmacopoeia of the People’s Republic of China(2020),the content of calycosin-7-O-β-D-glucoside in A.mongholicus with different growth patterns was determined by HPLC,and the relative quantification of formononetin,ononin,calycosin,and calycosin-7-O-β-D-glucoside was carried out by using the peak area of metabonomics.4.The c DNA transcriptome database of A.mongholicus was constructed by the Iso-seq and RNA-seq,and the key enzyme genes in isoflavone biosynthesis pathway were screened.Finally,the expression value was predicted with FPKM,and the Spearman correlation was used to analyze the results.5.The m RNA expression levels of isoflavone-related genes were detected by q RT-PCR in A.mongholicus with different growth patterns,and the results were analyzed by Spearman correlation and trend analysis.6.The nucleotide and protein sequence of Am UCGT were predicted and analyzed by MEGA 5.0,NCBI(https://www.ncbi.nlm.nih.gov/orffinder/),Signal P-5.0 and TMHMM Server.Then,the full length of Am UCGT was amplified by OE-PCR.Results: 1.The research group has conducted two field sampling of A.mongholicus in Hunyuan(Shanxi,Datong)and Ying County(Shanxi,Shuozhou).A total of 216 samples about 1-6-year-old wild A.mongholicus and 1-2-year-old transplanted A.mongholicus were collected.For the dried samples,the diameter range of A.mongholicus in each age was divided for HPLC experiments;For fresh samples,the results of RNA extraction indicated that all RNA was not degraded and could be used for q RT-PCR.2.The profile analysis of metabolomics showed that the difference between2-year-old transplanted A.mongholicus and 4-6-year-old wild A.mongholicus was the most obvious.After multivariate statistical analysis and identification of differential compounds,14 differential metabolites were found including primary metabolites(amino acids,glucose and other)and secondary metabolites(flavonoids,saponins and other).3.Four isoflavones were quantified including formononetin,formonoside,calycosin,and calycosin-7-O-β-D-glucoside.It was found that the isoflavones in 6-year-old wild A.mongholicus were significantly higher than those in 2-year-old transplanted A.mongholicus.The trend analysis showed that the changes of ononin and calycosin,formononetin and calycosin-7-O-β-D-glucoside were consistent.4.Transcriptome profile analysis showed that the most obvious difference was between 1-2 years old transplanted A.mongholicus and 4-6 years old wild A.mongholicus.The enrichment results of KEGG pathway showed that the differential genes were mainly involved in phenylpropanoid pathway,and isoflavone biosynthesis pathway was an important branch of phenylpropanoid pathway,which suggested that the differential expression of genes related to isoflavone biosynthesis pathway caused the difference between A6 and B2.5.Based on the q RT-PCR of isoflavone synthesis related genes,combined with the trend analysis and Spearman correlation results,it is concluded that the regulation of PAL and CHS,the two rate limiting enzymes,is one of the main reasons for the high accumulation of isoflavones in the wild A.mongholicus.6.The function of Am UCGT was predicted through software and online tools,it is found that Am UCGT was a hydrophilic protein,which might be calycosin-7-O-glycosyltransferase in A.mongholicus.Finally,the full-length of Am UCGT was cloned.Conclusion: In this study,we found that the trend of metabolite profile and transcriptome profile of the wild and transplanted A.mongholicus was basically the same.The isoflavones in 6-year-old wild A.mongholicus were significantly higher than those in2-year-old transplanted A.mongholicus.The regulation of PAL and CHS is one of the main reasons for the high accumulation of isoflavones in A.mongholicus.