| Stevia rebaudiana Bertoni is a perennial herb of the Asteraceae family,originated from the South America.Stevia rebaudiana Bertoni is mainly known for the accumulation of high sweet tasting mixture of diterpene glycosides,known as steviol glycosides in its leaves.Steviol glycosides(SGs)are used as non-calorific sweetener into food and beverage fields.More than 30 SGs have been detected in leaf extracts of Stevia rebaudiana Bertoni,of which the most abundant are stevioside(St),rebaudioside A(RA)and rebaudioside C(RC).However,the difference in composition of SGs may have an impact on sensory properties that might be relevant for industrial applications.St and RA are 250～300 times sweeter than sucrose but possess a bitter lingering aftertaste.The less abundant rebaudioside D(RD)and rebaudioside M(RM)have a sweetness potency up to 350 times that of sucrose while having a greatly reduced lingering bitterness.RM,in particular,provides a fast,clean sweet taste and is the steviol glucoside most similar to sucrose in organoleptic properties.These properties render RD and RM superior targets as high potency natural sweeteners.RD and RM are only present in the leaves of Stevia rebaudiana Bertoni in minute quantities making it impractical and costly to purify these two compounds from the Stevia rebaudiana Bertoni plant for industrial use.Several studies have reported that extracts of Stevia rebaudiana Bertoni exert signifificant antimicrobial,anti-hypertensive,antitumor,anti-inflammatory and hepatoprotective activities,with those biological effects being closely related to its high abundance in phenolic compounds.Therefore,the research on dynamic accumulation and molecular mechanism of biosynthesis and regulatory on SGs and phenolic compounds in Stevia rebaudiana Bertoni has vital significance of theory and practice for promoting the development of industry.The main results were as follows:1.By a high performance liquid chromatography(HPLC)-tandem quadrupole mass spectrometric(MS)analyzer,total sixteen phenolic compounds were positively identified by comparing retention times and mass spectra of samples with reference substances and publications.A method for simultaneous determination of sixteen phenolic compounds in flower and leaf of Stevia rebaudiana Bertoni was established by HPLC.The results showed that the major phenolic acids were neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid,1,3-dicaffeoylquinic acid,isochlorogenic acid B,isochlorogenic acid A and isochlorogenic acid C,and isochlorogenic acid A with the highest content of 9.76 mg/g and 41.88 mg/g in flower and leaf of of cultivar SR-11 of Stevia rebaudiana Bertoni.The major flavonoid glycosides were rutin,quercetin-7-O-glucoside,luteolin-7-O-glucoside,luteolin-7-O-rutinoside,quercetin-3-O-xyloside,quercetin-3-O-rhamnoside,apigenin-7-O-glucoside,kaempferol-3-O-arabinoside and kaempferol-3-O-rhamnoside,and quercetin-3-O-rhamnoside with the highest content of 2.44 mg/g and 8.19 mg/g in the flower and leaf.Total contents of phenolic compounds reached 22.31 mg/g and 91.11 mg/g in the flower and leaf during flowering phase.Flower can be developed as a health food,and leaf can be used as raw materials for industrial production of chlorogenic acids in Stevia rebaudiana Bertoni.The developed method could be used for the rapid analysis of phenolic compounds and quality evaluation of the raw material of Stevia rebaudiana Bertoni.2.The differences of SGs,chlorogenic acids and flavonoids in the leaves of seven cultivars of Stevia rebaudiana Bertoni were compared,so as to provide the basis for the breeding of new cultivars and expanding reproduction of improved cultivars.HPLC analysis showed that five kinds of SGs were detected,including RD,RM,RA,St and RC.The contents of five SGs in the leaves of seven cultivars were 35.15～111.48 mg/g(RA),2.87～70.18 mg/g(St),9.32～52.14 mg/g(RC),4.40～33.36 mg/g(RD)and 3.36～3.07 mg/g(RM),respectively.The total content of five SGs was 115.97～171.86 mg/g.The contents of main chlorogenic acids in the leaves of seven cultivars were 3.01～35.71 mg/g(isochlorogenic acid A),2.40～21.22 mg/g(isochlorogenic acid C),2.07～14.79 mg/g(chlorogenic acid),0.29～6.08 mg/g(neochlorogenic acid),0.41～2.93 mg/g(cryptochlorogenic acid)and 0.38～2.20 mg/g(isochlorogenic acid B),respectively.The total content of chlorogenic acids was 8.60～76.39 mg/g.The contents of main flavonoids in the leaves of seven cultivars were 2.97-9.23 mg/g(quercetin-3-O-rhamnoside),1.25-2.82 mg/g(rutin),0.44～1.35 mg/g(luteolin-7-O-glucoside),0.15-1.08 mg/g(luteolin-7-O-rutinoside),0.22～0.97 mg/g(kaempferol-3-O-rhamnoside),0.36～0.81 mg/g(apigenin-7-O-glucoside),0.18～0.71 mg/g(quercetin-7-O-glucoside),0.08～0.47 mg/g(quercetin-3-O-xyloside)and 0.00～0.36 mg/g(kaempferol-3-O-arabinoside),respectively.The total content of flavonoids was 7.45～14.76 mg/g.There were significant cultivar differences in the contents of SGs,chlorogenic acids and flavonoids.In the breeding of new cultivar and expanding reproduction of improved cultivar,the corresponding cultivar SR-7 rich in RD,RM and chlorogenic acids should be selected to develop of the advantages and commercial value of the cultivars.To study the general law of dynamic accumulation of the effective components and to explore the optimal harvest period of Stevia rebaudiana Bertoni,the contents of SGs,chlorogenic acids and flavonoids were investigated during the growth periods.The leaf samples of cultivar SR-6 from July 24th 2018 to October 16th 2018(growth period from 70 d to 154 d)were used as the subjects of HPLC analysis.The results indicated that biomass showed an increased trend,and the contents of SGs showed a small fluctuation.The contents of chlorogenic acids and flavonoids presented increased-decreased-increased-stabilized trend during the growth periods.The effective components and the production trended to be stable from the middle of September to the early of October,which meant that it was the optimal time for harvesting and consistent with the local traditional harvest period in Bazhou of Xinjiang.3.In order to study the mechanism of biosynthesis and accumulation of SGs,chlorogenic acids and flavonoids in Stevia rebaudiana Bertoni,leaves of three different cultivars(SR-8,SR-9 and SR-10)and the five different growth periods(52 d,73 d,94 d,115 d and 136 d)of cultivar SR-11 were used as test materials in transcriptome sequencing,and the protein abundance changes were analyzed to screen out important difference proteins by Tandem Mass Tag(TMT)-labeled quantitative proteomics LC-MS/MS techniques in leaves of Stevia rebaudiana Bertoni at the 73 d,94 d and 115 d periods.Twelve important proteins were verified by parallel reaction monitoring(PRM)technique.The main content in cultivar SR-10 was RA withl74.35 mg/g,RC as the main content with 82.77 mg/g in cultivar SR-9 and St as the main content with 101.89 mg/g in cultivar SR-8.Total contents of SGs,chlorogenic acids and flavonoids were the highest(222.70 mg/g,27.63 mg/g and 13.32 mg/g)in cultivar SR-10,compared with total contents of that(161.95 mg/g,12.15 mg/g and 10.20 mg/g)in cultivar SR-9 and total contents of that(168.06 mg/g,13.83 mg/g and 8.41 mg/g)in cultivar SR-8.There was a significant difference.The cultivar SR-11 contained higher contents of RD and RM,the contents of RD,RM and RA reached the highest levels(24.35 mg/g,20.86 mg/g and 56.98 mg/g)at 94 d,136 d and 115 d,respectively,and the total content of SGs reached the highest level(129.42 mg/g)at 94 d.The contents of chlorogenic acids and flavonoids presented increased-decreased-increased-decreased trend during the growth periods,and the total contents of chlorogenic acids and flavonoids reached the highest levels(71.89 mg/g and 13.84 mg/g)at 115 d and 94 d,respectively.By real-time fluorescent quantitative PCR,15 differentially expressed genes related to biosynthesis of SGs,chlorogenic acids and flavonoids were detected,and the results verified the reliability of the transcriptome data.It was found that the change trend of 12 differentially expressed proteins using PRM target verification was consistent with that of TMT quantitative analysis.4.AACT,DXS,DXR,MCS,HDS,HDR,IDI,GGPPS,CPPS,KS,KO,KAH,UGT85C2,UGT91D1,UGT74G1 and UGT76G1 involved in SGs biosynthesis showed a very high expression,and FPKM values of DXS,HDR,CPPS,KO,UGT85C2,UGT91D1 and UGT76G1 were more than 1 000.Differential expression of HMGR,DXS,DXR,GGPPS,CPPS,KO,UGT85C2,UGT91D1,UGT74G1 and UGT76G1 at the gene level and AACT,DXS,DXR,GGPPS,UGT85C2,UGT91D1,UGT74G1 and UGT76G1 at the protein level regulated SGs biosynthesis in Stevia rebaudiana Bertoni.UGT76G1 played a key regulation in the biosynthesis of RA and RM,and UGT91D1 might play a key regulation in the biosynthesis of RD.5.DAHPS,DHQS,DHD/SDH,CS,PPA-AT and ADT involved in phenylalanine biosynthesis showed a very high expression,and FPKM value of DAHPS was more than 1 000.The differential expression of DAHPS,DHD/SDH and ADT at the gene level and DAHPS,DHQS,SK,PPA-AT and ADT at the protein level regulated the flow of biosynthetic substrates into biosynthesis branches of phenylalanine and chlorogenic acid,and tyrosine and phenylalanine were converted by arogenate route in Stevia rebaudiana Bertoni.PAL,C4H,4CL,HCT/HQT and C3’H involved in the chlorogenic acid biosynthesis showed a very high expression,and FPKM value of PAL was more than 1 000.The differential expression of PAL,C4H,C3’H and HCT/HQT at the gene level and PAL,C4H and 4CL at the protein level regulated the biosynthesis of chlorogenic acid.CHS,CHI,FNS,FLS/F3H and F3’H showed a very high expression,and FPKM value of CHS was more than 2 000.The differential expression of CHS,CHI,FLS/F3H and F3’H at the gene level and CHI,FNS,FLS/F3H and F3’H at the protein level regulated the biosynthesis of flavonoids,and FLS/F3H played a key regulation in the biosynthesis of flavonol.The expression of COMT,CCoAOMT,CCR and CAD at the levels of gene and protein could have impact on the biosynthesis of chlorogenic acids and flavonoids,and UGT78D2 played a key role in the glucosylation of flavonol in Stevia rebaudiana Bertoni.6.Transcription factors including WD40,MYB,bHLH,WRKY,C3H,bZIP and GRAS were closely related to the biosynthesis of SGs,chlorogenic acids and flavonoids.The expression of ABC transporter,GST and VSR at the levels of gene and protein regulated biosynthesis,transport and accumulation of secondary metabolites in Stevia rebaudiana Bertoni. |
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