| Isatis tinctoria L.(woad)is the biennial herbaceous plant of Brassicaceae family.Its dried roots(Ban-Lan-Gen,Radix isatidis)has been used in Traditional Chinese Medicine(TCM)for hundreds of years.Nowadays,there is a contradiction that exists between the cultivation of 7.tinctoria and crops for land occupation.Generally,I.tinctoria needs to grow 2-3 years before harvesting its roots.So,the field cultivation is difficult to meet its normal growth period,thus resulting in the low levels of active ingredients(e.g.alkaloids and flavonoids)accumulated in I.tinctoria.Meanwhile,the quantity of active ingredients in field-grown I.tinctoria is often fluctuating due to the locality differences,climate changes,invasion of pests and diseases,soil heavy metals,pesticide residues and other unfavorable factors,thus leading to the un-uniform quality of.tinctoria roots in the market,poor clinical treatment effect resulting in undesirable consequences,and difficulty of meeting the high-standard demand in the domestic and international markets.In order to addressing the above problems,one way is to develop plant cell/tissue culture technology to replace the traditional field cultivation for the large-scale production of active secondary metabolites in I.tinctoria,and the other way is to use genetic engineering technology via clarifying the biosynthesis regulation mechanism for the efficient production of target active ingredients in I.tinctoria.In this study,a LC-MS/MS method for the qualitative and quantitative analysis of alkaloids and flavonoids was initially established.The developed LC-MS/MS method was used to guide the establishment of I.tinctoria hairy root cultures(ITHRCs)with the high productivity of alkaloids and flavonoids,which could act as a model platform for studying their biosynthesis mechanisms.Subsequently,ITHRCs were treated by the exogenous physicochemical and abiotic elicitors,and the target active ingredients accumulation was used as an indicator to optimize the optimal operational conditions of different elicitors.Moreover,the candidate enzyme genes involved in alkaloid and flavonoid biosynthetic pathways were initially screened from the existing genetic resources and transcriptome sequencing data of I.tinctoria.Eventually,the key regulatory enzyme genes involved in alkaloid and flavonoid biosynthetic pathways were preliminarily elucidated through the analysis of accumulation patterns of target ingredients and the expression profiles of candidate enzyme genes in ITHRCs before and after different elicitor treatments.The main research contents and results are summarized as follows:1.Development of LC-MS/MS method for the determination of principal active alkaloids and flavonoids in I.tinctoria roots.(1)LC-MS/MS conditions for the determination of six alkaloids:Chromatographic conditions:Phenomenex Gemini C18 110A reversed-phase column(250 mm × 4.6 mm I.D.,5 μm);mobile phase consisted of acetonitrile(A)and aqueous solution(B);gradient elution was 0-8 min 45-55%(A),8-9 min 55-85%(A),9-14 min 85%(A),14-15 min 85-45%(A),15-17 min 45%(A);column temperature was 30 ℃,flow rate was 1.0 mL/min,injection volume was 10 μL.MS conditions:electrospray ionization in negative ion mode(ESI);MS transitions of six target ingredients under the optimal selective reaction monitor(SRM)conditions were as follows:epigoitrin m/z 127.8→58.0,isatin m/z 145.6→118.0,indole-3-carboxaldehyde m/z 143.9→115.1,tryptanthrin m/z 247.2→218.9,indigo m/z 261.0→217.0,and indirubin m/z 261.2→157.0.(2)LC-MS/MS conditions for the determination of eight flavonoids:Chromatographic conditions:Phenomenex Gemini C18 110A reversed-phase column(250 mm × 4.6 mm I.D.,5 μm),mobile phase consisted of acetonitrile(A)and 0.001%formic acid aqueous solution(B);gradient elution is 0-5 min 40-50%(A),5-13 inin 50-60%(A),13-15 min 60-68%(A),15-16 min 68-40%(A),16-18 min 40%(A);column temperature was 30 ℃,flow rate was 1.0 mL/min,injection volume was 10 μL.MS conditions:electrospray ionization in negative ion mode(ESI-);MS transitions of the target ingredients under the optimal SRM conditions were as follows:rutin&neohesperidin m/z 609.3→301.4,buddleoside m/z 591.4-→283.1,liquiritigenin m/z 255.9→119.0,quercetin m/z 301.0→151.0,isorhamnetin m/z 315.0→300.1,kaempferol m/z 285.3→183.1,and isoliquiritigenin m/z 255.4→118.9.The proposed LC-MS/MS methods not only could effectively eliminate the interference of other non-target impurities,but also could quali-quantitatively determine fourteen target ingredients in I.tinctoria roots quickly,sensitively and accurately.So the developed LC-MS/MS methods were suitable for the accurate analysis of trace ingredients in ITHRCs.2.Establishment of ITHRCs system for the efficient production of alkaloids and flavonoids(1)An optimal transformation rate(76.67%)was obtained when 3 week-old petiole explants were co-cultured with Agrobacterium Rhizogenes LB A 9402 for 2 days with the supplementation of 125 μM acetosyringone and 1.5 mM arginine in the cultivation medium.Moreover,it is worth mentioning that the duration of hairy root induction was only 16 days under the optimized conditions.Based on the biomass production and the accumulation of active ingredients,the high-productive alkaloid root line III(ITHRL III)and high-productive flavonoid root line V(ITHRL V)were successfully selected and confirmed by PCR amplification.(2)Culture parameters of high-productive hairy root line were systematically optimized by Box-Behnken design(BBD).The optimal culture conditions of ITHRL III obtained were as follows:liquid culture medium 1/2MS,initial pH 5.8,culture temperature 24.7 ℃,sucrose concentration 3.14%,inoculum size 0.75%and harvest time 23 days.The optimal culture conditions of ITHRL V obtained were as follows:liquid culture medium 1/2MS,initial pH 5.8,culture temperature 24.7 ℃,sucrose concentration 3.06%,inoculum size 0.75%and harvest time 24 days.Under the optimal conditions,ITHRCs produced total alkaloid(521.77 ± 6.94μg/g DW)and total flavonoid(438.10 ± 3.46[μg/g DW),which were significantly superior to those of 2-year-old field-cultured I.tinctoria roots(464.69 ± 9.25 μg/g DW of alkaloid and 341.73 ± 4.85 μg/g DW of flavonoid).3.Enhanced production of alkaloids and flavonoids in ITHRCs elicited by exogenous plant signal molecules and its regulation mechanism(1)In this study,the biosynthesis of alkaloids and flavonoids in IITHRCs was elicited by methyl jasmonate(MJ),acetylsalicylic acid(ASA)and salicylic acid(SA).The results showed that SA was the most effective elicitor for alkaloid production,and MJ was the most effective elicitor for flavonoid production.SA and MJ elicitation conditions were systematically optimized by CCD,respectively.The highest enhancement of alkaloid accumulation was achieved in 23 day-old ITHRCs III elicited by 142.61 μM SA for 28.18 h.The resulting alkaloid yield was 3146.55 ± 97.63 μg/g DW,which was 5.89-fold higher than that of non-treated control(533.84 ± 13.02 μg/g DW).The optimal enhancement of flavonoid production was obtained in 24 day-old ITHRC V elicited by MJ under the concentration of 179.54 μM and the exposure time of 41.87 h.The resulting flavonoid yield was 4963.15 ± 110.18 μg/g DW,which tremendously increased 11.21-fold relative to that of non-treated control(442.63 ± 9.46μg/g DW).(2)Eleven candidate enzyme genes involved in alkaloid and flavonoid biosynthetic pathways were screened from the existing genetic resources and transcriptome sequencing data of.tinctoria.Transcriptional levels of eleven genes i were assayed by qRT-PCR.Accumulation patterns of alkaloids and flavonoids were detected by LC-MS/MS.Analysis of the relationship between gene transcription and active ingredient content preliminarily clarified that YUCCA was the potential key enzyme gene controlling alkaloid biosynthesis in response to SA elicitation,and CHI and F3’H were the potential key enzyme genes in flavonoid biosynthetic pathway following MJ elicitation.4.Enhanced production of alkaloids and flavonoids in ITHRC elicited by UV radiation and its regulation mechanism(1)In this study,ITHRCs were exposed to ultraviolet radiation(UV-A,UV-B,and UV-C)for promoting alkaloid and flavonoid biosynthesis.Results showed that UV-B was found to be the most effective elicitor for AG and IF production among all elicitor treatments.The highest enhancement of alkaloid accumulation was achieved in 23 day-old ITHRCs III treated by 162 kJ/m2 dose of UV-B radiation.Under the optimal condition,the resulting alkaloid yield was 773.35 + 28.19 g/g DW,which was 1.47-fold higher relative to control(526.09 ± 9.54 pg/g DW).The optimal enhancement for flavonoid production was achieved in 24 day-old ITHRCs V treated by 108 kJ/m2 dose of UV-B radiation.The resulting flavonoid yield was 7259.12 ±198.19 μg/g DW,which was 16.51-fold higher as compared to the level in non-treated control(439.68 ±8.27 μg/g DW).(2)Transcriptional levels of eleven genes involved in alkaloid and flavonoid biosynthetic pathways were assayed by qRT-PCR.Accumulation patterns of alkaloids and flavonoids were detected by LC-MS/MS.Analysis of the relationship between gene transcription and active ingredient content preliminarily clarified that YUCCA was the potential key enzyme gene controlling alkaloid biosynthesis in response to UV-B elicitation,and CHS was the potential key enzyme gene in flavonoid biosynthetic pathway following UV-B elicitation.In addition,activities of antioxidant enzymes and antioxidant effects of extracts from ITHRCs were significantly increased following UV-B radiation,which demonstrated that ITHRCs showed a significant oxidative stress defense in response to UV-B elicitation.5.Enhanced production of alkaloids and flavonoids in ITHRCs elicited by the inunobilized edible Aspergillus and its regulation mechanism(1)In this study,the biosynthesis of alkaloids and flavonoids in ITHRCs was induced by calcium alginate immobilized A.niger spores(IAN)and inmmobilized As.oryzae spores(IAO).Results showed that IAN possessed the best induction effect on the biosynthesis of the target ingredients.The optimal conditions for alkaloid accumulation in 23 day-old ITHRCs III elicited by IAN were as follows:105 spores per flask,co-cultivation temperature 30 ℃,pH 7.0 and co-cultivation time 48 h.Under the optimal condition,the resulting alkaloid yield was 1378.89 ± 33.47 μg/g DW,which was 2.57-fold higher relative to control(535.83 ± 15.16 μg/g DW).The optimal enhancement for flavonoid production in 24 day-old ITHRCs V elicited by IAN were as follows:106 spores per flask,co-cultivation temperature 30℃,pH 7.0 and co-cultivation time 72 h.Under the optimal condition,the resulting flavonoid yield was 3018.31 ±48.66 μ.g/g DW,which was 6.83-fold higher as compared to the level in non-treated control(441.91 ± 7.35 μg/gDW).(2)IAN could induce the sequential accumulation of endogenous NO,SA and JA signaling molecules in ITHRCs.NO rapidly generated and accumulated in ITHRCs elicited by IAN and reached its peak value(67.35 ± 5.61 μmol/g FW)at 12 h,which was 7.21-fold higher as compared to the level in non-treated control.The accumulation of SA slightly lagged behind NO and reached its peak value(202,22 ± 17.98 ng/g FW)at 24 h,which was 8.15-fold higher relative to control.The accumulation of JA was slightly lagged behind SA,and reached its peak value at 48 h(20.74 ± 1.66 ng/g FW),which was about 6.50-fold higher than that of non-treated control.(3)Transcriptional levels of eleven genes involved in alkaloid and flavonoid biosynthetic pathways were assayed by qRT-PCR.Accumulation patterns of alkaloids and flavonoids were detected by LC-MS/MS.Analysis of the relationship between gene transcription and active ingredient content further elucidated that YUCCA may be the key enzyme gene controlling AK biosynthesis in response to IAN,which was mediated by endogenous SA signaling molecules.And CHI and F3’H may be the key enzyme genes in FL biosynthetic pathways in response to-IAN,which was mediated by endogenous JA signaling molecules.Moreover,after five recycles,IAN beads could still maintain its good elicitation effect,which indicated IAN beads had a satisfactory reusability.In this work,a rapid and efficient I.tinctoria hairy root induction system for production of alkaloids and flavonoids was successfully established.On one hand,this work lays the theoretical foundation for the industrialized production of allkaloids and flavonoids in the future.On the other hand,it provides an ideal platform for studying the biosynthetic regulation mechanism of alkaloids and flavonoids in non-model plant(I.tinctoria).Additionally,elicitor technique not only enhanced the production of target active ingredients in ITHRCs,but also preliminarily clarified some key enzyme genes involved in alkaloid and flavonoid biosynthetic pathways,which provided an important theoretical basis for the use of metabolic engineering technology to efficiently produce target active ingredients in I.tinctoria.Meanwhile,this study also provides valuable references for the efficient production of active secondary metabolites and clarification of their biosynthesis regulation mechanisms in other plant cell/tissue culture systems by elicitor technique. |
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