| ObjectTo explore the effects of different kinds of endophytic fungal elicitors on Pulsatilla callus under different culture conditions,prepare endophytic fungal elicitors and Pulsatilla callus for suspension co-culture,and screen to promote the synthesis of Anemoside B4.The endophytic fungal elicitors and culture conditions were optimized to elucidate the physiological and biochemical mechanism of endophytic fungal elicitors promoting Anemoside B4 synthesis under different conditions,and the correlation analysis of physiological and biochemical indicators was carried out.Screening and identification of terpenoid differential metabolites such as Anemoside B4 under the regulation of endophytic fungal elicitors based on targeted metabolomics and enrichment of pathways.It is beneficial to realize the large-scale and industrialized development of Anemoside B4 under the suspension culture of endophytic fungal elicitors and Pulsatilla callus,and promote the sustainable utilization of Pulsatilla resources.Method1.Isolation,identification,and purification of endophytic fungi: Wild Pulsatilla chinensis(Bunge)Regel was used as experimental material.The root,stem,and leaf tissue blocks of P.chinensis were surface sterilized using a mixed sterilization method of 75 % ethanol and 2 % sodium hypochlorite.The sterilized tissue blocks were then inverted onto potato dextrose agar(PDA)culture medium and incubated at a constant temperature of 28 ℃ until fungal hyphae appeared.The endophytic fungi were purified using a tip hyphae purification method,and pure single colonies were obtained by multiple passages.The pure strains were stored in 25 % glycerol solution at-80 ℃.The species of endophytic fungi were identified by macroscopic observation and ITS-r DNA molecular identification.The colonization rate,isolation rate,similarity coefficient,and diversity index of endophytic fungi in P.chinensis were further analyzed.2.Screening of elicitors of endophytic fungi and optimization of culture conditions: Endophytic fungi were isolated and prepared as either mycelium-inducing or liquid-inducing agents,and were co-cultured with suspension-cultured Whitehead’s monkshood callus tissue.Inducing agents that significantly promoted the accumulation of Anemoside B4 without significantly inhibiting the fresh weight of callus tissue were screened.Furthermore,the co-culture conditions of inducing agents and callus tissue were optimized,including different concentrations(0 μg,20 μg,50μg,80 μg,110 μg),different addition times(early exponential phase at 15 d,middle exponential phase at 21 d,and late exponential phase at 27 d),and different co-culture times(1 d,2 d,3 d).The content of Anemoside B4 in callus tissue co-cultured with different strains of fungi was measured by high-performance liquid chromatography(HPLC),and the effect of Anemoside B4 on callus tissue growth was analyzed.3.Physiological and biochemical mechanisms of Anemoside B4 accumulation in callus induced by endophytic fungi: Under optimized culture conditions,different endophytic fungal inducers were evaluated for their effects on physiological and biochemical indicators in callus tissue,including the activities of the defensive enzymes superoxide dismutase(SOD),peroxidase(POD),and catalase(CAT),as well as the contents of malondialdehyde(MDA),electrical conductivity and p H.Pearson correlation analysis was performed to determine the relationship between physiological and biochemical indicators and the content of Anemoside B4,in order to clarify the physiological and biochemical mechanisms by which endophytic fungal inducers promote the accumulation of Anemoside B4.4.Research on the regulatory mechanism of triterpenoid synthesis in the callus of Anemone based on targeted metabolomics induced by endophytic fungi: The raw mass spectrometry data of the samples were obtained using a Triple-TOF 5600+ highresolution mass spectrometer.The data were preprocessed in Marker View software for peak extraction,alignment,filtering,and normalization of the raw mass spectrometry data in both positive and negative ion modes.The preprocessed data were imported into SIMCA14.1 software for multivariate statistical analysis.Differential metabolites were identified using Peakview software,human metabolome database(HMDB),Massbank,and other databases.Pathway enrichment analysis of differentially metabolites such as Anemoside B4 was conducted based on the Kyoto Encyclopedia of Genes and Genomes(KEGG)to provide important basis for subsequent mechanism research.Result1.Isolation,identification,and purification of endophytic fungi: A total of 62 strains of endophytic fungi were isolated and purified from 92 pieces of root,stem,and leaf tissue of Pulsatilla chinensis(Bunge)Regel.After macroscopic observation and molecular identification,they were classified into 11 genera and 18 species.Among them,two strains of endophytic fungi,Phoma sp.and Dothideomycetes sp.,were only identified to the genus level.The total colonization rate,isolation rate,and diversity index of endophytic fungi in A.capillaris were 58.70 %,67.39 %,and2.242,respectively,classified as level III.2.Screening of elicitors of endophytic fungi and optimization of culture conditions: 18 strains of endophytic fungi were isolated and prepared into inactivated mycelial and inactivated liquid inducers,totaling 36 kinds.Three strains of endophytic fungi were preliminarily screened,which can significantly promote the accumulation of Anemoside B4 without significantly inhibiting the growth of callus tissue.They are inactivated liquid inducers of Alternaria alternata(J-1-Y),Colletotrichum graminicola(J-10-Y),and inactivated mycelial inducer of Arcopilus cupreus(G1-8-S).After optimizing the culture conditions,all three strains of endophytic fungal inducers can significantly promote the accumulation of Anemoside B4.At a concentration of 50 μg/ml and after co-culturing with callus tissue at the exponential phase for 3 days,the content of Anemoside B4 induced by J-1-Y was2.28 times that of the control group.At a concentration of 110 μg/ml and after coculturing with callus tissue at the exponential phase for 2 days,the content of Anemoside B4 induced by J-10-Y was 2.18 times that of the control group.At a concentration of 80 μg/ml,and after co-culturing with callus tissue at the exponential phase for 2 days,the content of Anemoside B4 induced by G1-8-S was 2.2 times that of the control group.The growth of callus tissue in all three treatment groups was inhibited at a concentration of 110 μg/ml,with J-1 group being the most significant.They were all significantly inhibited when added and co-cultured for 2 or 3 days in the exponential phase,and there was no significant change in the fresh weight of callus tissue when the inducers were added in the middle or late exponential phase.3.The physiological and biochemical mechanisms of Anemoside B4 accumulation in callus induced by endophytic fungi were investigated.Results showed that within the concentration range of 0 μg/m L to 110 μg/m L of the different concentrations of endophytic fungi inducers,the MDA content and POD activity of the J-1,J-10,and G1-8 groups increased first and then decreased with increasing concentration,while the SOD activity of the J-1 and G1-8 groups increased first and then decreased,and that of the J-10 group decreased first and then increased.The CAT activity of the J-1 and G1-8 groups decreased first and then increased with increasing concentration,while that of the J-10 group decreased first and then increased.The electrical conductivity of the callus culture medium of the three treatment groups reached its peak at a concentration of 110 μg/m L,and the p H of the J-1 group culture medium reached its peak at 50 μg/m L.With the increase of the concentration of the endophytic fungi inducers,the Anemoside B4 content in the J-1group was positively correlated with the MDA content,SOD activity,and culture medium p H,while the fresh weight of the callus tissue was negatively correlated with the inducer concentration and electrical conductivity.The Anemoside B4 content in the J-10 group was positively correlated with SOD activity,while the fresh weight of the callus tissue was negatively correlated with both Anemoside B4 content and SOD activity.The Anemoside B4 content in the G1-8 group was positively correlated with both SOD and CAT activities and negatively correlated with the MDA content,while the fresh weight of the callus tissue was negatively correlated with the inducer concentration.The experimental results of different addition times and cultivation times of endophytic fungal inducers on the growth index,defense enzyme activity,and malondialdehyde(MDA)content of callus tissue showed that adding three types of endophytic fungal inducers in the early and middle stages of the callus tissue growth index and cultivating for different days(1 d,2 d,3 d)led to significant changes in defense enzyme activity and MDA content,while adding inducers in the late stage of the growth index and cultivating for different days(1 d,2 d,3 d)led to slightly slower but still synergistic changes in defense enzyme activity and MDA content.There was no significant change in electrical conductivity in the culture solution of the three treatment groups during the growth index period.Compared with early addition,the effect of inducers added in the middle and late stages on the p H value of the culture solution was more significant.Adding inducers in the early stage of the growth index showed a negative correlation between fresh weight and MDA content of callus tissue in the J-1 group,and a negative correlation between Anemoside B4 and MDA content in the G1-8 group.Adding inducers in the middle stage of the growth index showed a positive correlation between Anemoside B4 and superoxide dismutase(SOD)activity as well as the p H value of the culture solution in the J-1 group.Adding elicitors at the late stage of the index,the content of Anemoside B4 in the J-10 treatment group was positively correlated with the MDA content and SOD activity,and the callus fresh weight in the G1-8 treatment group was negatively correlated with the culture time.4.Study on the regulation mechanism of terpenoid synthesis in the healing tissue of Anemone induced by endophytic fungi based on targeted metabolomics: The experimental results show that 10 differential metabolites of terpenoids,including momordin A,jujuboside IV,Anemoside B4,and hedera saponin C,were preliminarily identified and up-regulated in group J-1.Group J-10 preliminarily identified 11 differential metabolites of terpenoids,of which 9 substances including soyasaponin I,Anemoside B4,and calenduloside A were up-regulated,while 2substances including cynaroside-2’-O-glucoside and 12-Hydroxy-11-methoxy-8,11,13-abietatrien-20-oic acid were down-regulated.Group G1-8 preliminarily identified 11 differential metabolites,of which 8 substances including soyasaponin II,Anemoside B4,and Ilex glycoside IV were up-regulated,while 3 substances including Annoglabasin F,dihydrocurcumin,and 12-Hydroxy-11-methoxy-8,11,13-abietatrien-20-oic acid were down-regulated.In addition,in the differential metabolite identification of group J-1,the precursor of the malic acid pathway,malic acid,was significantly increased.ConclusionThe study found that 11 genera and 18 species of endophytic fungi were isolated from Pulsatilla chinensis plants in the Taiyuan Tai gang suburban forest park,indicating a rich biodiversity that provides a good foundation for screening inducers of endophytic fungi and promoting the accumulation of Anemoside B4.After screening inducers and optimizing culture conditions,it was found that the inducers of three endophytic fungi could promote the synthesis and accumulation of Anemoside B4.The optimized co-culture conditions were: J-1-Y inducer at a concentration of 50μg/ml,added in the late exponential phase and co-cultured with callus suspension for3 days;J-10-Y inducer at a concentration of 110 μg/ml,added in the late exponential phase and co-cultured with callus suspension for 2 days;and G1-8-S inducer at a concentration of 80 μg/ml,added in the late exponential phase and co-cultured with callus suspension for 2 days.The J-1-Y inducer had the best effect.The physiological and biochemical mechanism by which the inducers promote the synthesis and accumulation of Anemoside B4 is as follows: The 110 μg/ml inducer has a certain inhibitory effect on callus tissue growth,and the conductivity also increases.At a specific concentration,there is a certain correlation between the accumulation of Anemoside B4 and the activity of some defense enzymes and MDA content.When the inducer is added in the early exponential phase,the defense enzyme system and MDA content change dramatically,inhibiting the growth of callus tissue and the accumulation of Anemoside B4.When the inducer is added in the middle and late exponential phases,and at specific concentrations and appropriate culture times,all three inducers can significantly promote the synthesis of Anemoside B4,and there is also a correlation between the accumulation of Anemoside B4 and the activity of some defense enzymes and MDA content.The conductivity and p H of the J-1 coculture medium are mainly affected by the concentration of the inducer and the number of culture days.Based on targeted metabolomics,the study investigated the regulatory mechanism of endophytic fungal inducers on the synthesis and accumulation of triterpenoid compounds,such as Anemoside B4.In the J-1 group,10 differentially expressed metabolites related to triterpenoids were upregulated,while in the J-10 and G1-8 groups,11 differentially expressed metabolites were either upregulated or downregulated.In the J-1 group,it is presumed that the accumulation of Anemoside B4 was promoted due to the significant increase of the precursor substance mevalonate.This study laid a theoretical basis for the large-scale cultivation and industrial production of Anemoside B4. |
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