Study On The Mechanism Of Accumulation Of Active Ingredients In Different Developmental Stages Of Gastrodia Elat

Gastrodia elata BI.is a perennial fungal nutritional herb in the genus Gastrodia R.Br.,Orchidaceae Juss.,which is a precious traditional Chinese medicine.The phenolic and glycoside compounds in the dried tubers are often used as medicine,which have various pharmacological effects.Till now,it is insufficient about the characteristics of different developmental stages of G.elata.There is little information about G.elata including changes in gene expression levels,the accumulation of active compounds,the physical and chemical properties of the soil and the structure of the microbial community.Therefore,it is important to uncover the accumulation pattern of active ingredients of G.elata,the synergistic relationship between it and soil microbial community for the scientific cultivation and management of this important medicinal plant resource and its further development and utilization.Ecology,phytochemistry,microbiology,molecular biology,genomics,bioinformatics,and other multidisciplinary methods were employed to study G.elata,including sequencing the transcriptome,determining the content of active compounds,determining soil physicochemical properties,and identifying soil microorganisms at different developmental stages of G.elata Bl.f.glauca S.Chow in Zhaotong county in Yunnan province,which investigated the accumulation pattern of active compounds in G.elata and its relationship with soil environmental factors,soil microbial communities and the gene expression level.The main results were the follows.(1)The contents of a total of 7 compounds of gastrodin(GAS),p-hydroxybenzyl alcohol(HA),p-hydroxybenzaldehyde(HBA),parishin A(PA),parishin B(PB),parishin C(PC),and parishin E(PE)in 5 different developmental stages of G.elata,including seed(A),protocorm(P),juvenile tuber(RG),immature tuber(WG),and mature tuber(SG)were determined.The results showed that 7 active compounds were found to be present at all 5 developmental stages,and the levels of each active compound varied significantly.The content of the total active compounds rised from the seed stage to the juvenile tuber stage and is the highest at the juvenile tuber stage(53.04±9.85 mg·g-1).And then it gradually decreased to only 20.55±2.81 mg·g-1 at the stage of SG,which was 1/2 of that at the RG stage.(2)Transcriptome sequencing of G.elata at five developmental stages yielded 21 562 high quality unigenes(N50=2 036 bp,Mapped Reads between 91.19%-96.40%).There were 12 751 differential unigenes,indicating significant differences in the expression of genes at different developmental stages(P<0.05),with the most abundant number of differential unigenes(7 817)being identified at the P stage.A total of 56 unigenes associated with 2 major metabolic pathways for the biosynthesis of 7 compounds were identified,encoding 12 enzymes.The expression patterns of 13 of these unigenes were significantly correlated with the synthesis of 6 key active compounds(P<0.05).And there were significant positive correlations between gastrodin and C4H(Cinnamate 4-hydroxylase),the AIM1/PhCHD(Abnormal inflorescence meristem 1/Petunia x hybrida cinnamoyl-CoA hydratase-dehydrogenase)gene family,and KAT1(3-ketoacyl thiolase 1).A high positive correlation was found between the synthesis of parishin C and aceE3(pyruvate dehydrogenase/pyruvate dehydrogenase complex),TE2(acyl-coenzyme A thioesterase 2),ADH4(alcohol dehydrogenase),ADH6,ADH10,and CCR5(cinnamoyl-CoA reductase 5)(P<0.05),respectively.A significant positive correlation was found between parishin A and KAT1(3-ketoacyl thiolase 1),DLAT1(dihydrolipoyllysine-residue acetyltransferase),and AIM1/PhCHD2(P<0.05),respectively.Parishin E was highly positively correlated with the expression of ADH14,C4H,KAT1,and AIM1/PhCHD gene family(P<0.05),respectively.Parishin B was significantly positively correlated with AIM1/PhCHD gene family,C4H,and KAT1(P<0.05).And p-hydroxybenzaldehyde was significantly positively correlated with ADH14,C4H,KAT1,4CL6(4-comarate coenzyme A ligase 6),and AIM1/PhCHDl(P<0.05),respectively.The results suggested that the above genes coordinated to promote the biosynthesis of ative compounds.(3)By examining the characteristics of the non-rhizosphere soil at 3 developmental stages of RG,WG and SG dynamically,it was found that the soil physicochemical properties changed with the growth of G.elata.TN,AN,SOM,EC,Cd,Cr,Hg in the non-rhizosphere soil increased with the age of G.elata,and the content of AP,pH,Cu,gradually decreased.Meanwhile,this study found that the soil of RG,WG and SG were contaminated with the heavy metal Cd which content were more than 0.3 mg·kg-1.(4)The second-generation sequencing techniques were used to sequencing 16S rDNA and ITS of the soil microorganisms in the non-rhizosphere and rhizosphere from 3 developmental stages of RG,WG,SG of G.elata.After bioinformatics analysis,1 740 OTUs were obtained from 16S rDNA sequences bacteria,and they were annotated to 26 phyla,51 classes,134 orders,239 families,382 genera,and 446 species of bacteria.A total of 1 947 OTUs were obtained from ITS sequences,and they were annotated to 10 phyla,45 classes,100 orders,227 families,504 genera,and 741 species of fungi.The abundance of soil microbial communities in G.elata differed significantly(P<0.05)at different developmental stages.Both the abundance of fungal communities in rhizosphere and non-rhizosphere soils increased significantly(P<0.05)with the maturation of G.elata.The abundance of bacterial communities in rhizosphere soils increased significantly(P<0.05)with the maturation of G.elata,while the abundance of bacterial communities in non-rhizosphere soils showed a trend of increasing firstly from RG stage to WG stage,and then decreasing at SG stage.The microbiota was complex at different developmental stages of G.elata.Burkholderia-Caballeronia-Paraburkholderia was the most abundant bacterial genus at RG stage.The genus of unclassified Xanthobacteraceae increased rapidly from RG stage to WG stage and became the highest abundance of genus at WG stage.Uncultured Acidobacteria bacterium increased rapidly and was the highest abundance of genus at SG stage.The fungus Trichoderma was the dominant genus at RG stage,and the genus Coprinellus increased in abundance as the G.elata matures and was the most dominant genus at WG and SG stages.The abundance of the germinating fungus Mycena decreased from RG stage to SG stage of G.elata,while which of Armillaria increased from RG stage to WG stage and then decreased at the stage SG of G.elata.A total of 30 important microbial groups was found among bacteria and fungi respectively which could be used as biomarkers and which could best explain the changes in soil microbes during the developmental stages of G.elata.(5)The relationship between soil components,soil microorganisms,key genes and the content of active components in G.elata:a.There were significant correlations(P<0.05)between soil physicochemical properties and microbial communities.The descending order of significant influence of soil composition on bacterial communities was the following,Hg(r=0.83),Cd(r=0.80),SOM(r=0.77),Cr(r=0.75),EC(r=0.75),AN(r=0.74)and pH(r=0.66)(P<0.05).And descending order of significant influence of soil composition on fungal communities was the following:Hg(r=0.79),Cr(r=0.76),EC(r=0.67)and SOM(r=0.66)(P<0.05).b.The content of active compounds in G.elata was regulated by the combination of soil bacterial community structure and soil composition.Among the bacteria,Acidobacteria,Actinomycetes,Aspergillus and unclassified Bacteria played key roles in the synthesis and accumulation of active compounds.Among the fungi,Glomeromycota and Olpidiomycota played key roles in the synthesis and accumulation of active compounds.Among the soil components,AN,Cd,Cr,EC,Hg,pH,and SOM were the key abiotic factors regulating the synthesis of active compounds in G.elata.c.Rhizosphere soil microorganisms and soil physicochemical properties showed a complex pattern in regulating the expression of genes related to the synthesis of active compounds in G.elata.There were 8 dominant genera of bacteria and 9 soil physicochemical properties that showed significant correlations with genes(P<0.05).EC was one of the key soil physicochemical property indicators for the synthesis of active compounds in G.elata,with 13 unigenes controlling the synthesis of 6 enzymes that were significantly negatively correlated with EC(P<0.05).ADH-related genes directly involved in the synthesis of p-hydroxybenzyl alcohol were negatively correlated with most of the soil physicochemical properties and positively correlated with pH(P<0.05).CS-related genes involved in citric acid synthesis showed a negative correlation with AN.Burkholderia-Caballeronia-Paraburkholderia was positively correlated with aceE(P<0.05),a gene involved in the parishin-like chemosynthetic pathway.There were 2 fungal dominant generas(Archaeorhizomyces,unclassified Ascomycota)that were extremely significantly correlated(P<0.01)with 8 unigenes controlling the synthesis of 8 unigenes(aceE6,TE2,TE3,DLAT3,ADH6,ADH8,ADH10,ADH11).Most fungi and soil physicochemical properties were extremely significantly negatively correlated with ADH(P<0.01).In summary,the content of active compounds in G.elata varies at different developmental stages of it,with higher level of the active compounds content in the stage of P and the stage of RG than other stages.Soil composition and soil microorganisms may have a regulatory role in the synthesis of active compounds and the expression of related genes.This study reveals how abiotic factors in soil(soil composition),biological factors(microbial community structure and diversity),and gene expression interacted to influence the accumulation of active components in G.elata.This study provided a basic scientific support and guidance for the selection of the actual harvesting period,inoculation,wild cultivation environment of G.elata,disease prevention and ecological protection and the production practice in the future.