Studies On The Effects Of Light Quality On The Accumulation Of Medicinal Compounds In Atractylodes Lancea Ana The Function Of AlHY5 Gene

Medicinal atractylodes is a dried rhizome of Atractylodes lancea（Thunb.）DC.and is a bulk herb commonly used in clinical practice.Its main medicinal components are sesquiterpene structured hinosel,β-eudesmol,atractylone and polyacetylene structured atractylin,all of which are volatile.It has the clinical efficacy of drying dampness and transporting spleen,dispersing dampness and removing paralysis,dispelling obscenity and relieving depression,strengthening stomach and nourishing deficiency,and nourishing liver and brightening the eyes,and its economic value has been increasing in recent years.The quality of herbs decreases when natural conditions are transplanted from the shaded wild to the full-light environment in the field.Light is one of the important environmental factors that cannot be ignored.The results of previous studies suggest that shade promotes the accumulation of the main medicinal components of A.lancea,and the mechanism of this promotion is not yet clear.Plants form a complex regulatory network in response to shade,and studies have shown that photoreceptors（phytochrome,cryptochromes）that absorb red and blue light sense the shade environment and then trigger a cascade response.Meanwhile,red and blue light are the main light sources for effective photosynthetic absorption.Therefore,in this study,red and blue light was chosen as the main light source treatment.In order to analyze the effect of light quality on the accumulation of sesquiterpenes and cryptophylls in the medicinal parts of A.lancea,it is crucial to understand the synthesis sites on the secondary metabolite pathway.Firstly,we analyzed the accumulation patterns of sesquiterpenes and atractylodin in different tissue parts of A.lancea and the expression of related genes in their biosynthetic pathways,and analyzed the consistency of synthesis and accumulation sites,so as to effectively target the key synthesis sites for the analysis of light quality-regulated synthesis of the medicinal components of A.lancea.Secondly,we established a transcriptome data platform for different tissue parts of A.lancea with different light quality regulation,and used chemical phenotypes combined with bioinformatics methods to analyze the data,aiming to explore the key enzyme genes and related transcription factors of terpenoids and polynylene biosynthesis pathway of A.lancea regulated by light quality.Finally,based on the results of transcriptome data screening and literature reports,the HY5 gene was selected for in-depth study.HY5 is a central transcription factor for light signal transduction,and studies have shown that HY5 can regulate the function of plant secondary metabolism.Therefore,we used the AlHY5 transcription factor of A.lancea as an entry point for the study of light quality regulation of the accumulation of major pharmacological components,and carried out the biological characterization and functional study of AlHY5,and predicted the AlHY5 binding site on the predicted region of synthase promoter in the synthesis pathway of major pharmacological components.The purpose of this study is to analyze the reasons for the changes of light quality on the content of the components of A.lancea,to lay the foundation for the study of the regulatory mechanism of light quality on the biosynthesis of sesquiterpenes and atractylodin in atractylodin,and finally to provide a reference for the study of shade in ecological cultivation.1 Research content（1）Study on the accumulation and synthesis of medicinal components in different tissue parts of A.lanceaThe volatile components of six tissue parts,namely rhizome,root,flower,bract,leaf and stem,were detected by GC-MS and quantified according to peak areas.By comparing the GC-MS chromatograms of different tissue parts and clustering heat map method,the accumulation pattern of volatile components and potent components was analyzed;the FPKM analysis of the synthesis enzymes on the synthesis pathway of potent components was performed by using the established transcriptome platform data of different tissue parts of wild A.lancea,and the expression pattern of synthesis enzymes of potent components was analyzed,and then the synthesis site of products was analyzed.The results of both analyses were combined to analyze the consistency of synthesis and accumulation sites.The possible transport relationship between roots and rhizomes was investigated by using the autofluorescence property of A.lancea.（2）Transcriptome analysis of different tissue sites in A.lancea regulated by light qualityIn this chapter,six groups of seedlings were treated with different ratios of red and blue light,and with the help of GC-MS instrument and RNA sequencing technology,the phenotypes of the contents of medicinal components in different treatments were obtained and the corresponding transcriptomic libraries were established.The transcriptome data were analyzed by PCA,differential gene screening,KEGG pathway and GO function enrichment,gene trend clustering,WGCNA,GSEA and other methods to screen and sort out the relevant enzyme genes and transcription factors involved in the pathway of light quality regulation of pharmacodynamic components.（3）Biological characterization and functional study of AIHY5 transcription factorsPhylogenetic clustering,multiple sequence alignment and genome annotation were used to determine and obtain the CDS sequence of AIHY5 gene;bioinformatics was used to analyze the physicochemical and spatial characteristics of the protein structure encoded by AIHY5;cloning vectors were used to clone the CDS of AIHY5;tobacco subcellular localization and arabidopsis hy5 mutant phenotype backfill experiments were conducted to study the nuclear localization and the function of AlHY5.The plantcare website predicts the AIHY5 binding site in the 2000 bp promoter region.upstream of terpenoid skeleton synthase.2 Results（1）Medicinal components has specific accumulation in different tissue of A.lancea,and the root,rhizome and leaf all have the potential to synthesize them.The results of the volatile components in different tissue parts of wild Atractylodes by GC-MS showed that there were obvious differences in the accumulation of specific pharmacodynamic components in six tissue parts:root,rhizome,stem,leaf,flower and bract.The rhizomes and roots were the main parts accumulating volatile components,and all of them accumulated the pharmacodynamic component atractylodin,while only the rhizomes specifically accumulated high amounts of the main pharmacodynamic component atractylon,and the stems,leaves,flowers and bracts accumulated little or no pharmacodynamic components.Genome annotation combined with KEGG mapped a clearer pathway of sesquiterpene biosynthesis in A.lancea,and initially screened 73 upstream genes and one ACC gene regulating sesquiterpene biosynthesis,which will lay the foundation for the study of sesquiterpene biosynthesis in A.lancea.The synthetases regulating the synthesis of sesquiterpene precursors were mainly expressed in the root and flower parts,but not in the rhizome.Combining chemical and transcriptional results,the synthesis of sesquiterpene precursors in A.lancea was not consistent with the site of sesquiterpene accumulation.Using laser confocal microscopy,the autofluorescence property of A.lancea substances was found,and the autofluorescence substances were initially identified to contain atractylon and atractylodin.Using this property,it was found that the transport process of pharmacological components existed in A.lancea plants,especially from roots to rhizomes,which has not been reported in the study of A.lancea.In summary,it is.speculated that the roots,flowers and leaves are the main sites for the production of sesquiterpene synthesis precursor substances or sesquiterpenes and atractylodin,and the components are transported to the rhizomes for storage through the transport mechanism of A.lancea itself,where the precursor substances of the pharmacodynamic components are further processed and eventually lead to higher accumulation of the pharmacodynamic components in the rhizomes.Therefore,the focus of research on the mechanism of light-regulated accumulation of potent ingredients cannot be limited to the rhizome,but the synthesis of potent ingredients in roots,leaves and flowers can be regulated by light,which affects the accumulation of potent ingredients in the rhizome.（2）A large number of genes involved in the regulation of light quality were screened by transcriptome data analysisThe GC-MS results showed that the light quality caused quantitative rather than qualitative changes in the differences of atractylenol and atractylenin contents in A.lancea;the participation of red light was beneficial to the accumulation of potent components,and the contents of atractylon and atractylodin showed an increasing trend with the increase of red light ratio.There were significant group differences between the white light group and the red-blue 3:1 group.The accumulation trends of atractylon and atractylodin in light quality treatment of white light>red light>blue light>red-blue 3:1 were used as the basis for gene screening,and the clustering branches with the same trend were screened in the gene trend change analysis.In the gene trend clustering analysis of light receptor genes and terpene skeleton synthase,the results showed that the blue light receptor and terpene skeleton part genes clustered into one branch in leaves,and the terpene skeleton part genes clustered into one branch with red and blue light receptors in rhizomes,and the terpene skeleton synthase genes and red and blue light receptors did not cluster together in roots.The leaves and rhizomes were more likely to respond to changes in light quality.Fifty-one enzyme genes and 35 transcription factors were screened in leaves by gene trend clustering,WGCNA,GSEA and other analysis methods.Among the enzyme genes,there was one for photosynthesis,PSAF;five for circadian rhythm,including three for GI,one for HD3A,and one for ADO3;three genes for fatty acid synthesis pathway,including two for MOD1 and one for S-ACP-DES6;19 genes for terpene skeleton,including HMG1（2）,HMGR2（2）,DXS2,ISPH,FLCY ICMEL2,Dehydrodolichyl diphosphate synthase 6,IPI2,At2g17570,Probable phytol kinase 3,Os07g0190000,dfrA,Os07g0190000,FTA,NUS1,At5g47720.Four sesquiterpene and triterpene synthesis pathways,including beta-amyrin synthase,Baccharis oxide synthase,PNA,dfrA;20 other pathways,including Ank3,RPL10,MSL2,BZR1,RPS11,Loxhd1,CSLC6,SHSP-1,CWINV1（2）,CSE,HSP23.6,HSP25.3,ARF16,D14,FLA2,AMPD,FAF2,SKIP27,PIP2-7.transcription factor genes in leaves included SHMT,HSP70（3 articles）,PK,PK,Beta_elim_lyase,Methyltransf₁1,20G-FeII_Oxy,AA_kinase,CCT（2）,ADK,zf-AN1,Aldose_epim,ChaC,CDF3,SAP12,NLP1,EF2,ERD2B,ERD2A,CMT3,THA1,CRF5,AVT1,PIF3,HY5（2）,APRR1,CHS2（2 articles）,and COP1.Twenty-three enzyme genes and 27 transcription factors were screened in the rootstock.Among the enzyme genes,15 were for fatty acid synthesis,including KAS1,LACS2,At3g45770,ARPC2A,S-ACP-DES6,fabZ,BCCP2,Picea abies,CAC3,S-ACP-DES6,KAS2,FATA,CAC3,ACC1,LACS4;two were for terpene skeleton synthesis.including ICMEL2,HMGR2;1 sesquiterpene and triterpene synthesis pathway,including PNA;and 5 pathways,including OS9,NAP1,CYCT1-3,NXT3,NPF4.3.Among them,transcription factor genes in rhizome include ATHB-12,BBP,SPA3,TOP2,COL5,MYB12,PIP5K7,MJ0531,AHK2,TTN,RDM3,TRRAP,CLPB1,SR45A,PRPF8,PMA1,ATX1,PHYB,HSPD,HSP70,HIPP26,TUBA,APRR5,ATHB-7,and CYP72A129.（3）AlHY5 has nuclear localization and light signal transduction functionsThe results of bioinformatics analysis showed that the two AlHY5 sequences were highly homologous,and both were homodimers in the three-dimensional structure.The results of physicochemical properties and three-dimensional structure showed that the two have similarity in protein properties and spatial structure.In addition,the sequences of the bZIP structural domain proteins bound to DNA were consistent.Tobacco subcellular localization results showed that both of the 2 AlHY5 transcription factors were localized in the nucleus,thus demonstrating that AIHY5 gene has a nuclear localization function,which is consistent with the reported species HY5 results.The phenotypic backfill results of the Arabidopsis hy5 mutant demonstrated that AlHY5 could function as a light signal transducer in Arabidopsis,thus proving that AlHY5 has functional activity.The subcellular localization of tobacco and the phenotypic complementation of abidopsis hy5 demonstrated that AlHY5 is a transcription factor that transmits light signaling function,which is consistent with the results of related studies.The key enzyme genes of terpene skeleton synthesis HMGS,HMGR,DXS and DXR containing the G-box binding site of HY5 were screened using the plantcare website,which provided a research basis for subsequent in-depth in vitro and in vivo binding validation experiments of AlHY5 transcription factor and sesquiterpene biosynthetic enzyme genes.3 Conclusion（1）There are obvious composition-specific differences among the six tissue parts:root,rhizome,stem,leaf,flower and bract.The rhizomes and roots stored abundant volatile products,which were dominated by terpenoids.The roots were dominated by the accumulation of non-oxygenated sesquiterpenes such as Modephene,α-Isocomene,β-Isocomene,and β-Caryophyllene,while the rhizomes were dominated by the accumulation of oxygenated sesquiterpenes such as Hinesol,β-eudesmol,and atractylon.All parts have the potential to synthesize sesquiterpenes,and the root is the main part for the synthesis of sesquiterpene components,and it is speculated that the accumulation of oxidized sesquiterpene components in the rhizome may be closely related to the root.（2）Compared with blue light,red light was favorable for the accumulation of atractylon and atractylodin.The expression of red light signals PHYB and HY5 had the same trend as the accumulation of atractylon and atractylodin,which has potential regulatory research value.（3）The two sequences of AlHY5-1 and AlHY5-2 cloned in A.lancea are structurally similar homodimeric structures with conserved functional structural domains and located in the nucleus,which can play the function of light signal transduction in plants,laying the foundation for the subsequent in vitro and in vivo functional verification of A.lancea.