Reveal Of Flavonoids And Their Biosynthesis Candidate Genes Of Ziziphora Bungeana

Ziziphorae herba is a traditional Chinese medicine and ethnic medicine in Xinjiang.It has many effects such as strengthening the heart and removing dampness,regulating qi and resolving phlegm,eliminating inflammation and dispersing nodules.It is mainly used for heart disease,cold with fever,palpitation,insomnia,etc..Animal experiments and clinical studies have shown that total flavonoids extract of Ziziphorae herba has significant efficacy on protecting anoxia-injured myocardial cells.Some products including Tianxiangdan,capsule and tea of Ziziphorae herba have been developed.There are abundant wild resources of Ziziphorae herba in Tianshan and Altai mountains.However,the comprehensive development and utilization of Ziziphorae herba have been limited due to the prohibiting excessive digging of wild resources,the lack of cultivated varieties,the controversy on the original plant,and the unclear medicinal parts and specific active ingredients.In addition,in order to produce the active components efficiently in the future,it is very necessary to investigate the biosynthetic pathways of the main active components in Ziziphorae herba.Therefore,we carried out systematic research on the herbal textual,flavonoids and other components,the mechanism of linarin,the biosynthetic pathways of flavonoids,cloning of key genes,and their expression and functional verification.The methods of literature research and original plant identification were used in the herbal research of the Ziziphorae herba.The method of high performance liquid chromatography tandem mass spectrometry(HPLC-MS/MS)was used in the study of components in different tissues of the Z.bungeana Juz.(Zb).The methods of combining the metabonomics with network pharmacology and cell experiment in vitro were used in the exploration of the mechanism of effective flavonoids of the Zb.These methods of illumina high-throughput sequencing and single molecular real time sequencing(SMRT),and cDNA cloning,real-time fluorescent quantitative PCR and prokaryotic expression of E.coli,which were used in the study of flavonoid biosynthesis pathway and related genes.The results are as follows.First,the origin of Ziziphorae herba was verified through their names,original plant and medicinal parts.The name of Ziziphorae herba is unified as Xintahua.The medicinal parts are aerial part of Z.bungeana Juz.and Z.tomentosa Juz.and collected in florescence,then cut into sections to dry in the air.Zbis widely distributed in Xinjiang and Eurasia,which should be used more.The study on chemical constituents in different tissues of the Zbby HPLC-MS/MS implies that the aerial part can be used to replace the whole plant.The flavonoids and phenolic acids of the different medicinal parts including root,stem,leaf,flower and fruit were analyzed.43 chemical constituents were identified,which include 29 flavonoids,12 phenolic acids and 2 others.22 components in which are discovered in Zbfor the first time.These components will provide a reference for the separation and purification of the active components in Zb.Using PCA analysis and variance analysis,9 tissue-specific components were screened out,which could effectively distinguish 4 different medicinal parts of Zb.The identification method can provide quality control for the precise application of the medicinal parts of Zb.Linarin was the most abundant one of the major 12 flavonoids and accounted for49.17%,and the highest proportion was found in inflorescence.Therefore,using the aerial parts of the Zbwithout destroying its root has little effect on the main components of Zb,which implies that the aerial part can be used to replace the whole plant.The experiment also provides the basis for the sustainable utilization and resource protection of Zb.Second,the mechanism of multiple components(7 components),multiple targets(12targets)and multiple pathways(72 pathways)about anti-myocardial ischemia of Zbflavonoids has been preliminarily explored through combining the metabonomics studies with network pharmacology studies and cell experiment in vitro.The protective effect of Zbon myocardial ischemia was speculated as follows: linarin is hydrolyzed into acacetin in gastrointestinal tract,and the acacetin protected myocardial tissue through arachidonic acid metabolism(ACM).Third,the Zbtranscriptome and differential expression platforms were established through analyzing the database of Illumina high-throughput sequencing and SMRT transcriptome,and the biosynthetic pathways of major flavonoids in Zbwere obtained.397,182 assembled unigenes and the full-length cDNA sequences of 14 key enzymes in flavonoid biosynthetic pathway of Zbwere screened and cloned.Combining metabolomics with tissue-specific qPCR,12 biosynthetic pathways of flavonoids in Zbwere proposed.It is proposed that ZbPALs,Zb4CLl3,ZbCHS1 and ZbCHI1 are mainly involved in the biosynthesis of flavonoid skeleton,ZbFNSII is mainly involved in the accumulation of linarin,ZbFLS is mainly involved in the accumulation of some flavone glycosides,ZbANS is mainly involved in the biosynthesis of anthocyanins.Unlike linarin in Chrysanthemum indicum L.,linarin in Zbmight be synthesized by adding a methoxyl group to apigenin-7-O-rutinoside,which further verified the diversity of secondary metabolite biosynthesis in plant.Fourth,the prokaryotic expression systems of Zb4CL1,Zb4CL2 and Zb4CL3 were constructed.The three proteins were successfully expressed in recombinant E.coli.The in vitro catalytic experiments show that Zb4CL1 can only catalyze sinapic acid and Zb4CL3 can catalyze caffeic acid.This is different from the previous reports,which indicates to some extent the functional diversity of 4CL family.This experiment laid a foundation for the construction of eukaryotic expression system and related functional verification of 4CLs of Zbin the future.In summary,this study ascertained the origin of Z.herba,the components of flavonoids and their tissue distribution,obtained the database of transcriptome and biosynthetic pathways of major flavonoids,preliminarily analyzed the activities of some enzymes.These results provide technical means for the Z.herb to reform radically and accurate quality control,and lay a foundation for the rational development and utilization of Zb.