High-throughput Sequencing Of Full-length Transcriptome And Expression Profile Of Evodia Rutaecarpa Var.officinalis And Key Enzymes For The Synthesis Of Effective Components

Euodiae Fructus refers to the dried and nearly mature fruit of Evodia rutaecarpa(Jusss.)Benth.,Evodia rutaecarpa(Juss)Benth var.officinalis(Dode)Huang or Evodia rutaecarpa(Juss.)Benth.var.bodinieri(Dode)Huang,which is a traditional Chinese medicine in China.It has the functions of relieving pain,relieving cold and relieving vomiting,and helping to stop diarrhea.It is the raw material of many kinds of Chinese medicine preparations,such as changkang tablets,xiang tablets,zuojin tablets,wuji tablets and evodia soup.Euodiae Fructus has a long history of application and has a wide range of medicinal effects.Its effevtive components are evodiamine,rutecarpine,limonin and volatile oil.At present,the research of Euodiae Fructus is mainly focused on cuLtivation techniques,pharmacological effects and effective components,while the biosynthesis pathway of effective components and their key enzymes are rarely studied.This experiment uses High Performance Liquid Chromatography(HPLC)and Gas Chromatography-Mass Spectrometry(GC-MS)technology to acquire the variation in content of its effective components.Then,PacBio Sequel high-throughput sequencer was uesd to sequence transcriptome and Gene Expression profile for E.rutaecarpa(Juss)Benth var.officinalis(Dode)Huang in different periods,which acquired a lot of gene information.Based on the chemical content,fuLl length of the transcriptome and gene expression profile total expression pattern,screened biosynthetic key enzyme genes for evodiamine,rutecarpine,limonin and volatile oil.It can lay a foundation for elucidating the biosynthesis pathway of evodia,and provided the possibility for the biological manufacture and molecuLar breeding of evodiae by means of transgenic,synthetic biology and fermentation engineering.The main research resuLts are as follows:1.Variation trend of the content of effective components of E.rutaecarpa var.officinalis in different periodsHPLC and GC-MS were used to detect E.rutaecarpa var.officinalis in 6 periods,and the contents of six key effective components including evodiamine,rutecarpine,limonin,linalool,myrcene and(Z)-ocimene were acquired.The variation trend of evodiamine and rutecarpine content was almost the same,and it was in the rising state in the five periods,but there was a brief decline in the last period.The change of limonin content was always in a declining state.The content changes of myrcene,linalool and(Z)-ocimene in the six periodss were complex and different,but the overall content changes were small.Four periods were selected for high-throughput sequencing by comprehensively considering the accumuLation ruLe,growth characteristics and production and harvesting requirements of E.rutaecarpa var.officinalis.2.FuLl-length transcriptome sequencing and bioinformatics analysis of E.rutaecarpa var.officinalisFuLl-length transcriptome sequencing of mixed 4 periods evoica was carried out,and 73 Gb of original data were obtained.After clustering and correction,63,744 consistent sequences were obtained,and the high quality sequences reached 63,550,reaching 99.7%.After redundancy removal,19044 Unigene were finally obtained.After comparison with various databases,18704 Unigene were annotated,with a total annotation rate of 98.21%.Among them,NR database had the most annotations,reached 18662 genes,while KEGG database had the least annotations reached 8502 genes.Based on the analysis of biosynthetic genes,53 candidate genes of volatile terpenes such as myrcene,(z)-ocimene and linalool were found.2 candidate genes were found in the biosynthesis pathway of limonin(excluding the MVA and MEP pathways).The biosynthesis pathway of evodiamine and rutecarpine also found 53 candidate genes.All there were 108 candidate genes were found.3.RNA-seq sequencing and bioinformatics analysis of E.rutaecarpa var.officinalis expression profile in different periodsRNA-seq alone was used to sequence the expression profile of evodia in four periods,and 69446626-93598438 different reads were obtained for each expression profile,and the gene annotation rate of the whole transcriptome reached 70.53%to 73.40%.According to the difference analysis,the genes expression difference between WZY1 and WZY6 is the biggest,reched 11996 genes,and the genes expression difference between WZY3 and WZY4 is the smallest,reached 5943 genes.4.Screening of key enzyme genes in the biosynthesis of effective components of E.rutaecarpa var.officinalisBased on the co-expression model of "chemical content+fuLl-length transcriptome+gene expression profile",29 candidate genes were screened out from 108 candidate genes that might be involved in biosynthesis of the above six components.Among them,8 key enzyme genes of(Z)-ocimene biosynthesis were screened out.Six key enzyme genes of linalool biosynthesis;Five key enzyme genes of myrcene biosynthesis;Five key enzyme genes of limonin biosynthesis(including MVA and MEP pathway);FIve genes of the key enzyme in the biosynthesis of evodiamine and rutecarpine were found.