Gentiana Transcriptome Sequencing And Gene Expression Pattern Research

QinJiao is widely used as Chinese herbal medicine in the treatment of diverse diseases,such as diabetes,apoplexy,paralysis,and rheumatism.It also can resolve damp-heat and facilitate defecation,and its ethanol extract has an inhibitory effect on anthrax bacillus,staphylococci,typhoid bacillus,pneumococci,bacillus dysenteriae,and vibrio comma.The dried roots of the plant Gentiana macrophylla(Gentianaceae)is the main medicinal source of QinJiao.G.macrophylla is distributed mainly in China and Siberia and has been classed as state-three-level emphasized protective wild medicinal plant.For many years,researches about it are focused on the studies of its medicinal active ingredients extraction,pharmacological effect,germplasm resources,and also so on.Little research is on its genetic.However,the lack of genetic resources has been severely limiting the development on biosynthesis pathway of active ingredients,and also limiting the protection and development of the resources of wild with cell engineering or genetic engineering.To solve this problem,we first mined G.macrophylla gene resources and gene profiles of different organs with the second-generation high-throughput sequencing technology.Then the effect of MeJA on the iridoid glycosides compounds biosynthesis and the gene change in G.macrophylla roots were also investigated.This study not only greatly enrich the Gentiana genetic resources and lay the foundation for the further cloning of related genes and producing transformation Gentiana with biological engineering means,but also provide reference for the protection and development of this medicinal plant.Detailed results are as follows:(1)To obtain detailed and general data,we employed Solexa deep RNA sequencing(RNA-seq)to evaluate the transcriptome of G.macrophylla root,stem,leaves and flowers in different parts of the mixed material.About 2.45 Gb nucleotides were obtained and were assembled into 42,918 unigenes,among which 7,316 unigenes have the completed ORFs.A total of 32,141 unigenes(74.89%)were annotated.About 4,413 uni genes belonged to 51 families have transcription factor activity by BlastX analysis against Arabidopsis thaliana transcription factor database.In total,2,339 unigene can be mapped to 24 different secondary metabolic pathways especially,24 unigenes involved in MVA pathway,17 unigenes involved in MEP pathway,63 unigenes involved in iridoid pathway.Therefore,this broad coverage by these secondary metabolic genes provides more abundant information for examining the biosynthesis process of secondary metabolites in G.macrophylla.About 6440 SSR were found in 5442 unigene among 42,918 G.macrophylla unigene,and 843 SSR appears more than once.We also designed 5953 primers according these SSRs.(2)The gene expression profiles of G.macrophylla different organs were detected using Solexa/Hiseq2000,and 12,891,19,351,13,954 and 14,213 reference unigenes were detected in roots,stems,leaves and flowers samples.Low abundance genes were very rich in these four samples,which indicated a high sensitivity.Some organ-specific expression genes were also screened,which laid the foundation for further screening of organ-specific promoter.Last,genes involved in iridoid glycosides biosynthesis were also analyzed.(3)After treatment of 200 ?M exogenous methyl jasmonate on G.macrophylla,little changes were observed on contents of swertiamarin,swertiamarin and loganic acid.But the content of the gentiopicroside had a significantly changes and reach 1.57 times on 10d after treatment comparing with untreated sample.(4)Most up-regulated genes detected by digital gene expression profiling are resistance genes or unknown function genes in G.macrophylla roots after MeJA treatment.Most genes involved in iridoid glycosides biosynthesis was no significant difference comparing with untreated sample.This may be due to that main parts of the gentiopicroside biosynthesis are completed in other organ not in roots of G.macrophylla.But we also screened two WRKY transcription factors,which had significant difference between MeJA used or not,and may be involved in the regulation of the biosynthesis of gentiopicroside.