Transcriptome Analysis Of Tetrastigma Hemsleyanum Fully Developed Tuberous Roots In Different Seasons And Induction Of Its Hairy Root

Sanyeqing(SYQ,Tetrastigma hemsleyanum Diels et Gilg.),a perennial climbing liana with tuberous roots,is used as Chinese folk medicine.Especially,its tuberous root has many pharmacological activities,such as anti-inflammatory,anti-virus,detoxification and antipyretic,and is mainly used for treating fevers,children convulsions,pneumonia,etc.Flavonoids are the main components in the SYQ fully developed tuberous roots(FD-TRs)and are the material basis for medicine.FD-TR refers to the slow-growth stage of tuberous root after being induced and rapidly expanded with little morphological changes;tuberous root in this stage mainly transfers the energical components into storage materials and accumulates secondary metabolites.SYQ’s FD-TRs can grow for many years under natural conditions.However,little is known about the molecular basis of the growth and development and flavonoid metabolism in SYQ FD-TRs.In addition,SYQ’s hairy root is scarcely known,either.These facts inhibit the improvement of the artificial cultivation technique and the possibility of further study on the basis of metabolite biosynthesis in SYQ.Thus,some experiments were carried out in this study:(1)The transcriptomic profiling of FD-TRs in four seasons(Sp:spring;Su,summer;Au,autumn:and Wi,winter)detected the genes related to the flavonoids biosynthesis pathway,and then confirmed the expression level of some genes by real time PCR;(2)Optimizing the methods for total-flavonoids extraction from ultra-low temperature stored fresh FD-TRs of SYQ,combining with ultraviolet spectrophotometer and high performance liquid chromatography to determine the total flavonoids and 11 compounds of flavonoids and phenolic acids;(3)Establishment of the induction system of SYQ hairy roots.Several results were obtained:(1)After RNA sequencing by Illumina,12 RNA libraries with a total 78.54 Gb of raw data(average:6.55 Gb)were obtained,65,578 unigenes were ultimately obtained after removal of adaptor and low-quality reads.The total clean reads of the 12 libraries were subsequently de novo assembled,resulted in 144,096 transcripts with N50 value of 1,627 nt and median length 552 nt;and 65,578 unigenes with N50 value of 1,450 nt and the median length of 393 nt.The unigenes were aligned to sequences in six public databases,including NR,GO,SwissProt,KEGG,eggNOG,and Pfam.40,979(62.49%)of the 65,578 unigenes were matched to eggNOG(57.79%),NR(55.65%),GO(47.76%),Pfam(45.01%),SwissProt(37.08%),and KEGG(25.60%).Pearson correlation analysis showed that the correlation among the three repeated samples at each sampling time was higher,and the sampling method was suitable for RNA sequencing.12 samples were classed into three groups using PCA analysis:group 1:Sp;group 2:Su and Au;group 3:Wi.This results indicated that the expression pattern of most genes in Su and Au are similar.The results of Venn diagram showed that most unigenes were specially expressed in Au.(2)Differential expressed genes(DEGs)analysis,Wi vs Su(6,953)and Wi vs Au(6,752)have the most enriched DEGs.The Venn diagram results show that the number of common DEGs while compared Su,Au,and Wi to Sp was only 1,809 DEGs were specific for Wi,following 1586 for Au,and 1,126 for Su.Through KEGG enrichment analysis,the significant enrichment of DEGs in terms such as plant pathogen interaction,starch and sucrose metabolism,plant hormone signal transduction and other three gene groups,and all kinds of DEGs showed dynamic changes with season.According to the KEGG enrichment of DEGs,we found DEGs may participate in the biosynthesis of flavonoids.(3)To evaluate the expression levels of DEGs detected by transcriptomic sequencing,6 genes(PAL3,4CL1,C4H1,C4H2,CHI2,CHS3)were selected for validating RNA-seq data through qRT-PCR.The expression levels of 5 genes calculated by qRT-PCR were consitent with those from RNA-sequencing data(p<0.05;C4H2(p=0.08),slightly above 0.05),which indicates that sampling method and RNA-seq are suitable for the study on the transcriptomic patterns and biological processes of SYQ FD-TRs.(4)Optimizing the methods total-flavonoids extraction from ultra-low temperature stored fresh root tubers of SYQ by orthogonal method and single-factor analysis.For solvent ethanol,the optimal extraction conditions were 10-fold 65%ethanol(v/v)for 3 times,1 h each extraction time under ultrasound condition;For solvent methanol,the optimal extraction conditions were 20-fold 70%methanol(v/v)for 3 times,1 h each extraction time under ultrasound condition.(5)Among the SYQ FD-TRs in different seasons,the accumulation patterns of total flavonoids and 5 of the 6 flavonoid components(catechin,orientin,isoquercitrin,nictoflorin,astragalin),had similar patterns as the expression changes of some genes in flavonoid biosynthesis pathway,that their contents increased from Sp to Au,but decreased in Wi.In addition,the content of 3 of the 5 phenolic acids(chlorogenic acid,polydatin,piceatannol)also had the same accumulation pattern as 5 flavonoid components,the highest contents appeared in Au.(6)In order to establish hairy root induction from SYQ leaves.Agrobacterium rhizogenes C58C1 and ATCC15834 strains were used here.Only the C58C1 strain could successfully induce the hairy roots in our lab,and the hairy roots grew well in MS liquid medium without adding auxin.The total content of flavonoids of hairy roots was about one third of the 28 months tuberous roots.The results provide the most comprehensive transcriptional sequence resource and accumulation patterns of some components according different seasons for SYQ FD-TRs for the first time,and improve the understanding of the molecular mechanisms of the TR development and metabolisms of some medicinal components;The hairy root induction system of SYQ provides a useful method for the production of medicinal active ingredients from SYQ transgenic roots.