Mining And Identification Of Verbascoside Biosynthesis Associated Genes Based On Transcriptome Sequencing Of Rehmannia Glutinosa

Rehmannia glutinosa is an important medicinal plant of the Scrophulariaceae family.Because of its important medicinal value and food value,it has been widely used in clinics for a long time.It contains catalpol,calorie glycosides,flavin and carbohydrates and other compounds.Among them,verbascoside as one of the index components in Rehmannia glutinosa,has anti-inflammatory,anti-oxidation,anti-tumor,neuroprotective and memory-enhancing and other physiological activities.Due to its widespread distribution in plants,few toxic side effects and strong biological activities,It is favored by researchers and becomes a new target for drug development.At present,verbascoside is mainly extracted from medicinal plants.However,medicinal plants' compoundsare very complex,so it is very difficult to extract and separate verbascoside from them,and its development and applicatins are seriously hindered,while medicinal plants resources for verbascoside production increasingly scarce due to human over development and uses for medicinal plants and medicinal plant-planting arable land reduction.Therefore,more attentionhas been paid to the pathway of verbascoside biosynthesis and its related enzyme gene as an important alternative to develop verbascoside.Meanwhile,the tentative pathway of verbascoside biosynthesis was proposed,and several verbascoside biosynthesis-related enzyme genes were reported.Nevertheless,So far its true pathway of verbascoside biosynthesis has not been reported,while more verbascoside biosynthesis-related enzyme genes remain to discover.In this study,the pathway of verbascoside biosynthesis was established,in which some verbascoside biosynthesis–associated enzyme genes were mined based on in Rehmannia glutinosa transcriptome sequencing.Eight of these genes in the Rehmannia glutinosa transcriptomic dataset were validated by RT-PCR,and their roles forverbascoside biosynthesis were verified by qRT-PCR.In addition,the over expression vector of cinnamic acid-4-hydroxylase gene RgC4 H,one of the above eight,was constructed by recombinant DNA technique.The main results are as follows:1 ? Ttranscriptome sequencing of a combined Rehmannia glutinosa tuberous roots from its multidevelopmental stages on the Illumina HiSeq2500 plat form generated a substantial EST data set composed of 1,498 million raw reads.Based on them,96,961 Final unigenes with the average length of 678.3bp were assembled,and 16294 EST-SSRs were screened.Based on sequence similarity searches against the public sequence databases such as Nr,COG,GO,KEGG,the sequences were annotatedfirstly and then were subjected to GO and KEGG based analysis.Out of 96,961 Final unigenes,42,785 was annotated with a percentage of 44.7%,11848 Final unigenes were mapped to 280 pathways containing 2019 Rehmannia glutinosa enzymes.2?Based on the comparison of 280 KEGG pathways and the tentative pathway of verbascoside biosynthesis proposed by animal isotope labeling test,4 candidate KEGG pathways were screened out,which are phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism way,tyrosine metabolic pathway map,phenylpropanoid biosynthesis pathway,and then the pathway of verbascoside biosynthesis was ablished,in which 19 candidate verbascoside biosynthesis-associated enzymes(chorismate mutase,arogenate/prephenate dehydratase,prephenate dehydrogenase,aspartate-prephenate aminotransferase,aspartate aminotransferase,chloroplastic,aspartate aminotransferase,mitochondrial,phenylalanine ammonia-lyase,4-coumarate--CoAligase,Cinnamate-4-hydroxylase,shikimate O-hydroxy cinnamoyl transferase,coumaroyl quinate 3'-monooxygenase,caffeoylshikimate esterase,cyclohexadienyl dehydratase,arogenate dehydrogenase,tyrosinase,tyrosine decarboxylase,dopamine beta-monooxygenase,primary-amine oxidase,aryl-alcohol dehydrogenase)were found out.According to the correlations between these candidate enzymes and Congtigs or Unigenes,19 candidate verbascoside biosynthesis associated enzymes corresponding genes were determind.3?Based on the correlations between the Congtigs or Unigenes and ORFs,their corresponding ORFs were determined.Then,primers for RT-PCR were designed and synthesized according to their base sequences.The following 8 candidate genes were validated by RT-PCR in the Rehmannia glutinosa data set prephenic acid dehydratase(ADT),aspartate aminotransferase(ASP5),4-coumarate: coenzyme A ligase(4CL),primary amine Oxidase(AOC3),Cinnamate-4-hydroxylase(C4H),shikimate O-hydroxycinnamoyl transferase(HCT),coumaroylquinate(coumaroylshikimate)3'-monooxygenase(C3'H)aspartate aminotransferase(GOT2).4?In order to verify the correlation between the eight selected genes and verbascoside biosynthesis,the functions of 8 candidate genes were predicted by bioinfomatics analysis,qRT-PCR was used to detect the expressions of eight genes in two different verbascoside content tuberous roots at two differentdevelopmental stages of cultivar Wen85-5,and in the same developmental stage'sdifferent tuberous roots and leaves of two different verbascoside content cultivars such as cultivar Wen85-5 and Beijing No.3.The differences in their expression levels suggest decisive roles for theseenzymes in verbascoside biosynthesis.5?In order to further verify the correlation between the eight genes and verbascoside biosynthesis,their overexpressions are going to be investigated.Here,based on the transcriptomic data of Rehmannia glutinosa,the complete CDS region of cinnamic acid-4-hydroxylation enzyme RgC4 H was successfully cloned and analyzed by bioinformatics.According to the multiple cloning sites of plant expression vector pCAMBIA1300,the primers with suitable restriction sites,either with one,for RT-PCR were designed and synthesized,and thenits full-length cDNA sequence,whose complete ORF was cloned intoplant expression vector pCAMBIA1300.Its sequencing and double digestionconfirmed that the recombinant vector was successfully constructed,named pCAMBIA1300-RgC4H-GFP.The results will accelerate the understanding of the genetic basis of verbascoside biosynthesis,and lay the foundation for genetic engineering breedingin R.glutinosa and the research and development of verbascoside via metabolic engineering in the future.