Construction Of Transcriptome Database Of Dioscorea Zingiberensis And Comparative Transcriptome Analysis Identifies Putative Genes Involved In Dioscin Biosynthesis

As a spirostanol-type steroidal saponin,dioscin has one glucose residue and two rhamnose groups linked to its aglycon diosgenin.Diosgenin has a wide range of pharmacological effectsincluding anti-tumor,anticancer and cholesterol-reducing properties.Moreover,diosgenin serves as a precursor of more than 300 steroidial drugs.Therefore,diosgenin has been called as“drug gold”because of its medical importance.To date,it is not completely clear how diosgenin is biosynthesized in plantsDiosgenin has been detected in many plant species while amoung the diosgenin-producing plants the Dioscorea zingiberensis contains the highest amount of diosgenin.In this study,the transcriptome database derived from the rhizome and leaf tissues of the Dioscorea zingiberensis under two developmental stages was constructed.By an extensive analysis on the D.zingiberensis transcriptomic data,the putative genes involved in disocin biosynthesis were identified.The main findings of this study were summarized as follows:?1?A total of 45367360,5579810 and 42564054 raw reads were obtained from the three cDNA libraries of Aug_R,Aug_L,and Nov_R,respectively.After filtering the low-quality sequences,a total of clean reads of 43234636 for the Aug_R,53330426for the Aug_L,and 40686226 for theNov_R were finally obtained..By assembling the three libraries,a total of 176406 transcripts and 143245 unigenes were acquired.The N₅₀0 value of the unigenes is 814bp and a total of 10655 unigenes?7.43%of the total unigenes?were successfully annotated by a blast searching against several public databases including NR,NT,PFAM,KOG,SwissProt,KEGG and GO databases;there were 74908 unigenes?52.29%?unigenes that were successfully annotated against at the least one of the above databases,and the searching of the NR database led to the successful annotation of 50797 unigenes which account for 35.46%of the total numbers of the unigenes.?2?Putative genes in the biosynthetic steps from FPP to 2,3-oxidosqualene in the upstream pathway to dioscin synthesis had been found in the transcriptome.For example,the unigene c66823_g1 was annotated as HMGR from the MVA pathway while the unigene c71143 probably codes for DXR from the MEP pathway.?3?In the pathway from from 2,3-oxidosqualene to chelosterol in disocin biosynthesis,five unigenes?c73551_g1,c70423_g1,c62143_g1,c57629_g1 and c56203_gl?with complete ORFs were detected and they putatively encodeCAS,SSR-2,SMO1,CPI and CYP51,respectively.?4?Cytochrome P450s catalyze site-specific oxidations in the pathway from cholesterol to diosgenin,A total of 485 unigennes from the transcriptome were annotated as CYP450 genes,of which twenty two P450 candidates were discovered to be potential genes that could play roles in the oxidations for diosgenin biosynthesis based on their positive correlations to diosgenin accumulation across the different tissues under different developmental stages.?5?Glucosyltransferases and rhamnosyltransferases might play roles in the conversion from diosgenin to disocin.From the transcriptome,there were six unigenes that were annotated as sterol 3-?-glucosyltransferases and two unigenes?c69762_g2 and c60525_g2?as the rhamnosyltransferase genes.Among the3-?-glucosyltransferase unigenes,only the c60958_g1 and c59129_gl unigenes were presented at a full length.The c60958_g1 and c59129_gl showed amino acid similarities of 42%and 46%to a previously published 3-?-glucosyltransferase which catalyzes the 3-?-glucosylation of diosgenin.?6?From the transcriptome,a total of 2491 unigenes were annotated as transcription factors,of which 299 unigenes were differentially expressed between the three cDNA libraries,including 50 unigenes belonging to ZinC family,18 unigenes to MYB family,11 unigenes tothe bHLH family,29 unigenes to the AP2/ERF family,15unigenes to the Bzipi and 18 unigenes to the MRKY family.