RNA-Seq For DEG Analysis About Triterpenoid Saponin Synthesis And Transcript Profiling Of Ardisia Crenata Sims

Ardisia crenata Sims (Myrsinaceae family) is an important traditional medicinal herbs that yields triterpene saponins. Recently, triterpene saponins are used to antihuman immunodeficiency virus, antiviral, anti-oxidative effects and against to human tumor cell. Most recently, we found that triterpenoid saponins is similar to paclitaxel in terms of anti-cancer texture and antitumor activity. Although the biosynthetic pathway of triterpenoid saponins has been extensively studied, the genes involved in the pathway remain prooly understood. Additionally, the Ardisia crenata Sims as a non-model species, remains uncharacterized. Then the recent technique of RNA-seq, provides an opportunity to expand the identification of Ardisia crenata Sims gene, In this study, we performed the transcriptome profiles of two genotypes (treatments by Salicylic acid and Methyl Jasmonate) of Ardisia crenata Sims using RNA-seq.Conclusion:1. We are completed the sequencing of the different sample Ml and SI. After data quality control and cleaning,We received 12.06 Gb Clean Data. The Clean Data of every samples are 5.69 Gb, Q30 base percentage at 88.42% or more, sequencing in good quality.2. We received 54601 Unigene, including 14659 Unigene which length more than 1 Kb by De novo assembly with Trinity software. Then we compared all 54601 Unigene in NR, Swiss Prot, KEGQ COG, KOG, GO and Pfam database, received 31585 Unigene annotation results.3. Based on unigene library we analyzed the genetic structure, through the SSR analysis, received 9190 SSR markers. Also we predicts the CDS and SNP analysis, and get a number of good data.4. We use EBSeq software to analyze gene differentially expression quantity, obtained the differentially expressed genes set between these two samples, then we use a well-established Benjamini-Hochberg methods selected 3010 differentially expressed genes, which include 1532 up-regulated gene,1478 down-regulated gene. And then do pattern clustering to clearly show.5.3010 different genes (DEG Unigene) for functional annotation, received 2581 unigene annotation. In each database respectively:the COG (1068 unigene), GO (1937 unigene), KEGG (701 unigene), KOG (1386 unigene), Pfam (2038 unigene), the Swiss-Prot (2000 unigene), nr (2547 unigene).6. In GO database for functional annotation and analysis, enrichment of the concentration on the GO:0006721 nodes and terpenoids metabolism (Terpenoid metabolic process) related unigene has 256 genes, DEG Unigene has 31 genes.7. Classification statistical analysis in COG database, received 93 differentially expressed genes in biosynthesis of secondary metabolites, transportation, and decomposition metabolism, they are related to secondary metabolites(triterpenoid saponin) directly in Ardisia crenata sim.8. The 3010 differentially expressed genes in KEGG database annotation, received 741 metabolic pathways. There are 22 pathways about polyketides and terpenoids Metabolism (Metabolism of terpenoids and polyketides) potential affects the synthesis of triterpenoid saponins, and 8 pathways belongs to the "Terpenoid backbone biosynthesis", After enrichment analysis of differentially expressed, genes were in enrichment in main 96 pathways, the enrichment factor of "Terpenoid backbone biosynthesis" KO:00900 is 0.68, The smaller of enrichment factor the significant enrichment. This result suggests that the "Terpenoid backbone biosynthesis" KO: 00900 is directly related to the synthesis of triterpenoid saponins.9. We are sorting out the "Terpenoid backbone biosynthesis" KO:00900 pathways, and use different colour to marked enzyme which is related to up-regulated gene and down-regulated gene, then we cut multiples calculated using EBSeq software upgrades. The results confirmed 7 DEG Unigene related to key enzyme: 1-deoxy-D-xylulose-5-phosphate reductoisomerase, hydroxymethylglutaryl-CoA reductase (NADPH), mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, dimethylallyltranstransferase, (2E,6E)-farnesyl diphosphate synthase, respectively c41539.graph_c0 (-1.18); c31761.graph_c0 (-2.08); c31761.graph_c1(-2.20); c41096.graph_c0 (-1.87); c22737.graph_c0 (-1.21); c11481.graph_c0 (-1.35); c20493.graph_c0 (-1.76). Based on the performance of these DEG Unigene in the pathway, we can deduced that these DEG Unigene are inhibition key enzyme’s activity, then provide the research design for us to following experiment.Our study yielded new insights into the process of triterpenoid saponins biosynthesis in Ardisia crenata Sims. And firstly comprehensive reported the transcriptome information and provided a comprehensive uderstand of Ardisia. crenata sim or other related species. For further analyze, the encoding enzymes potentially involved in triterpenoid saponins biosynthesis could be rapidly identified by these candidate genes. This study plays an important role for depth development of Ardisia crenata sim’s medicinal value, and demonstrated the feasibility of using a combination of RNA-seq and DEG to indentify and study the genes involved in secondary metabolism for non-model medicial herb plant.