Screening Of Candidate Genes Related To Biosynthesis Of Ursane-Type Triterpenoid Saponins In Llex Pubescens

Aquifoliaceae plant Ilex pubescens (Chinese name Maodongqing), containing a large variety of ursane-type triterpenoid saponins, is used as a folk medicine in certain areas and provinces of South China. Modern pharmacological experiments have demonstrated the saponins of I. pubescens exhibit various important medicinal properties, including anti-tumor, anti-thrombotic and hepatoprotective activities. Despite their important properties, further studies on these triterpenoids are retarded because of insufficient supply and difficulties in purificatioa Synthetic biology, which has emerged in recent years, sheds a light on the production of the triterpenoids in an engineered microbial host Yet one of the key challenges is to obtain the BioBrick parts related to their biosynthesis. Until now, little has been known about the biosynthetic pathway of triterpenoid saponins in I.pubescens, especially the key enzymes involved in downstream of triterpenoid biosynthetic pathway, i.e. Cytochrome P450 monooxygenases (P450s) and Uridine diphosphate glycosyltransferases (UGTs). In this study, transcriptomic analysis and comparative transcriptomic analysis are applied to screen genes potentially involved in triterpenoid saponin biosynthesis in I.pubescens.I. asprella (Chinese name Gangmef), a sibling plant of I, pubescens, also contains abundant triterpenoid saponins similar as in I.pubescens. Andrographis paniculata (Burm.f) Nees, also known as Chuangxinlian, produces and stores a number of diterpeneoids and flavonoids but no triterpenoids. Comparative transcriptomic profiling of I. asprella and I.pubescens revealled ortholog pairs and specific genes. Further comparison of the P450 ortholog pairs with those of A. paniculata resulted in Ilex genus-specific P450s, narrowing down the number of candidate P450s underlying the biosynthesis of characteristic triterpenoid saponins. The results are listed as follows.1. Transcriptome sequencing output of I. pubescens.RNA extracted from various parts of I. pubescens was subjected to next-generation sequencing using Illumina HiseqTM 2500 platform, which generated a total of 6.14 Gb of clean reads and produced 68,688 unigenes. Of these,32,184 unigenes (46.86% of the total unigenes) were successfully annotated by comparison with the sequences in the major public databases Nr, Nt, Swiss-Prot and KEGG.2. Enrichment of secondary metabolite biosynthetic pathwaysIn I. pubescens transcriptome,128 unigenes (1.53% of yield) were assigned to five terpene-like pathways. While 16 and 18 unigenes were annotated to be involved in MYA and MEP pathway, respectively,18 unigenes were designated to be related to several intermediates (DMAPP, GPP, GGPP and FPP) in the upstream of triterpenoids biosynthesis pathway. As for the downstream,11,228 and 269 unigenes were annotated to OSCs,P450s and UGTs, respectively. By homo logy-based BLAST and phylogenetic analysis with well-characterized genes related to triterpenoid saponin biosynthesis,2 OSCs,18 P450s and 1 UGT were further screened as the most potential candidate genes.Unigenes encoding the key enzymes of flavone compounds biosynthesis were also found in I. pubescens transcriptome, which is consistent with the fact that the plant contains some flavonoids like daidzein and genistin.Moreover, transcription factors were detected in the transcriptome as well. Most of the transcription factors were annotated to AP2/ERF, BHLH, Myb and WRKY families, which are related to secondary metabolic biosynthesis.3. Comparative transcriptomic analysis12,756 putative ortholog pairs were identified between I. pubescens and I.asprella, while only 836and 1,183 unigenes were specific for each plant In triterpenoids biosynthetic pathway, only some HMGR and P450 genes were categorized to specific genes. In flavone biosynthetic pathway,2 CHS genes were annotated as special genes of I. pubescens,4 FLS are annotated as special genes of I.asprella.Further comparative analysis between P450s ortholog pairs andP450s from A. paniculata,38 unigenes of I. pubescens and 33 of I. asprella are classified as Ilex-genus specific P450 genes, which might be involved in the oxidation of triterpenoid skeleton.4. Identification of a mixed amyrin synthaseUnigene0045736 (named as IpASl), one of the 11 genes annotated as OSCs in I. pubescens, was successfully cloned and expressed in Saccharomyces cerevisiae INVScl. The analysis of the yeast extract by GC and GC-MS proved IpAS1 is a mixed amyrin synthase, producing α-amyrin and β-amyrin at ratio of 5:1, which is similar as its ortholog IaAS1 from I. asprella.This study first peeked into the transcriptome of I. pubescens using transcriptome sequencing and generated an extensive database, which contains enormous bioinformation about physiology and metabolism of I. pubescens. By the means of comparative transcriptomic analysis, a series of candidate unigenes involved in the biosynthetic pathway of triterpenoid saponins were identified, providing new insight into their biosynthesis at transcriptome level.