Comprehensive Analysis of the Triterpenoid Saponins Biosynthetic Pathway in Anemone flaccida by Transcriptome and Proteome Profiling

Abstract

Background: Anemone flaccida Fr. Shmidt (Ranunculaceae), commonly known as ‘Di Wu’ in China, is a perennial herb with limited distribution. The rhizome of A. flaccida has long been used to treat arthritis as a tradition in China. Studies disclosed that the plant contains a rich source of triterpenoid saponins. However, little is known about triterpenoid saponins biosynthesis in A. flaccida.Results: In this study, we conducted the tandem transcriptome and proteome profiling of a non-model medicinal plant, A. flaccida. Using Illumina HiSeq 2000 sequencing and iTRAQ technique, a total of 46,962 high-quality unigenes were obtained with an average sequence length of 1,310 bp, along with 1473 unique proteins from A. flaccida. Among the A. flaccida transcripts, 36,617 (77.97%) showed significant similarity (E-value < 1e-5) to the known proteins in the public database. Of the total 46,962 unigenes, 36,617 open reading frame (ORFs) were predicted. By the fragments per kilobases per million reads (FPKM) statistics, 14,004 isoforms/unigenes were found to be upregulated, and 14,090 isoforms/unigenes were down-regulated in the rhizomes as compared to those in the leaves. Based on the bioinformatics analysis, all possible enzymes involved in the triterpenoid saponins biosynthetic pathway of A. flaccida were identified, including cytosolic mevalonate pathway (MVA) and the plastidial methylerythritol pathway (MEP). Additionally, a total of 126 putative cytochrome P450 (CYP450) and 32 putative UDP glycosyltransferases were selected as the candidates of triterpenoid saponins modifiers. Among them, four of them were annotated as the gene of CYP716A subfamily, the key enzyme in the oleanane-type triterpenoid saponins biosynthetic pathway. Furthermore, based on RNA-Seq and proteome analysis, as well as quantitative RT-PCR verification, the expression level of gene and protein committed to triterpenoids biosynthesis in the leaf versus the rhizome was compared.Conclusion: A combination of the de novo transcriptome and proteome profiling based on the Illumina HiSeq 2000 sequencing platform and iTRAQ technique was shown to be a powerful method for the discovery of candidate genes, which encoded enzymes that were responsible for the biosynthesis of novel secondary metabolites in a non-model plant. The transcriptome data of our study provides a very important resource for the understanding of the triterpenoid saponins biosynthesis of A. flaccida.

Anemone medicinal plants: ethnopharmacology, phytochemistry and biology

“…Transcriptome sequencing and proteomic techniques were combined to comprehensively analyze the triterpenoid saponin biosynthetic pathway in Anemone flaccida 49 . A total of 126 putative cytochromes P450 (CYP) and 32 UDP glycosyltransferases were selected from 46,962 unigenes as candidates for triterpenoid saponin modifiers.…”

Proteomic Analysis of Chicken Skeletal Muscle during Embryonic Development

“…Our previous study identified differentially expressed chicken mRNAs and long non-coding RNAs (lncRNA) during embryonic skeletal muscle development, and provides a functional interaction network between lncRNAs and protein-coding genes (Li et al, 2017 ). However, proteins and mRNA levels do not often directly correlate and proteins also provides post-translational modified information and more direct evidence (Greenbaum et al, 2003 ; Zhan et al, 2016 ). Thus, we aimed to assess the protein expression profiles during embryonic skeletal muscle development for this study.…”

Proteomic Contributions to Medicinal Plant Research: From Plant Metabolism to Pharmacological Action

“…Improved and accurate identification of proteins via proteomics is available for various medicinal plants [ 34 ]. Jointly used with RNA sequencing, isobaric tags for relative and absolute protein quantitation (iTRAQ) helped identify four proteins belonging to the CYP718A subfamily of enzymes in Anemone flaccida that are essential for triterpenoid saponin biosynthesis [ 35 ]. The choice of sequence database is important for biosynthetic pathway analysis, as is indicated by a report that ascertained a series of enzymes playing a role in the final stages of artemisinin biosynthesis in Artemisia annua [ 36 ].…”