Protein sequences of 4 characterized CYP450s and 4 UGTs, which were reported to be involved in the triterpenoid saponin biosynthetic pathway, were selected as objectives to run BLASTp analysis against the protein sequences that were translated from the raw reads data (E value threshold set at 10−5).
Triterpenoid saponins were isolated from various tissues of I. aprella like roots and leaves, but this study is restricted to root tissue. A more thorough analysis of different plant tissues coupled with metabolomic data would help in building a global picture of Ilex triterpenoid biosynthesis and perhaps find novel candidate genes which would otherwise be difficult with sequence-homology-based searches.
The transcriptome of the I. asprella root was obtained using RNA-Seq, resulting in many unigenes. The unigene dataset that was generated in this study provides a significant resource for further molecular studies of I. asprella, especially for characterising candidate genes in the biosynthetic pathways of triterpenoid saponins. Using appropriate approaches, a series of candidate genes were identified and were consequently analysed for expression patterns and phylogenetic relationships. A comprehensive bioinformatics analysis contributed to a better understanding of the candidate genes and to a reliable design for further research. The putative genes identified in I. asprella will be cloned and characterised in further studies.
Unlike OSCs, the identification of new CYP450s and UGTs involved in the biosynthesis of triterpenoid saponins is beset with difficulties owing to the poor relationship between gene homology and functions of these two gene families. In this study, gene annotation provided a great number of putative CYP450s and UGTs. The candidate number was narrowed down to a few homologous unigenes by applying direct, homology-based screening of characterised genes. However, it is unknown whether these candidates are actually involved in the biosynthesis of triterpenoid saponins. Therefore, additional strategies should be engaged to identify credible candidate CYP450s and UGTs. The combination of exlicitor-induced expression regulation and co-expression analysis with OSC [,,] would contribute to identification of the targeting CYP450s and UGTs in I. aprella.
The identification of genes involved in the biosynthetic pathway of terpenoid saponins has been achieved by using many different techniques, including the next-generation sequencing technology (NGS). A recently developed technique called RNA Sequencing (RNA-Seq) for transcriptome profiling using NGS techique has shown great potential for functional gene mining for non-model plants [,] and can help in the discovery of rare transcripts in the transcriptome owing to its great sequencing depth. Since no appropriate reference is available for the non-model plants, de novo assembly is the only option for sequence assembly . Therefore, RNA-seq utilising Illumina next-generation sequencing was used for the transcriptomic study of the I. asprella root and the detection of candidate genes involved in the triterpenoid saponin biosynthetic pathway as presented in this study.
Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins.