AUTHOR=Wen Lingling , Yun Xiaoyun , Zheng Xiasheng , Xu Hui , Zhan Ruoting , Chen Weiwen , Xu Yaping , Chen Ye , Zhang Jie 

TITLE=Transcriptomic Comparison Reveals Candidate Genes for Triterpenoid Biosynthesis in Two Closely Related Ilex Species

JOURNAL=Frontiers in Plant Science

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.00634

DOI=10.3389/fpls.2017.00634

ISSN=1664-462X

ABSTRACT=<p>Native to Southern China, <italic>Ilex pubescens</italic> and <italic>Ilex asprella</italic> are frequently used in traditional Chinese medicine. Both of them produce a large variety of ursane-type triterpenoid saponins, which have been demonstrated to have different pharmacological effects. However, little is known about their biosynthesis. In this study, transcriptomic analysis of <italic>I. pubescens</italic> and comparison with its closely related specie <italic>I. asprella</italic> were carried out to identify potential genes involved in triterpenoid saponin biosynthesis. Through RNA sequencing (RNA-seq) and <italic>de novo</italic> transcriptome assembly of <italic>I. pubescens</italic>, a total of 68,688 UniGene clusters are obtained, of which 32,184 (46.86%) are successfully annotated by comparison with the sequences in major public databases (NCBI, Swiss-Prot, and KEGG). It includes 128 UniGenes related to triterpenoid backbone biosynthesis, 11 OSCs (oxidosqualene cyclases), 233 CYPs (cytochrome P450), and 269 UGTs (UDP-glycosyltransferases). By homology-based blast and phylogenetic analysis with well-characterized genes involved in triterpenoid saponin biosynthesis, 5 <italic>OSC</italic>s, 14 <italic>CYP</italic>s, and 1 <italic>UGT</italic> are further proposed as the most promising candidate genes. Transcriptomic comparison between two <italic>Ilex</italic> species using blastp and OrthoMCL method reveals high sequence similarity. All <italic>OSC</italic>s and <italic>UGT</italic>s as well as most <italic>CYP</italic>s are classified as orthologous genes, while only 5 <italic>CYP</italic>s in <italic>I. pubescens</italic> and 3 <italic>CYP</italic>s in <italic>I. asprella</italic> are species-specific. One of <italic>OSC</italic> candidates, named as <italic>IpAS</italic>1, was successfully cloned and expressed in <italic>Saccharomyces cerevisiae</italic> INV<italic>Sc</italic>1. Analysis of the yeast extract by gas chromatography (GC) and gas chromatography–mass spectrometry (GC-MS) shows <italic>IpAS</italic>1 is a mixed amyrin synthase, producing α-amyrin and β-amyrin at ratio of 5:1, which is similar to its ortholog <italic>IaAS</italic>1 from <italic>I. asprella</italic>. This study is the first exploration to profile the transcriptome of <italic>I. pubescens</italic>, the generated data and gene models will facilitate further molecular studies on the physiology and metabolism in this plant. By comparative transcriptomic analysis, a series of candidate genes involved in the biosynthetic pathway of triterpenoid saponins are identified, providing new insight into their biosynthesis at transcriptome level.</p>