De novo Sequencing and Transcriptome Analysis Reveal Key Genes Regulating Steroid Metabolism in Leaves, Roots, Adventitious Roots and Calli of Periploca sepium Bunge.

Jian Zhang,Guangjian Han,Yunhe An,Shanying Wang,Lin Ma,Xiankuan Li,Fuping Lu,Xinglin Li,Xiaoxing Su

doi:10.3389/fpls.2017.00594

Abstract

Periploca sepium Bunge is a traditional medicinal plant, whose root bark is important for Chinese herbal medicine. Its major bioactive compounds are C21 steroids and periplocin, a kind of cardiac glycoside, which are derived from the steroid synthesis pathway. However, research on P. sepium genome or transcriptomes and their related genes has been lacking for a long time. In this study we estimated this species nuclear genome size at 170 Mb (using flow cytometry). Then, RNA sequencing of four different tissue samples of P. sepium (leaves, roots, adventitious roots, and calli) was done using the sequencing platform Illumina/Solexa Hiseq 2,500. After de novo assembly and quantitative assessment, 90,375 all-transcripts and 71,629 all-unigenes were finally generated. Annotation efforts that used a number of public databases resulted in detailed annotation information for the transcripts. In addition, differentially expressed genes (DEGs) were identified by using digital gene profiling based on the reads per kilobase of transcript per million reads mapped (RPKM) values. Compared with the leaf samples (L), up-regulated genes and down-regulated genes were eventually obtained. To deepen our understanding of these DEGs, we performed two enrichment analyses: gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Here, the analysis focused upon the expression characteristics of those genes involved in the terpene metabolic pathway and the steroid biosynthesis pathway, to better elucidate the molecular mechanism of bioactive steroid synthesis in P. sepium. The bioinformatics analysis enabled us to find many genes that are involved in bioactive steroid biosynthesis. These genes encoded acetyl-CoA acetyltransferase (ACAT), HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MDD), isopentenylpyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), cycloartenol synthase (CAS), sterol C-24 methyltransferase (SMT1), sterol-4alpha-methyl oxidase 1 (SMO1), sterol 14alpha-demethylase (CYP51/14-SDM), delta(14)-sterol reductase (FK/14SR), C-8,7 sterol isomerase (HYD1), sterol-4alpha-methyl oxidase 2 (SMO2), delta(7)-sterol-C5(6)-desaturase (STE1/SC5DL), 7-dehydrocholesterol reductase (DWF5/DHCR7), delta (24)-sterol reductase (DWF1/DHCR24), sterol 22-desaturase (CYP710A), progesterone 5beta-reductase (5β-POR), 3-beta-hydroxysteroid dehydrogenase (3β-HSD). This research will be helpful to further understand the mechanism of bioactive steroid biosynthesis in P. sepium, namely C21 steroid and periplocin biosynthesis.

Highlights

IntroductionThe dried root bark of P. sepium, known as “Cortex Periplocae,” is used as a Chinese medicinal herb for the treatment of rheumatism, cancer, inflammation, and cardiac failure (Xu et al, 1990; Umehara et al, 1995; Yin et al, 2009; Ding et al, 2014)
Periploca sepium Bunge is a traditional medicinal plant of the Asclepiadaceae family that is widely distributed in the northern temperate regions of China
Our analysis revealed a general absence of up-regulated genes in roots; only a few genes were significantly upregulated in roots (Table S14), a result similar to that for upstream biosynthesis

Summary

Introduction

The dried root bark of P. sepium, known as “Cortex Periplocae,” is used as a Chinese medicinal herb for the treatment of rheumatism, cancer, inflammation, and cardiac failure (Xu et al, 1990; Umehara et al, 1995; Yin et al, 2009; Ding et al, 2014). Both C21steroids and periplocin, a kind of cardiac glycoside, are the major bioactive constituents occurring in the root bark of P. sepium. Some genes are related to the biosynthesis of pregnane derivatives, such as the genes encoding cholesterol monooxygenase/side chain-cleaving enzyme (SCCE), 5βPOR, 3βHSD, delta 5-delta 4-ketosteroid isomerase (KSI), pregnane 14βhydroxylase (P14β), and others (Kreis and Müller-Ur, 2012; Zheng et al, 2014)

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