Comprehensive transcriptome profiling of Salvia miltiorrhiza for discovery of genes associated with the biosynthesis of tanshinones and phenolic acids

Wei Zhou,Qiang Huang,Guoyin Kai,Fenfen Huang,Shen Li,Zewen Zhou,Xiao Wu,Min Shi,Yao Wang,Mingquan Ding

doi:10.1038/s41598-017-10215-2

Abstract

Tanshinones and phenolic acids are crucial bioactive compounds biosynthesized in Salvia miltiorrhiza. Methyl jasmonate (MeJA) is an effective elicitor to enhance the production of phenolic acids and tanshinones simultaneously, while yeast extract (YE) is used as a biotic elicitor that only induce tanshinones accumulation. However, little was known about the different molecular mechanism. To identify the downstream and regulatory genes involved in tanshinone and phenolic acid biosynthesis, we conducted comprehensive transcriptome profiling of S. miltiorrhiza hairy roots treated with either MeJA or YE. Total 55588 unigenes were assembled from about 1.72 billion clean reads, of which 42458 unigenes (76.4%) were successfully annotated. The expression patterns of 19 selected genes in the significantly upregulated unigenes were verified by quantitative real-time PCR. The candidate downstream genes and other cytochrome P450s involved in the late steps of tanshinone and phenolic acid biosynthesis pathways were screened from the RNA-seq dataset based on co-expression pattern analysis with specific biosynthetic genes. Additionally, 375 transcription factors were identified to exhibit a significant up-regulated expression pattern in response to induction. This study can provide us a valuable gene resource for elucidating the molecular mechanism of tanshinones and phenolic acids biosynthesis in hairy roots of S. miltiorrhiza.

Highlights

Salvia miltiorrhiza Bunge (Dan shen in Chinese) is a traditional Chinese herb with significant medicinal and economic values
It is reported that some cytochrome P450 proteins, decarboxylase, dehydrogenase and reductase probably participate in the catalytic reaction steps in the downstream biosynthesis of tanshinones through analyzing the structure traits of downstream compounds
S. miltiorrhiza hairy root cDNA libraries generated 37,866,226 raw reads from the non-elicitor treated control, 42,574,704 from Methyl jasmonate (MeJA)-1h (Sample was induced by MeJA for one hour), 42,514,138 from MeJA-6h, 49,822,250 from yeast extract (YE)-1h and 54,383,514 from YE-12h

Summary

Introduction

Salvia miltiorrhiza Bunge (Dan shen in Chinese) is a traditional Chinese herb with significant medicinal and economic values. Genetic manipulation of active ingredients biosynthesis pathway in plants or hairy roots provide us a promising strategy[5], which rely on the detail understanding of the biosynthesis pathway and regulation mechanism. Some key enzyme genes in the upstream of biosynthesis pathways of bioactive compounds in S. miltiorrhiza have been cloned and characterized by conventional cloning methods with slow speed[8,9,10,11,12]. The exact information about the downstream enzymes catalyzing the several steps from ferruginol to tanshinones in tanshinone biosynthesis pathway and from rosmarinic acid to lithospermic acid in phenolic acids biosynthesis pathway is still unknown (Supplementary Fig. S1)

Objectives

Methods

Results