Complex interplay among DNA modification, noncoding RNA expression and protein-coding RNA expression in Salvia miltiorrhiza chloroplast genome.

Haimei Chen,George Yuan,Jianhui Zhang,Chang Liu,Rogerio Margis

doi:10.1371/journal.pone.0099314

Abstract

Salvia miltiorrhiza is one of the most widely used medicinal plants. As a first step to develop a chloroplast-based genetic engineering method for the over-production of active components from S. miltiorrhiza, we have analyzed the genome, transcriptome, and base modifications of the S. miltiorrhiza chloroplast. Total genomic DNA and RNA were extracted from fresh leaves and then subjected to strand-specific RNA-Seq and Single-Molecule Real-Time (SMRT) sequencing analyses. Mapping the RNA-Seq reads to the genome assembly allowed us to determine the relative expression levels of 80 protein-coding genes. In addition, we identified 19 polycistronic transcription units and 136 putative antisense and intergenic noncoding RNA (ncRNA) genes. Comparison of the abundance of protein-coding transcripts (cRNA) with and without overlapping antisense ncRNAs (asRNA) suggest that the presence of asRNA is associated with increased cRNA abundance (p<0.05). Using the SMRT Portal software (v1.3.2), 2687 potential DNA modification sites and two potential DNA modification motifs were predicted. The two motifs include a TATA box–like motif (CPGDMM1, “TATANNNATNA”), and an unknown motif (CPGDMM2 “WNYANTGAW”). Specifically, 35 of the 97 CPGDMM1 motifs (36.1%) and 91 of the 369 CPGDMM2 motifs (24.7%) were found to be significantly modified (p<0.01). Analysis of genes downstream of the CPGDMM1 motif revealed the significantly increased abundance of ncRNA genes that are less than 400 bp away from the significantly modified CPGDMM1motif (p<0.01). Taking together, the present study revealed a complex interplay among DNA modifications, ncRNA and cRNA expression in chloroplast genome.

Highlights

Salvia miltiorrhiza, known as Chinese sage, tan shen, or danshen, is highly valued in traditional Chinese medicine for its roots or rhizomes [1]
This ideogram provides an overall picture of the transcriptome and DNA modifications in the S. miltiorrhiza chloroplast, which are described in detail below
As demonstrated in the current study, strand-specific RNASeq technology enables the detection of polycistrons and antisense noncoding RNA (ncRNA) (asRNA) from chloroplast transcriptome, which is essential for interpreting the functional elements of chloroplast genomes

Summary

Introduction

Known as Chinese sage, tan shen, or danshen, is highly valued in traditional Chinese medicine for its roots or rhizomes [1]. S. miltiorrhiza extracts are the main components of two of the ten best-selling traditional Chinese medical products, each with a total sale of around 100 million US dollars. More than 70 active components have been isolated from S. miltiorrhiza [2,3]. These active ingredients can be classified into two groups: lipid-soluble tanshinones and watersoluble phenolic acids. Compounds from both groups exhibit antioxidant, antitumor, anti-inflammatory, and antimicrobial properties [1]. A detailed understanding of the chloroplast genome structure, gene contents, and gene expression regulation is a prerequisite for the development of effective chloroplast-based transgenic systems

Methods

Results

Conclusion