Transcriptomic and physiological analysis of atractylodes chinensis in response to drought stress reveals the putative genes related to sesquiterpenoid biosynthesis.

BACKGROUND: Atractylodes chinensis (DC) Koidz., a dicotyledonous and hypogeal germination species, is an important medicinal plant because its rhizome is enriched in sesquiterpenes. The development and production of A. chinensis are negatively affected by drought stress, especially at the seedling stage. Understanding the molecular mechanism of A. chinensis drought stress response plays an important role in ensuring medicinal plant production and quality. In this study, A. chinensis seedlings were subjected to drought stress treatment for 0 (control), 3 (D3), and 9 days (D9). For the control, the sample was watered every two days and collected on the second morning after watering. The integration of physiological and transcriptomic analyses was carried out to investigate the effects of drought stress on A. chinensis seedlings and to reveal the molecular mechanism of its drought stress response. RESULTS: The malondialdehyde, proline, soluble sugar, and crude protein contents and antioxidative enzyme (superoxide dismutase, peroxidase, and catalase) activity were significantly increased under drought stress compared with the control. Transcriptomic analysis indicated a total of 215,665 unigenes with an average length of 759.09 bp and an N50 of 1140 bp. A total of 29,449 differentially expressed genes (DEGs) were detected between the control and D3, and 14,538 DEGs were detected between the control and D9. Under drought stress, terpenoid backbone biosynthesis had the highest number of unigenes in the metabolism of terpenoids and polyketides. To identify candidate genes involved in the sesquiterpenoid and triterpenoid biosynthetic pathways, we observed 22 unigene-encoding enzymes in the terpenoid backbone biosynthetic pathway and 15 unigene-encoding enzymes in the sesquiterpenoid and triterpenoid biosynthetic pathways under drought stress. CONCLUSION: Our study provides transcriptome profiles and candidate genes involved in sesquiterpenoid and triterpenoid biosynthesis in A. chinensis in response to drought stress. Our results improve our understanding of how drought stress might affect sesquiterpenoid and triterpenoid biosynthetic pathways in A. chinensis.

Comparative transcriptome analysis reveals sesquiterpenoid biosynthesis among 1-, 2- and 3-year old Atractylodes chinensis.

BACKGROUND: Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood. RESULTS: In this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson's correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. CONCLUSION: The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

Cytogenetic Diversity of Simple Sequences Repeats in Morphotypes of Brassica rapa ssp. chinensis.

A significant fraction of the nuclear DNA of all eukaryotes is comprised of simple sequence repeats (SSRs). Although these sequences are widely used for studying genetic variation, linkage mapping and evolution, little attention had been paid to the chromosomal distribution and cytogenetic diversity of these sequences. In this paper, we report the distribution characterization of mono-, di-, and tri-nucleotide SSRs in Brassica rapa ssp. chinensis. Fluorescence in situ hybridization was used to characterize the cytogenetic diversity of SSRs among morphotypes of B. rapa ssp. chinensis. The proportion of different SSR motifs varied among morphotypes of B. rapa ssp. chinensis, with tri-nucleotide SSRs being more prevalent in the genome of B. rapa ssp. chinensis. We determined the chromosomal locations of mono-, di-, and tri-nucleotide repeat loci. The results showed that the chromosomal distribution of SSRs in the different morphotypes is non-random and motif-dependent, and allowed us to characterize the relative variability in terms of SSR numbers and similar chromosomal distributions in centromeric/peri-centromeric heterochromatin. The differences between SSR repeats with respect to abundance and distribution indicate that SSRs are a driving force in the genomic evolution of B. rapa species. Our results provide a comprehensive view of the SSR sequence distribution and evolution for comparison among morphotypes B. rapa ssp. chinensis.