De novo transcriptome sequencing and gene expression profiling of Magnolia wufengensis in response to cold stress

Magnolia wufengensis is a new species of Magnolia L. and has considerable ornamental and economic value due to its unique characteristics. However, because of its characteristic of poor low temperature resistance, M. wufengensis is hardly popularization and application in the north of China. Furthermore, the mechanisms of gene regulation and signaling pathways involved in the cold-stress response remained unclear in this species. In order to solve the above-mentioned problems, we performed de novo transcriptome assembly and compared the gene expression under the natural (25 °C) and cold (4 °C) conditions for M. wufengensis seedlings.

More than 46 million high-quality clean reads were produced from six samples (RNA was extracted from the leaves) and were used for performing de novo transcriptome assembly. A total of 59,764 non-redundant unigenes with an average length of 899 bp (N50 = 1,110) were generated. Among these unigenes, 31,038 unigenes exhibited significant sequence similarity to known genes, as determined by BLASTx searches (E-value ≤1.0E-05) against the Nr, SwissProt, String, GO, KEGG, and Cluster of COG databases. Based on a comparative transcriptome analysis, 3,910 unigenes were significantly differentially expressed (false discovery rate [FDR] < 0.05 and |log₂FC (CT/CK)| ≥ 1) in the cold-treated samples, and 2,616 and 1,294 unigenes were up- and down-regulated by cold stress, respectively. Analysis of the expression patterns of 16 differentially expressed genes (DEGs) by quantitative real-time RT-PCR (qRT-PCR) confirmed the accuracy of the RNA-Seq results. Gene Ontology and KEGG pathway functional enrichment analyses allowed us to better understand these differentially expressed unigenes. The most significant transcriptomic changes observed under cold stress were related to plant hormone and signal transduction pathways, primary and secondary metabolism, and photosynthesis. In addition, 113 transcription factors, including members of the AP2-EREBP, bHLH, WRKY, MYB, NAC, HSF, and bZIP families, were identified as cold responsive.

We generated a genome-wide transcript profile of M. wufengensis and a de novo-assembled transcriptome that can be used to analyze genes involved in biological processes. In this study, we provide the first report of transcriptome sequencing of cold-stressed M. wufengensis. Our findings provide important clues not only for understanding the molecular mechanisms of cold stress in plants but also for introducing cold hardiness into M. wufengensis.