Transcriptomic response to cold of thermophilous medicinal plant Marsdenia tenacissima

Abstract

Extracts from Marsdenia tenacissima, involving tenacissoside H, I and G, have been used as remedies of cancer, inflammation and asthma. Low temperature serves as one of the main factors constrain the planting expansion and quality of M. tenacissima, but its functional mechanism has been known scarcely for the lack of genomic information and transcriptional profile. Here we investigated the transcriptomic responses of M. tenacissima under cold stress to gain insight into the molecular mechanism of low temperature sensitivity. Total RNAs were collected from samples obtained at 4-time points (after 0, 3, 6 and 48 h cold treatments with 4 °C, respectively), then used for library construction and sequenced on the Illumina Hiseq™ 4000 platform. Passing quality assessments, 500794 transcripts, and 206137 unigenes were de novo assembly out in Trinity v2.4.0, holding contig N50 of 2566 bp and unigene mean length of 754 bp. 44.20% of assembled unigenes were annotated to the well-known public protein database on a basis of sequence similarity. Using statistical comparison of the fragments per kilo base of transcript per million reads mapped (FPKM) values between conditions, 6082 group-specific differentially expressed genes (DEGs) were identified and considered as cold-responsive genes, which contained copious transcription factors and active secondary metabolism. Among them, 43 unigenes were constantly up-regulated expression along with cold time, which mainly implicated in the biosynthesis of secondary metabolites, carbon metabolism, RNA and DNA metabolism. Conversely, 21 unigenes involved in photosynthesis, cell wall, protein degradation, and transporters were downregulated continually with cold timescale. Experimentally, MtEF1α was chosen as the best housekeeping gene. Functional enrichments found that damaging of cold stress on M. tenacissima may be ascribed to inability of photosynthesis, ribsome processing, flavonoid biosynthesis and terpenoids degradation. Correlation analysis between cold induced transcription factors and tenacissoside biosynthesis-related genes indicated that 3β-HSD significant positively correlated with bHLH51, and 4-MSO with NF-YB, GRAS3, Trihelix, FAR1, MYB60, MYBS1, bZIP43. Further promoter clone found MYB-binding site in the promoter of 4-MSO. In view of the reported cold tolerance of MYB60, it is recommended as a potential candidate suitable for future molecular design of exaptation cultivation with high bioactive constituents.

Keywords: Cold stress; Marsdenia tenacissima; Polyoxypregnane glycosides; Transcription factor.