Identification of cold stress responsive microRNAs in two winter turnip rape (Brassica rapa L.) by high throughput sequencing

Abstract

Background: Low temperature is a major abiotic stress affecting the production of rapeseed in China by impeding plant growth and development. A comprehensive knowledge of small-RNA expression pattern in Brassica rapa under cold stress could improve our knowledge of microRNA-mediated stress responses.

Results: A total of 353 cold-responsive miRNAs, 84 putative novel and 269 conserved miRNAs, were identified from the leaves and roots of two winter turnip rape varieties 'Longyou 7' (cold-tolerant) and 'Tianyou 4' (cold-sensitive), which were stressed under - 4 °C for 8 h. Eight conserved (miR166h-3p-1, miR398b-3p, miR398b-3p-1, miR408d, miR156a-5p, miR396h, miR845a-1, miR166u) and two novel miRNAs (Bra-novel-miR3153-5p and Bra-novel-miR3172-5p) were differentially expressed in leaves of 'Longyou 7' under cold stress. Bra-novel-miR3936-5p was up-regulated in roots of 'Longyou 7' under cold stress. Four and five conserved miRNAs were differentially expressed in leaves and roots of 'Tianyou 4' after cold stress. Besides, we found two conserved miRNAs (miR319e and miR166m-2) were down-regulated in non-stressed roots of 'Longyou 7' compared with 'Tianyou 4'. After cold stress, we found two and eight miRNAs were differentially expressed in leaves and roots of 'Longyou 7' compared with 'Tianyou 4'. The differentially expressed miRNAs between two cultivars under cold stress include novel miRNAs and the members of the miR166 and miR319 families. A total of 211 target genes for 15 known miRNAs and two novel miRNAs were predicted by bioinformatic analysis, mainly involved in metabolic processes and stress responses. Five differentially expressed miRNAs and predicted target genes were confirmed by quantitative reverse transcription PCR, and the expressional changes of target genes were negatively correlated to differentially expressed miRNAs. Our data indicated that some candidate miRNAs (e.g., miR166e, miR319, and Bra-novel-miR3936-5p) may play important roles in plant response to cold stress.

Conclusions: Our work indicates that miRNA and putative target genes mediated metabolic processes and stress responses are significant to cold tolerance in B. rapa.

Keywords: Brassica rapa; Cold stress; Target gene; Turnip; microRNA.

Fig. 1

The responses of MDA content and POD activity to cold stress

Fig. 2

Sequence length distribution of sRNAs in winter turnip rape

Fig. 3

Analysis of identified conserve microRNAs in leaves and roots from two winter turnip rape varieties

Fig. 4

Secondary structures of differentially expressed novel miRNA precursors

Fig. 5

Differential expression of miRNAs under cold stress

Fig. 6

Gene Ontology (GO) categories and sub-categories of differentially expressed miRNA targets between the following samples: 7LCK versus7LTR, 4LCK versus 4LTR (A); 4RCK versus 4RTR, 7RCK versus 7RTR (B)

Fig. 7

qRT-PCR analysis of differentially expressed miRNAs and the target genes