A set of miRNAs from Brassica napus in response to sulphate deficiency and cadmium stress

Summary MicroRNAs (miRNAs) are a class of short endogenous non-coding small RNAs that can base pair their target mRNAs to repress their translation or induce their degradation in organisms. However, whether miRNAs are involved in the global response to sulphate deficiency and heavy metal stress is unknown. In this study, we constructed a small RNA library from rapeseed (Brassica napus) treated with sulphate deficiency and cadmium (Cd2+), respectively. Sequencing analysis revealed 13 conserved miRNAs representing nine families, with five new miRNAs that have not been cloned before. Transcriptional analysis with RT-PCR showed the differential expression of these miRNAs under sulphate deficiency and Cd exposure. We have cloned five genes BnSultr2;1 and BnAPS1-4, which encode a low-affinity sulphate transporter and a family of ATP sulphurylases in B. napus, respectively. BnSultr2;1, BnAPS3 and BnAPS4 were first cloned from B. napus, and BnSultr2;1, BnAPS1, BnAPS3 and BnAPS4 were identified as the targets of miR395. Analysis with 5′-RACE and transformation of MIR395d into B. napus confirmed that all of them were the authentic targets of miR395. Our results support the importance of miRNAs in regulating plant responses to abiotic stresses and suggest that identification of a set of miRNAs would facilitate our understanding of regulatory mechanisms for plant tolerance to sulphate deficiency and heavy metal stress.