Genome-wide identification of Cd-responsive NRAMP transporter genes and analyzing expression of NRAMP 1 mediated by miR167 in Brassica napus

In plants, metal transporters are responsible for metal uptake, translocation and homeostasis. These metals include essential nutrients such as zinc (Zn) and manganese (Mn) or non-essential metals like cadmium (Cd) and lead (Pb). Although a few metal transporters have been well characterized in model plants, little is known about their functionality in rapeseed (Brassica napus). In the study, 22 NRAMP transporter genes from B. napus genome were identified and annotated using bioinformatics and high-throughput RNA-sequencing (RNA-seq). Based on the sequence identity, these NRAMP transporters can be classified into 6 subfamilies. RNA-seq analysis revealed that 19 NRAMP transporters were detected and some of the genes were well confirmed by qRT-PCR. Ten NRAMP transporters (45.5%, 10/22) were found to be differentially expressed (> 2 fold change, p < 0.05) under Cd exposure. As an example, we specified expression of BnNRAMP1b under Cd exposure. BnNRAMP1b is a constitutive gene expressing throughout all development stages including seedlings, vegetative tissue, flowers and siliques. Expression of BnNRAMP1b can be strongly induced in seedlings exposed to 80, 160 and 240 mu M Cd. To define whether BnNRAMP1b was specific for Cd transport, a yeast (wild-type, BY4741) system with its mutants (ycf1, zrc1, and smf1) defective in transport activity of Cd, Zn and Mn, respectively were tested. Compared to empty vectors (pYES2), cells carrying BnNRAMP1b can rescue the transport functions. As a consequence, excess Cd, Zn and Mn were taken in the cells, which led to metal toxicity, suggesting that BnNRAMP1b is responsible for transport of these metals in B. napus. Using our previously created degradome datasets, we found that BnNRAMP1b could be cleaved by miR167, suggesting that BnNRAMP1b is a target of miR167 in B. napus. The contrasting expression pattern of BnNRAMP1b and miR167 under Cd stress supported the post-transcriptional regulation of BnNRAMP1b by miR167.