Transcript Analysis Of Cd-Regulated MiRNAs From Brassica Napus And Mir395D Vector Construction And Transformation

MicroRNAs (miRNAs) are a new class of endogenous non-coding small RNAs in eukaryotic organisms. miRNAs play multiple roles in biological processes, including development, cell proliferation and stressful responses. Compared with animal miRNAs, the discovery of plant miRNAs is much later. Moreover, plant miRNAs studies have been confined mainly to Arabidopsis and rice, two plant model species with fully sequenced genomes. Recently, several miRNAs have been identified to regulate plant response to different stresses including drought, salinity and nutrient deficiency. Our recent studies identified a set of conserved and non-conserved miRNAs from rice, Medicago truncatula and Brassica napus in response to heavy metals. These results suggest that miRNAs that play an important role in regulation of plant responses to heavy metal stress in addition to other specific abiotic stresses. We previously constructed a library of small RNAs from B. napus exposed to cadmium. Sequencing resulted in identification of 9 known miRNAs including miR156a, miR159, miR167a, miR167b, miR167c, miR168, miR169m, miR393 and miR395.Additionally,6 novel miRNAs were detected. On the base of sequence complementarity, the targets of new miRNAs were predicted. To get insights into the regulatory role of these miRNAs, we performed experiments focusing on trasncription analysis of the miRNAs under Cd stress. Expressions of these miRNAs were shown to be differentially regulated by Cd stress. To relate Cd-induced toxicity to sulfate deficiency, we cloned a novel gene (BnSultr2;1)coding a B. napus low-affinity sulfate transporter. The full-length cDNA, with an open reading frame (ORF) of 2033 bp, encodes a 667-amino acid protein with a predicted molecular weight of 74 kD and isoelectric point of 9.1. Interestingly, BnSultr2;1 was found to be the target of miR395.Finally, the overexpression vector:pCAMBIA1304-pre-miR395d was constructed to explore the function of miR395 in sulphate assimilation and allocation.