Functional Identificaiotn Of Brassica Napus MiR167 And Its Target Under Cadmium Stress

microRNAs(miRNAs)are a class of non-coding,20-24 nt,single-stranded endogenous small RNAs,which play important roles in plant development,signal transduction and responses environmental stresses by post-transcriptional regulation of their target genes.NRAMP proteins is a class of metal transporters positioned on Biomembrane,that play an important role in plant uptake and transport of metal ions such as Fe,Mn and Cd.In this study,we took advantage of the early degradome sequencing datasets of Brassica napus exposed to Cd to identify miR167 and its target genes BnNRAMPl in regulating plant response to Cd.Firstly,we investigated the responses of miR167 and BnNRAMP1 to Cd stress.Our analysis showed that miR167 precursors(Pri-miR167a,b,c)were downregulated under Cd exposure;mature miR167 expression in roots was downregulated,while in shoots it was upregulated.Its target gene BnNRAMPl was always induced under Cd stress.Analysis of tissue expression pattern showed that both miR167 and BnNRAMPl were found to be differentially expressed at various developing stages.BnNRAMPl mainly expressed in roots and leaves.Secondly,we further bioinformatically analyzed correlation of NRAMP familys between Brassica napus and Arabidopsis.The sequence similarity BnNRAMP1 and Arabidopsis AtNRAMPl was up to 91%.We than constructed the vectors with BnNRAMPl and m8BnNRAMPl(resistant to miR167 cleavage)and transformed them into Arabidopsis Avild-type(Col).A mutant defective in AtNRAMP1 gene(nrampl-1)was used to identify plant response to Cd stress.Finally,we used yeast to identify uptake of Mn,Zn and Cd in the cell expressing the BnNRAMPl.It is shown these yeast can transport and showed sensitivity to the heavy metals.