Functional Identification Of Bnmir158 And Bnrh6 Ectopic Expressing In Arabidopsis Under Cd Stress

microRNAs(miRNAs)are a class of non-coding,20-24 nt,single-stranded and endogenous small RNA,which play important roles in plant development,signal transduction and responses to environmental stresses.miRNAs silence port-transcription or translation of their target genes.In this study,we identified miR158 and its target gene DEAD-Box RNA Helicase 6(BnA04g26450D)in Brassica napus.RNA helicases involve the functions of RNA transcription,translation initiation,RNA assembly and nuclear mRNA export processes such as mRNA splicing,mRNA degradation,rRNA processing and ribosome.They particularly play important roles in regulation of gene expression.Using bioinformatics technology and based on homologues of DEAD-Box RNA Helicase family members in Arabidopsis,we identified 157 DEAD-Box helicase members in Brassica napus.Accoding to the sequence similarity,we selected a gene BnRH6 for further functional identification.Our analysis was summarized as follows:First,BnRH6 is a homologous gene of yeast Dhhl and human In plants,BnRH6 belongs to a DEAD-Box RNA Helicase family member in Arabidopsis.Alignment of protein sequence,conserved motifs,and tertiary structure was analyzedSecond,precursor sequence pre-RNA158 and its target gene BnRH6 were cloned from Brassica napus.Analysis of tissue expression pattern showed that both miR158 and BnRH6 were found to be differentially expressed at various developing stages.In shoots,miR158 was induced by Cd,whearas expression of BnRH6 was suppressed.In roots,expression of miR158 was depressed,while BnRH6 expression was increased.Third,to identify the biological function,we constructed the transgenic Arabidopsis expressing BnRH6.Our analysis showed that BnRH6 expression lines OE1#and OE2#were sensitive to 10?25?50 ?M Cd;in contrast,the Arabidopsis rh6 mutants were tolerance to the Cd treatment as compared to wild-type.The phenotypes were shown in biomass,primery root elongation,chlorophyll content,and oxidative response.We further analyzed GSH,PC and NBT contents in the OE1#and OE2#lines and rh6 muatnts,found that rh6 accumulated more contents of GSH,PC and NBT,whereas both transgenic lines accumulated less.These results indicate that BnRH6 negatively regulated plant response to Cd toxicity.