Bioinformatic Identification Of Micrornas In Brassica Family And Cloning Of Micrornas From Brassica Napus

MicroRNAs(miRNAs) are a newly discovered class of non-protein-coding small RNAs.Growing evidence has demonstrated that miRNAs play multiple roles in a variety of biological processes,including development,cell proliferation and stressful responses.The computational approach has greatly improved the efficiency of finding novel miRNAs. Thus far,a number of miRNAs with specific targets in Brassica napus have been identified by computational methods.However,in the other species of the Brassica family,such as Brassica rapa,Brassica oleracea,B.juncea,B.nigra and B.carinata,no miRNAs have been described.In this study,we blasted the expressed sequence tags(EST) and genomic survey sequence(GSS) databases of B.rapa,B.oleracea,B.juncea,B.nigra and B.carinata to search for potential miRNAs,using previously known miRNA sequences from Arabidopsis, rice and other plant species.Following a range of stringent filtering criteria,67 potential miRNAs in B.rapa and 95 in B.oleracea were detected.Using these potential miRNA sequences,we found 62 potential targets in B.rapa and 48 in B.oleracea.According to the mRNA target information,provided by NCBI(http://www.ncbi.nlm.nih.gov/),most of the target mRNAs appeared to be involved in plant growth,development and stress responses. To identify novel abiotic stress-regulated miRNAs in Brassica family,we constructed a library of small RNAs from B.napus,which were treated with phosphate and sulfate deficiency,as well as cadmium.Sequencing 472 clones of the library and subsequent analysis revealed 17 previously reported miRNAs,representing 9 families including miR156,miR159,miR160,miR164,miR167,miR168,miR169,miR393 and miR403. Additionally,3 novel miRNAs were detected.On the base of sequence complementarity, the targets of new miRNAs were predicted.