Abstract

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition. The negative regulation of miRNAs has been found in response to stress in Arabidopsis, rice and other plants. The sequence of the maize genome has been completed very recently; however, only a few maize miRNAs have been identified and deposited in the miRNA registry database, and the details of their regulation of target genes remain to be elucidated. We identified 21 maize miRNAs using bioinformatics computation and small RNA cloning. They are designated as zma-miR161, zma-miR397, zma-miR446, zma-miR479, zma-miR530, zma-miR776, zma-miR782, zma-miR815a, zma-miR818a, zma-miR820, zma-miR828, zma-miR834, zma-miR1, zma-miR2, zma-miR3, zma-miR4, zma-miR5, zma-miR6, zma-miR7, zma-miR8 and zma-miR9. The characterization of these miRNAs reveals a length of 19–24 nt and the typical secondary structure. Using reverse transcriptional polymerase chain reaction (RT-PCR), 13 of these miRNAs were amplified and validated from total small RNA samples of maize. zma-miR1, zma-miR3, zma-miR6, zma-miR479, zma-miR782, zma-miR815a and zma-miR820 were found to be expressed specifically in drought-tolerant inbred line 87-1 under simulated drought stress, whereas the other six were amplified in line 87-1 and in the sensitive inbred line 200B under drought stress and in a well-watered control. The seven differentially expressed miRNAs are suggested to be associated with the regulation of gene expression under drought stress. Using the plant miRNA target finder software miRU and miRU2, 14 of the 21 miRNAs were identified to target genes encoding 21 proteins, most of which are metabolic enzymes. Seven of them are involved in signal transduction, transcription regulation, and bio- or abio-stress responses. The identification and characterization of 21 miRNAs enrich the maize miRNA database and provide valuable information for functional studies of miRNAs.

Key words:    MicroRNA,  maize,  bioinformatics,  gene cloning,  differential expression,  drought stress.

Abbreviation

miRNAs, MicroRNAs; PCR, polymerase chain reaction; GSS,genomic survey sequence; EST, expressed sequence tags; NCBI, National Center for Biotechnology Information; BLAST, Basic Local Alignment Search Tool; SDS-PAGE, sodium dodecyl sulfate-polyacrylaminde gel electrophoresis; MFEs,minimal free energies; MFEIs, minimal free energy indices.