The MicroRNA Libraries Analysis And Verification Of Maize Seedlings Under Co-stress

Drought, salt and alkali are three of the major abiotic stresses adversely affecting plant growth, development and productivity. Maize is one of the world’s most important crop. The current research of drought, salt and alkali stresses in maize have been previously focused on the individual stress while co-stress has rarely been studied. MicroRNAs (miRNAs) are classes of non-coding small RNAs that widely exist in plants and play an important role in abiotic stress responses. However, there is no research on co-stress of drought, salt and alkali in maize. In this study, we identified 58 co-stress significant response miRNAs of by constructing two libraries. In order to understand the function of these miRNAs, qRT-PCR was used to analyze and validate.In total,112 conserved miRNAs belonging to 31 miRNA families and 10 novel miRNAs were identified by analyzing the data of miRNA libraries.58 miRNAs were identified as differentially expressed. Of these miRNAs,17 miRNAs were up-regulated,41 miRNAs were down-regulated. We obtained 2224 predicted target genes for all conserved and novel miRNAs.In this study, we provided evidence that the expression of miRNAs were more stable than other reference genes by using geNorm, NormFinder and BestKeeper, in order to ensure the accuracy of the qRT-PCR. Finally, zma-miR172 and zma-miR171a were used as internal reference genes in next experiment.11 co-stress responsive miRNAs were randomly selected for qRT-PCR. The expression of 10 miRNAs in the co-stress treatments had a similar regulation tendency between deep sequencing and qRT-PCR except for novel 8. All 11 miRNAs were significantly responsive to co-stress of drought, salt and alkali. qRT-PCR was used to detect the dynamic expression profile of 10 target genes for 9 conserved miRNAs in different tissues (shoot and root) and treatments (co-stress of mild drought, salt and alkali, co-stress of moderate drought, salt and alkali and co-stress of severe drought, salt and alkali). From the results, in different tissues, most of miRNAs had higher response in roots than in shoots; in different treatments, main miRNAs had a increase tendency of expression with the rising of degree of treatments; target genes had highly response in moderate and severe treatment and the expression of main target genes had a down-regulation tendency with gradually strengthened and this indicated an approximately negative correlation between the expression of miRNAs and their corresponding targets.The cis-acting elements of miRNAs and their target genes were analysed using PlantCare. The results showed that a large number of stress responsive and hormone responsive elements were present in the promoter region.