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. 2015 May 22;11(7):781-93.
doi: 10.7150/ijbs.11619. eCollection 2015.

Identification and Characterization of Novel Maize Mirnas Involved in Different Genetic Background

Lei Sheng  1 , Wenbo Chai  1 , Xuefeng Gong  1 , Lingyan Zhou  1 , Ronghao Cai  1 , Xiaoyu Li  1 , Yang Zhao  1 , Haiyang Jiang  1 , Beijiu Cheng  1
Affiliations

Identification and Characterization of Novel Maize Mirnas Involved in Different Genetic Background

Lei Sheng et al. Int J Biol Sci. .

Abstract

MicroRNAs (miRNAs) are a class of small, non-coding regulatory RNAs that regulate gene expression by guiding target mRNA cleavage or translational inhibition in plants and animals. At present there is relatively little information regarding the role of miRNAs in the response to drought stress in maize. In this study, two small RNA libraries were sequenced, and a total of 11,973,711 and 14,326,010 raw sequences were generated from growing leaves of drought-tolerant and drought-sensitive maize seedlings, respectively. Further analysis identified 192 mature miRNAs, which include 124 known maize (zma) miRNAs and 68 potential novel miRNA candidates. Additionally, 167 target genes (259 transcripts) of known and novel miRNAs were predicted to be differentially expressed between two maize inbred lines. Of these, three novel miRNAs were up-regulated and two were down-regulated under drought stress. The expression of these five miRNAs and nine target genes was confirmed using quantitative reverse transcription PCR. The expression of three of the miRNAs and their putative target genes exhibited an inverse correlation, and expression analysis suggested that all five may play important roles in maize leaves. Finally, GO annotations of the target genes indicated a potential role in photosynthesis, may therefore contribute to the drought stress response. This study describes the identification and characterization of novel miRNAs that are the differentially expressed in drought-tolerant and drought-sensitive inbred maize lines. This provides the foundation for further investigation into the mechanism of miRNA function in response to drought stress in maize.

Keywords: Drought stress; High-throughput sequencing; Maize; MicroRNA; Target genes; qRT-PCR.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Length distribution of small RNAs from sequencing of the two inbred maize libraries. (A) Size distribution of total sequences. (B) Size distribution of unique sequences.
Figure 2
Figure 2
Abundance of conserved miRNA families in the two inbred maize libraries.
Figure 3
Figure 3
Relative nucleotide bias at each position of the known maize miRNAs.
Figure 4
Figure 4
Validation of 18 novel miRNAs which low abundance and more than 24 nt in length using stem-loop real-time PCR. (A) different expression levels of miRNAs compared with the sequencing data. (B) similar expression levels of miRNAs compared with the sequencing data. Ordinates indicate relative expression levels.
Figure 5
Figure 5
Secondary structure of five novel miRNA precursors. Mature miRNA sequences are shown in yellow.
Figure 6
Figure 6
Quantitative real-time RT-PCR analysis of five novel miRNAs and their target genes. Expression levels of miRNAs were normalized against 18S rRNA. Fold changes in expression level were estimated using the 2-ΔΔCT method. Data are reported as mean ± SE for three independent experiments. (A) The inverse relationship between three novel miRNAs (PC-3p-190, PC-3p-552502 and PC-5p-139812) and their putative target genes. (B) The uniform relationship between two novel miRNAs (PC-3p-104764 and PC-3p-129630) and their putative target genes.
Figure 6
Figure 6
Quantitative real-time RT-PCR analysis of five novel miRNAs and their target genes. Expression levels of miRNAs were normalized against 18S rRNA. Fold changes in expression level were estimated using the 2-ΔΔCT method. Data are reported as mean ± SE for three independent experiments. (A) The inverse relationship between three novel miRNAs (PC-3p-190, PC-3p-552502 and PC-5p-139812) and their putative target genes. (B) The uniform relationship between two novel miRNAs (PC-3p-104764 and PC-3p-129630) and their putative target genes.
Figure 7
Figure 7
Expression profiles of nine target genes. A heat map was generated by hierarchical clustering using a dedicated heat map package . Expression data were normalized and hierarchically clustered with average linkage. The color scale in the top right corner represents the relative gene expression level, where red, yellow and blue indicate high, medium and low levels of gene expression, respectively.
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