Analysis of miRNAs and their target genes in five Melilotus albus NILs with different coumarin content

Abstract

MicroRNAs (miRNAs) exhibit diverse and important roles in regulation of various biological processes at the post-transcriptional level in plants. In this study, Melilotus albus miRNA and their target genes were elucidated from five M. albus near-isogenic lines which differ in coumarin content to construct small RNA libraries through high-throughput sequencing. A total of 417 known miRNAs and 76 novel miRNAs were identified in M. albus. In addition, 4155 different target genes for 114 known miRNA families and 14 target genes for 2 novel miRNAs were identified in M. albus. Moreover, mtr-miR5248 and mtr-miR7701-5p target c35498_g3 and gma-miR396a-3p target c37211_g1 involved in coumarin biosynthesis were identified by using the differential expression of the miRNAs and their target genes correlation analysis. The abundance of miRNAs and potential target genes were validated by qRT-PCR analysis. We also found that there were both positive and negative expression changing patterns between miRNAs and their related target genes. Our first and preliminary study of miRNAs will contribute to our understanding of the functions and molecular regulatory mechanisms of miRNAs and their target genes, and provide information on regulating the complex coumarin pathway in M. albus for future research.

Figure 1

The expression of differential miRNAs in M. albus. (A) The number of up- and down-regulated genes in comparisons of N47 vs N46, N49 vs N48, N48 vs N46 and N49 vs N47. (B) The Venn diagrams of differential expression miRNAs from N47 vs N46, N49 vs N48, N48 vs N46 and N49 vs N47.

Figure 2

Expression profiles of miRNAs in the five different genotypes of M. albus. Validation of the expression of 28 miRNAs using qRT-PCR. The blue bar graph indicates the small RNA sequencing results, and red line graph represents the qRT-PCR results. Data are mean ± SE from three biological replicates.

Figure 3

Gene ontology (GO) classification of miRNAs target genes. The results are summarized under three main GO categories: BP-biological process, CC-cellular component and MF-molecular function.

Figure 4

Top of 20 pathways assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Figure 5

A combined view of correlation expressions between miRNA and its target gene compared in N48 vs N46 and N49 vs N47. The left side of heat map show miRNA expression level, and the right side show corresponding target gene expression levels of both N48 vs N46 and N49 vs N47. Up and down regulation in the expression were based on normalize data (color bar at the top) generated by Cluster 3.0 software.

Figure 6

Validation of the expression of miRNAs and their target genes using qRT-PCR in five genotypes of M. albus. The blue lines indicate the miRNAs relative expression, and red represents the target genes relative expression. The relative expression was calculated using 2^−∆∆CT method.

Figure 7

The expression of three miRNAs and their target HCT genes in five genotypes of M. albus. The red color represents high expression levels, and the green color represents low level expression.