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. 2019 May 10;9(5):182.
doi: 10.3390/biom9050182.

Identification of miRNAs and Their Response to Cold Stress in Astragalus Membranaceus

Merhaba Abla  1 , Huigai Sun  2 , Zhuyun Li  3 , Chunxiang Wei  4 , Fei Gao  5 , Yijun Zhou  6 , Jinchao Feng  7
Affiliations

Identification of miRNAs and Their Response to Cold Stress in Astragalus Membranaceus

Merhaba Abla et al. Biomolecules. .

Abstract

Astragalus membranaceus is an important medicinal plant widely cultivated in East Asia. MicroRNAs (miRNAs) are endogenous regulatory molecules that play essential roles in plant growth, development, and the response to environmental stresses. Cold is one of the key environmental factors affecting the yield and quality of A. membranaceus, and miRNAs may mediate the gene regulation network under cold stress in A. membranaceus. To identify miRNAs and reveal their functions in cold stress response in A. membranaceus, small RNA sequencing was conducted followed by bioinformatics analysis, and quantitative real time PCR (qRT-PCR) analysis was performed to profile the expression of miRNAs under cold stress. A total of 168 conserved miRNAs belonging to 34 families and 14 putative non-conserved miRNAs were identified. Many miRNA targets were predicted and these targets were involved in diversified regulatory and metabolic pathways. By using qRT-PCR, 27 miRNAs were found to be responsive to cold stress, including 4 cold stress-induced and 17 cold-repressed conserved miRNAs, and 6 cold-induced non-conserved miRNAs. These cold-responsive miRNAs probably mediate the response to cold stress by regulating development, hormone signaling, defense, redox homeostasis, and secondary metabolism in A. membranaceus. These cold-corresponsive miRNAs may be used as the candidate genes in further molecular breeding for improving cold tolerance of A. membranaceus.

Keywords: Astragalus membranaceus; cold stress; miR390; miRNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Length distribution of small RNAs in the library of A. membranaceus. X-axis, size group of small RNA; Y-axis, corresponding percentage of raw reads.
Figure 2
Figure 2
The distribution of the identified distinct miRNA sequences of each conserved miRNA family in A. membranaceus.
Figure 3
Figure 3
The read counts of each conserved miRNA family in A. membranaceus.
Figure 4
Figure 4
The secondary structure of the miRNA precursors of ame-miRN1 (a), ame-miRN8 and ame-miRN9 (b), and ame-miRN-14 (c). The mature sequences of the miRNAs were shown in uppercase. These graphs were generated by using mfold web server.
Figure 5
Figure 5
Expression of selected miRNAs in A. membranaceus leaves and roots. The expression level of each miRNA was normalized to that of U6. Error bars indicate SD between replicates.
Figure 6
Figure 6
Gene ontology terms and numbers of the predicted miRNA targets.
Figure 7
Figure 7
The expression patterns of conserved miRNAs under cold stress in A. membranaceus leaves. A. membranaceus U6 was used as an internal control. Error bars represent ±SD from three independent experiments.
Figure 8
Figure 8
The expression patterns of six selected non-conserved miRNAs under cold stress in A. membranaceus leaves. A. membranaceus U6 was used as an internal control. Error bars represent ±SD from three independent experiments. * P < 0.05 compared to the control group, ** P < 0.01 compared to the control group.
Figure 9
Figure 9
The expression patterns of selected targets of cold-responsive miRNAs under cold stress in A. membranaceus leaves. A. membranaceus 18S rRNA was used as an internal control. Error bars represent ±SD from three independent experiments. * P < 0.05 compared to the control group, ** P < 0.01 compared to the control group.
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