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. 2020 Apr 14;21(8):2708.
doi: 10.3390/ijms21082708.

Identification and Characterization of microRNAs in the Developing Seed of Linseed Flax (Linum usitatissimum L.)

Tianbao Zhang  1 , Zhen Li  1 , Xiaxia Song  1 , Lida Han  1 , Limin Wang  2 , Jianping Zhang  2 , Yan Long  1 , Xinwu Pei  1
Affiliations

Identification and Characterization of microRNAs in the Developing Seed of Linseed Flax (Linum usitatissimum L.)

Tianbao Zhang et al. Int J Mol Sci. .

Abstract

Seed development plays an important role during the life cycle of plants. Linseed flax is an oil crop and the seed is a key organ for fatty acids synthesis and storage. So it is important to understand the molecular mechanism of fatty acid biosynthesis during seed development. In this study, four small RNA libraries from early seeds at 5, 10, 20 and 30 days after flowering (DAF) were constructed and used for high-throughput sequencing to identify microRNAs (miRNAs). A total of 235 miRNAs including 114 known conserved miRNAs and 121 novel miRNAs were identified. The expression patterns of these miRNAs in the four libraries were investigated by bioinformatics and quantitative real-time polymerase chain reaction (qPCR) analysis. It was found that several miRNAs, including Lus-miRNA156a was significantly correlated with seed development process. In order to confirm the actual biological function of Lus-miRNA156a, over-expression vector was constructed and transformed to Arabidopsis. The phenotypes of homozygous transgenic lines showed decreasing of oil content and most of the fatty acid content in seeds as well as late flowering time. The results provided a clue that miRNA156a participating the fatty acid biosynthesis pathway and the detailed molecular mechanism of how it regulates the pathway needs to be further investigated.

Keywords: fatty acid synthesis; linseed flax; miRNA156; microRNA; seed development.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The length of miRNAs in the four libraries.
Figure 2
Figure 2
Comparison of the expression of miRNAs in the four libraries. (A). The numbers of miRNAs expressing in each of the library. (B). Differentially expressed miRNAs during seed development. M5 library was as a control.
Figure 3
Figure 3
Gene ontology classifications of miRNA targets and differentially expressed targets in seed development. (A) M5 vs. M10, (B) M5 vs. M20, (C) M5 vs. M30.
Figure 4
Figure 4
Expression patterns of miR156a, miR172e, miR159b, miR397a, Lus-miR-10 and Lus-miR-24 in seed developmental stages (M5, M10, M20 and M30) of qPCR and Next-generation sequencing (NGS) data. Error bars indicated standard deviation of three replicates. (A)The results of qPCR, (B) The results from NGS data.
Figure 5
Figure 5
Mapping target mRNA cleavage sites by 5’-RLM-RACE. The arrows indicate the cleavage sites and the numbers show the frequency of clones sequenced.
Figure 6
Figure 6
Number of rosette leaves and flowering time of the transgenic lines and wild type. (A) Number of rosette leaves, (B) Flowering time.
Figure 7
Figure 7
Expression levels of SPL genes and seed oil synthesis genes in the over-expression Arabidopisis transgenic lines and WT. Line 9: Lus-MIR156a-OX9, Line 5: Lus-MIR156a-OX5, Line 3: Lus-MIR156a-OX3. * p < 0.05 and ** p < 0.01 indicated significant differences with WT.
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