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

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

doi:10.3390/ijms21082708

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.

Highlights

MiRNAs are non-coding RNAs about 20–25 nucleotide in length and they are encoded by endogenous genes, from which a primary non-protein coding message is transcribed [1]
Huo et al found that the suppression of DELAY OF GERMINATION1 (DOG1) gene could enable seed germination at high temperature in associated with reduced miR156 and increased miR172 levels [10]
BBna-miR156b, bna-miR156g, bna-miR159, bna-miR395b, bna-miR6029 and 19 novel miRNAs were found to be involved in fatty acid biosynthesis [12]

Summary

Introduction

MiRNAs are non-coding RNAs about 20–25 nucleotide (nt) in length and they are encoded by endogenous genes, from which a primary non-protein coding message is transcribed (pri-miRNA) [1]. MiRNAs are found involving in the regulation of seed development process, such as miR156, miR397, miR396 and miR408. MiR396 regulates seed size and yield via its’ target gene OsGRF4 in rice [9]. Several miRNAs have been discovered via high-throughput sequencing during the seed development in different oil crops, such as soybean, rapeseed. BBna-miR156b, bna-miR156g, bna-miR159, bna-miR395b, bna-miR6029 and 19 novel miRNAs were found to be involved in fatty acid biosynthesis [12]. MiR156 could play important roles in the modulation of grape berry development and ripening [14,15]. Previous studies have confirmed that miR156 regulating developing processed through the SPL gene family [16]. There were too many researches about miR156 but researches involving in fatty acids metabolism of miR156 are limited

Methods

Results

Conclusion