Identification of new miRNAs in rapeseed (Brassica napus L.) and their role in suppressing target genes

MicroRNAs(miRNAs) are a group of small non-coding RNAs of approximately 18 - 24 nucleotides that play a negative role in post-transcriptional changes in eukaryotes. The miRNAs are then loaded onto the argonate family proteins (AGO) to form a protein complex (RISC). The primary activity of the RISC complex is to direct the mature miRNA to the target RNA and to stop protein production (Megah et al., 2018). miRNAs are involved in response to abiotic stresses in plants such as drought; several miRNA families have been reported in response to drought stress in rice, tomato, Arabidopsis, Medicago truncatula, peach, barley and wheat (Akdogan et al., 2015). The purpose of this study was to identify new microRNAs and their role in suppressing and preventing expression of some of their target genes in rapeseed.

A total of 38589 known mature miRAN sequences were downloaded from the miRBase database. The miRNA sequences were used as known sequences to find conserved miRNAs based on homology search for miRNAs with rapeseed GSS sequences.103369 GSS for rapeseed was downloaded from NCBI database. Mature miRNA sequences were uploaded to the BLASTn algorithm to search for homology with rapeseed GSSs in Linux. The miRNA sequences as known sequences and the GSS sequences as sequences were compared with each other for homology search. GSSs with mature miRNA sequences up to four mismatches were selected as candidates (Zhang, 2005). GSS sequences were used instead of EST sequences because miRNAs can generate GSS sequences in addition to EST sequences. Consequently, GSSs were sequenced between the BLASTx miRNA sequences and the GSS coding sequences were deleted and only non-coding GSS sequences remained (Karimi et al., 2017). Mfold software was used to predict the secondary structure of candidate miRNAs (Vivek, 2018). ath-miR5021 and ath-miR8175 from Arabidopsis, bol-miR9410 and bol-miR9411 from wild cabbage and cas-miR11592 from Camelina sativa were selected from the psRNATarget website (Dai and Zhao, 2011.

For miR5021: The NST1 target gene encodes a protein called Stress response protein NST1, which plays a role in regulating secondary wall thickness in plants and preventing its destruction against a variety of stresses (Mitsuda et al., 2005). For miR9410: The HST gene is one of the target genes that encodes an enzyme called Shikimate O-hydroxycinnamoyltransferase in plants, which participates in the phenylpropanoid biosynthesis and heat stress control in plants, propanoids as secondary metabolites during developmental stages. The plant is synthesized in response to stress conditions (Lukasik et al., 2013).

Types of microRNAs and their role in suppressing target genes during live and abiotic stresses in barley, wheat, soybean, cucumber, alfalfa, olive, rice have been reported (Ozhuner et al., 2013). In this study, we tried to identify new microRNAs and their role in suppressing target genes for the first time in rapeseed. The results of this study showed that among the newly identified microRNAs, miR5021 and miR9410 families play an important role in suppressing NST1 and HST target genes, respectively, especially during stress in canola.. Therefore, identifying the molecular mechanism of these microRNAs and their target genes can help us in selecting drought and heat resistant varieties for rapeseed. A study of microRNAs for boron stress tolerance in leaves and roots of barley showed that of the four new microRNAs identified, miR408 was more involved in regulating cell signaling in leaves than the other three microRNAs (Ozhuner et al., 2013). Also, no response has been reported in hybrid and maize inbred lines for miR172 under drought and salt stress conditions (Kong et al., 2010). Therefore, understanding the cellular regulation mechanism for new microRNAs, including how to regulate the activity pathway of antioxidant enzymes such as superoxide dismutase in organs such as leaves, roots and shoots of canola, requires further investigation.

MicroRNAs can be used as a new molecular tool alongside existing classical breeding methods to improve the genetic status of plants to promote tolerance to a variety of biotic and abiotic stresses in plant breeding.