Molecular cloning and characterization of miR399 and its target UBC24 in Dn1 winter wheat
The precursor of miR399 (pre-tae-miR399 ) from the tillering nodes of Dn1 winter wheat was composed of 126 nt, which contained a 19 nt mature sequence. tae-miR399 was located on A chromosome of wheat (Fig. S1A). There was no mRNA between upstream and downstream 1kb of miR399, which belonged to intergenic miRNA (Fig. S1B). The mature and precursor miR399 sequences of monocots and eudicots were obtained from the miRbase database. The conservation of miR399 was analysed by the WebLogo software (Fig. 1A). The miR399 sequences were found to be highly conserved and have two motifs (5p and 3p). The difference between miR399-3p and miR399-5p was at base 17 (Fig. 1B). According to the prediction by RNAfold, the secondary structure of precursor formed a complex but stable stem-loop (Fig. 1C). The line in Fig. 1C showed the mature sequence of tae-miR399 (tae-miR399: 5’-UGCCAAAGGAGAAUUGCCC-3’) (Fig. 1C). A phylogenetic tree was constructed from miRNA precursors using MEGA6. The phylogenetic tree of miR399 showed that the precursor of miR399 was present in both monocots and eudicots, meaning that miR399 was evolutionarily conserved in the common plants listed above (Fig. S1C). To explore the regulation role of tae-miR399, we extracted 2.0 kb of genomic sequence upstream of the transcription start site of thepre-tae-miR399 and searched for cis-acting elements in the PlantCARE database. We found that the transcription start site of the tae-miR399 precursor contained basic promoter elements, transcription factor such as MYC binding site, MYB binding site and a variety of cis-acting and response elements. It showed that both the expression of tae-miR399 might be induced or inhibited by environmental response factors such as light, low temperature stress and hormones (Fig. 1D).
The target of tae-miR399 in Dn1 was predicted by the psRNATarget software to be the highly reliable TaUBC24(TRIAE_CS42_1DL_TGACv1_062114_AA0208970.2). The 2532 bp CDS sequence of TaUBC24 was cloned from the tillering nodes of Dn1 (Fig. S2A). The ORF of the TaUBC24 gene encoded a 843aa protein with a predicted molecular weight of 93.774 kDa and a predicted isoelectric point of 4.78. To investigate the orthologous relationship of UBC24 in different species, we obtained UBC24 protein sequences from 16 species, respectively. It could be seen from the phylogenetic tree and multiple sequence alignments that UBC24 from different species was highly conserved, and TaUBC24 was most closely related to TuUBC24, which was considered the ancestral progenitor of the wheat A genomes(Ling et al., 2018) (Fig. S2B C). The subcellular localization analysis showed that TaUBC24 was located in the cytoplasm and nuclear (Fig. S2D). Moreover, we screened the upstream promoter ofTaUBC24 for cis-acting elements with PlantCARE and found that it contained a variety of cis-acting and response elements (Fig. 1D) that responded to light, low temperature stress and hormones. These results are similar to those for the corresponding miRNA precursors. It was indicated that both tae-miR399 and TaUBC24 could respond to the same environmental factors.