Genome-Wide Identification and Characterization of microRNAs in Developing Grains of <i>Zea mays</i> L.

<div><p>The development and maturation of maize kernel involves meticulous and fine gene regulation at transcriptional and post-transcriptional levels, and miRNAs play important roles during this process. Although a number of miRNAs have been identified in maize seed, the ones involved in the early development of grains and in different lines of maize have not been well studied. Here, we profiled four small RNA libraries, each constructed from groups of immature grains of <i>Zea mays</i> inbred line Chang 7–2 collected 4–6, 7–9, 12–14, and 18–23 days after pollination (DAP). A total of 40 known (containing 111 unique miRNAs) and 162 novel (containing 196 unique miRNA candidates) miRNA families were identified. For conserved and novel miRNAs with over 100 total reads, 44% had higher accumulation before the 9^th DAP, especially miR166 family members. 42% of miRNAs had highest accumulation during 12–14 DAP (which is the transition stage from embryogenesis to nutrient storage). Only 14% of miRNAs had higher expression 18–23 DAP. Prediction of potential targets of all miRNAs showed that 165 miRNA families had 377 target genes. For miR164 and miR166, we showed that the transcriptional levels of their target genes were significantly decreased when co-expressed with their cognate miRNA precursors <i>in vivo</i>. Further analysis shows miR159, miR164, miR166, miR171, miR390, miR399, and miR529 families have putative roles in the embryogenesis of maize grain development by participating in transcriptional regulation and morphogenesis, while miR167 and miR528 families participate in metabolism process and stress response during nutrient storage. Our study is the first to present an integrated dynamic expression pattern of miRNAs during maize kernel formation and maturation.</p></div

Northern blot analysis of selected maize miRNAs.

<p><b>Maize</b><i>U6</i> RNA was used as an internal control. DAP: days after pollination.</p

Coexpression of miRNA precursors and their target genes in a transient expression system in <i>N</i>. <i>benthamiana</i> cells.

<p>(A) Relative expression levels of NAM gene coexpressed with miR164 precursor in <i>N</i>. <i>benthamiana</i>. (B) Relative expression levels of unknown gene coexpressed with miR166 precursor in <i>N</i>. <i>benthamiana</i>. Target genes were also coexpressed with an unrelated <i>GFP</i> construct as a control. Tobacco 18s rRNA was used as an internal control for normalization.</p

Summary of Signatures that Match Various RNAs.

<p>Summary of Signatures that Match Various RNAs.</p

Relative expression levels of 14 miRNAs and their target genes at four time points during maize seed development.

<p><i>U6</i> and <i>TUBULIN</i> were used as internal controls for miRNAs and target genes, respectively. DAP: days after pollination. Maize miRNAs are indicated by dark gray bars; target genes are indicated by light gray bars.</p

Expression Pattern of Known miRNAs in Maize Grain Development.

<p>Expression Pattern of Known miRNAs in Maize Grain Development.</p

Two-dimensional gel electrophoretic separation of extracts from 100 fourth instar larvae and 100 pupae of <i>An. gambiae</i>.

<p>The gel was stained with colloidal Coomassie Brilliant Blue and all the spots migrating with apparent molecular weight lower than 24(coverage by aminoacid sequence up to 61.87%), found in several spots (red circles). In larvae we could also detect OBP21 (Entry code in Uniprot Q8I8S3; coverage by aminoacid sequence 9.16%) and SAP3 (coverage by aminoacid sequence up to 18.25%), present in spots where also OBP9 was identified. Molecular weight markers are, from the top: Phosphorylase b, from rabbit muscle (97 kDa), Bovine serum albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa), α-Lactalbumin (14 kDa).</p

Expression of <i>An. gambiae</i> OBP5 in <i>E. coli</i>. SDS-PAGE of bacterial pellets before (Pre) and after (Ind) induction of the culture with Isopropyl-1-thio-β-D-galacto-pyranoside.

<p>Molecular weight markers are, from the top: Bovine serum albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa), α-Lactalbumin (14 kDa). OBP5: purified protein.</p

Western-blot of crude antennal extracts of male and female <i>An. gambiae</i>, using polyclonal antisera against OBPs 9, 4 and 5.

<p>Left panels: SDS-PAGE of crude extracts (Ex) and sample of purified OBPs as indicated by their numbers. Right panels: Western-blot analysis of crude extracts (Ex) performed with the three antisera. A sample of OBPs 9, 4 and 5 (0.5 µg of each protein) utilised for raising the antibodies was also loaded on the same gel. OBP4 and 5 are not detectable in our experimental conditions, while OBP9 is present in both sexes, in agreement with the shotgun experiment results. Molecular weight markers are, from the top: Bovine serum albumin (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa), α-Lactalbumin (14 kDa).</p

Abundance of OBPs, CSPs and other proteins in the antennae of <i>An. gambiae</i> males and females, as reported in Table 1.

<p>The evaluation of relative abundance (in arbitrary units) is based on the values produced by MaxQuant (see text). The values are the averages of three sets of analyses. Error bars represent standard error of the mean. By far the most abundant proteins in male antennae are OBP9, SAP1 and SAP3, in agreement with the results of the 2D-gel (Figure 1).</p