Schematic representation of the DNA structures of <i>Dpps</i>.

<p>The legend presents the pattern of untranslated regions, introns, and exons. Some introns of <i>Dpp</i> have been partially abridged in this figure for their partial sequence. The length of the genomic DNA of <i>Dpps</i> is loaded from the NCBI database, and their GeneBank accession numbers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149117#pone.0149117.s005" target="_blank">S4 Table</a>.</p

Expression of <i>AccDpp</i> in <i>Transetta</i> (DE3) chemically competent cells.

<p>Recombinant AccDpp was separated by SDS-PAGE, and then stained with Coomassie brilliant blue. Lane 1–4, expression of AccDpp after IPTG induction for 4, 5, 6, and 7 h. Lane 5 and Lane 6, non-induced of recombinant AccDpp and induced overexpression of pET-30a (+) vector for 7 h. The box shows the site of recombinant AccDpp.</p

Characterization of a Decapentapletic Gene (<i>AccDpp</i>) from <i>Apis cerana cerana</i> and Its Possible Involvement in Development and Response to Oxidative Stress

<div><p>To tolerate many acute and chronic oxidative stress-producing agents that exist in the environment, organisms have evolved many classes of signal transduction pathways, including the transforming growth factor β (TGFβ) signal pathway. Decapentapletic gene (<i>Dpp</i>) belongs to the TGFβ superfamily, and studies on <i>Dpp</i> have mainly focused on its role in the regulation of development. No study has investigated the response of <i>Dpp</i> to oxidative pressure in any organism, including <i>Apis cerana cerana</i> (<i>A</i>. <i>cerana cerana</i>). In this study, we identified a <i>Dpp</i> gene from <i>A</i>. <i>cerana cerana</i> named <i>AccDpp</i>. The 5΄ flanking region of <i>AccDpp</i> had many transcription factor binding sites that relevant to development and stress response. <i>AccDpp</i> was expressed at all stages of <i>A</i>. <i>cerana cerana</i>, with its highest expression in 15-day worker bees. The mRNA level of <i>AccDpp</i> was higher in the poison gland and midgut than other tissues. Furthermore, the transcription of <i>AccDpp</i> could be repressed by 4°C and UV, but induced by other treatments, according to our qRT-PCR analysis. It is worth noting that the expression level of <i>AccDpp</i> protein was increased after a certain time when <i>A</i>. <i>cerana cerana</i> was subjected to all simulative oxidative stresses, a finding that was not completely consistent with the result from qRT-PCR. It is interesting that recombinant <i>AccDpp</i> restrained the growth of <i>Escherichia coli</i>, a function that might account for the role of the antimicrobial peptides of <i>AccDpp</i>. In conclusion, these results provide evidence that <i>AccDpp</i> might be implicated in the regulation of development and the response of oxidative pressure. The findings may lay a theoretical foundation for further genetic studies of <i>Dpp</i>.</p></div

Expression of <i>AccDpp</i> under different stress conditions.

<p>The transcript levels of <i>AccDpp</i> were analysed via qRT-PCR. Untreated 15-day worker bees and the <i>β-actin</i> gene were used as controls and an internal control, separately. The data are the mean ± SE of three independent experiments. Significant differences (p<0.001) were represented by different letters on the bar based on Duncanʾs multiple range tests.</p

Characterization of <i>Dpp</i> from various species.

<p>The amino acid sequences of Dpp were all downloaded from the NCBI database (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149117#pone.0149117.s005" target="_blank">S4 Table</a>). A, alignment of the deduced <i>AccDpp</i> protein sequence with other Dpp proteins. B, Conserved domain of Dpp. The conserved domains are marked by different shapes.</p

Partial nucleotide sequences and prediction transcription factor binding sites in the promoter region of <i>AccDpp</i>.

<p>The transcription start site and putative transcription factor binding sites mentioned in this paper are marked with arrows and boxes, respectively. The sequence was deposited in GenBank, and the GenBank accession no. is KT750953.</p

Phylogenetic analysis and the tertiary structure of <i>AccDpp</i>.

<p>A, phylogenetic analysis of AccDpp from different species. The species source of the above analysis is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149117#pone.0149117.s005" target="_blank">S4 Table</a>. B, the tertiary structure of AccDpp. Helices, sheets, and coils are presented in different colours.</p

The cDNA sequence of <i>AccDpp</i> and its amino acid sequence.

<p>The top line shows the nucleotide sequence of <i>AccDpp</i>, and the second line shows the deduced amino acid sequence. The start codon (ATG) and stop codon (TAG) are boxed. The polyadenylation signal (AATAA) sequence is marked by an oval. The underlined region indicates the signal peptide, and the shaded amino acid sequence denotes the predicted antimicrobial peptide. The sequence was deposited in GenBank, and the GenBank accession no. is KT750952.</p

Disc diffusion assays of overexpressed recombinant <i>AccDpp</i>.

<p>The selected reagents are HgCl₂, CdCl₂, paraquat, and cumene hydroperoxide. The numbers on the filter discs from 2–5 represent the concentration of reagents from small to large, and the number 1 indicates the control. The data are the mean ±SE of three independent experiments.</p

Expression profile of <i>AccDpp</i> and Western blot analysis of <i>AccDpp</i> at different developmental stages and different tissues.

<p>A, and B, the mRNA level of <i>AccDpp</i> at different developmental stages: egg (E), one-day to seven-day larvae (L1-L7), pre-pupal phase pupae (Po), pupae (white-eyed (Pw), pink-eyed (Pp), brown-eyed (Pb) and dark-eyed (Pd) pupae)), 1-day worker bees (A1), 15-day worker bees (A15), and 30-day worker bees (A30), and different tissues: leg (Le), wing (Wi), muscle (Ms), midgut (Mi), haemolymph (He), rectum (Re), poison gland (Pg), honey sac (Hs), antennae (An) and epidermis (Ep), respectively. The data are the mean ± SE of three independent experiments. The letters above the columns suggest significant differences (P<0.0001) according to Duncan’s multiple range tests. C, and D, the expression level of AccDpp protein at different tissues and stages of development, separately.</p