Shotgun Proteomic Analysis on the Diapause and Non-Diapause Eggs of Domesticated Silkworm <i>Bombyx mori</i>

<div><p>To clarify the molecular mechanisms of silkworm diapause, it is necessary to investigate the molecular basis at protein level. Here, the spectra of peptides digested from silkworm diapause and non-diapause eggs were obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) and were analyzed by bioinformatics methods. A total of 501 and 562 proteins were identified from the diapause and non-diapause eggs respectively, of which 309 proteins were shared commonly. Among these common-expressed proteins, three main storage proteins (vitellogenin precursor, egg-specific protein and low molecular lipoprotein 30 K precursor), nine heat shock proteins (HSP19.9, 20.1, 20.4, 20.8, 21.4, 23.7, 70, 90-kDa heat shock protein and heat shock cognate protein), 37 metabolic enzymes, 22 ribosomal proteins were identified. There were 192 and 253 unique proteins identified in the diapause and non-diapause eggs respectively, of which 24 and 48 had functional annotations, these unique proteins indicated that the metabolism, translation of the mRNA and synthesis of proteins were potentially more highly represented in the non-dipause eggs than that in the diapause eggs. The relative mRNA levels of four identified proteins in the two kinds of eggs were also compared using quantitative reverse transcription PCR (qRT-PCR) and showed some inconsistencies with protein expression. GO signatures of 486 out of the 502 and 545 out of the 562 proteins identified in the diapause and non-diapause eggs respectively were available. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed the Metabolism, Translation and Transcription pathway were potentially more active in the non-dipause eggs at this stage.</p> </div

KEGG pathways of non-diapause unique proteins.

<p>KEGG pathways of non-diapause unique proteins.</p

Table 2 the unique expressed proteins of ND with functional annotation.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060386#pone-0060386-t002" target="_blank">Table 2</a> the unique expressed proteins of ND with functional annotation.</p

Theoretical pI and Mw distribution of the identified proteins.

<p>(a) Distribution of pI. (b) Distribution of Mw. The theoretical isoelectric point (pI) and molecular weight (Mw) of the proteins were calculated using the Compute pI/Mw tool (<a href="http://cn.expasy.org/tools/pi_tool.html" target="_blank">http://cn.expasy.org/tools/pi_tool.html</a>) according to the predicted amino acid sequences.</p

Venn diagram shows the numbers of identified proteins from diapause and non-diapause eggs of the silkworms.

<p>Each number with no overlap of circles shows the number of proteins uniquely observed in that sample, while overlapping circle shows the numbers of identified proteins common to two of the analyzes.</p

Table 1 the unique expressed proteins of D with functional annotation.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060386#pone-0060386-t001" target="_blank">Table 1</a> the unique expressed proteins of D with functional annotation.</p

Table 3 KEGG pathways of diapause unique proteins.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060386#pone-0060386-t003" target="_blank">Table 3</a> KEGG pathways of diapause unique proteins.</p

SDS-PAGE patterns of the diapause (D) and non-diapause (ND) eggs protein samples respectively.

<p>The samples were separated by 12.5% resolving gel in triplicate. The three batches for both ND and D were pooled into two sets of eight prior to in-gel digest. Then eight slices were combined to four groups for each sample (comprising slices 1 and 2, slices 3 and 4, slices 5 and 6, slices 7 and 8 for both ND and D) prior to the LC separation and MS detection.</p

Differentially expressed microRNAs in diapausing versus HCl-treated <i>Bombyx</i> embryos

<div><p>Differentially expressed microRNAs were detected to explore the molecular mechanisms of diapause termination. The total small RNA of diapause-destined silkworm eggs and HCl-treated eggs was extracted and then sequenced using HiSeq high-throughput method. 44 novel miRNAs were discovered. Compared to those in the diapause-destined eggs, 61 miRNAs showed significant changes in the acid-treated eggs, with 23 being up-regulated and 38 being down-regulated. The potential target genes of differentially expressed miRNAs were predicted by miRanda. Gene Ontology and KEGG pathway enrichment analysis of these potential target genes revealed that they were mainly located within cells and organelles, involved in cellular and metabolic processes, and participated in protein production, processing and transportation. Two differentially expressed genes, <i>Bombyx mori SDH</i> and <i>Bmo-miR-2761-3p</i>, were further analyzed with qRT-PCR. <i>BmSDH</i> was significantly up-regulated in the HCl-treated eggs, while <i>Bmo-miR-2761-3p</i> was down-regulated. These results suggested that these two genes were well coordinated in silkworm eggs. Dual luciferase reporter assay demonstrated that <i>Bmo-miR-2761-3p</i> inhibited the expression of <i>BmSDH</i>.</p></div

Expressions pattern of <i>Bmo-miR-2761-3p</i> and <i>BmSDH</i> from 3 to 7 day old eggs of silkworm, <i>B</i>. <i>mori</i>.

<p>Each time point was replicated three times using independently collected samples. Error bar = 1 SD. (A) Relative <i>Bmo-miR-2761-3p</i> expression level; (B) Relative <i>BmSDH</i> expression level.</p