Expression Profiling and Regulation of Genes Related to Silkworm Posterior Silk Gland Development and Fibroin Synthesis

Abstract

The posterior silk gland (PSG) is the most important suborgan responsible for the synthesis and secretion of silk core fibroin proteins in silkworm. Here, we performed genome-scale expression profiling analysis of silkworm PSG at the fourth molting (M4) and at day 1 (V1), day 3 (V3), day 5 (V5), and wandering stage (W) of the fifth instar by microarray analysis with 22 987 probes. We found that the five genes of silk proteins secreted from PSG including fibroin heavy (H) and light (L) chains, P25, seroin 1, and seroin 2 basically showed obvious up-regulation at V3 which lasted to V5, while slight down-regulation at W. The expression of translation-related genes including ribosomal proteins and translation initiation factors generally remained stable from M4 to V5, whereas it showed clear down-regulation at W. Clustering analysis of the 643 significantly differentially expressed transcripts revealed that 43 of the important genes including seroin 1 and sugar transporter protein had co-expression patterns which were consistent with the rate changes of fibroin synthesis and PSG growth. Pathway analysis disclosed that the genes in different clusters might have co-regulations and direct interactions. These genes were supposed to be involved in the fibroin synthesis and secretion. The differential expression of several hormone-related genes also suggested their functions on the regulation of PSG development and fibroin synthesis. 2D gel-based proteomics and phosphoproteomics profiling revealed that the phosphorylated proteins accounted for no more than one-sixth of the total proteins at each stage, which was much lower than the level in normal eukaryotic cells. Changes in the phosphorylation status and levels of several proteins such as actin-depolymerizing factor 1 and enolase might be deeply involved in fibroin secretion and tissue development. Shotgun proteomic profiling combined with label-free quantification analysis on the PSG at V3, V5, and W revealed that many small heat shock proteins (sHSP) were specially expressed at W, which was substantially consistent with the results from 2-DE analysis, and implied the close correlations of sHSP with the physiological states of PSG at W. A majority of significantly up-regulated proteins at V5 were related to ribosome pathway, which was different from the microarray results, implying that the translation-level regulation of ribosomal proteins might be critical for fibroin synthesis. In contrast, the ubiquitin-proteasome pathway related proteins appeared obviously up-regulated at W, suggesting that the programmed cell death process of PSG cells might be started before cocooning.

Comparative proteomic analysis of the silkworm middle silk gland reveals the importance of ribosome biogenesis in silk protein production

“…Protein extraction from the MSG-A, − M, and -P was performed as described previously [24,25]. The extracted protein samples were quantified using the 2-D Quant Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's instructions.…”

“…The MSG proteins were separated by SDS-PAGE, and the gels were sliced into 12 sections followed by in-gel digestion and mass spectrometry (MS) analysis, according to our previously described methods [24,25]. Briefly, the digested peptide sample was re-suspended and subjected to an Ettan MDLC nanoflow/capillary LC system (GE Healthcare, Pittsburgh, PA) coupled to a linear ion trap Orbitrap mass spectrometer (LTQ-Orbitrap XL TM , Thermo Fisher Scientific, Bremen, Germany).…”

“…The retrieved MS/MS data was searched against the same database which was previously used, containing 1739 entries of silkworm protein sequences from NCBI Refseq and 14,623 entries of the predicted silkworm genome coding sequences [24]. The MS/MS data were automatically submitted to the in-house Mascot server for database search using Mascot Daemon software (version 2.2, Matrix Science, London, U.K.).…”

“…The MS/MS data were automatically submitted to the in-house Mascot server for database search using Mascot Daemon software (version 2.2, Matrix Science, London, U.K.). The parameters for database searching were the same as in our previous study with minor changes [24]. Briefly, the parent and fragment ion mass tolerances were set at 50 ppm and 0.6 Da, respectively.…”

“…Differential expression of proteins from different sections of the silk gland has been analyzed [22,23]. The PSG expression profiles at transcriptional, translational, and post-translational modification levels during the fifth instar have been characterized [24]. However, the proteome of the MSG is not well understood, and proteomic differences among the three subsections of the MSG have not been deeply analyzed.…”