| The colleterial gland of the silkworm, Bombyx mori, is referred to the female accessory system. The gland developed from the imaginal disc located in the ninth abdominal segment. Morphological and cytological observations have shown that the colleterial gland is differentiated into two parts, a tubular secretory region and a reservoir lobe. During certain developing stage, glue-like substance will be secreted by the secretory region of the gland and stored in the reservoir lobe. In silkworm, like some other insects, the glue like proteins are released to the surface o f eggs when egg-laying, then the glue-like substance will be hardened to fix the eggs on the surface of plants or other substrata, such that they aren't easily detached even in a strong wind or a heavy rain. The colleterial gland of silkworm grows gradually until day 8 after pupal ecdysis, then enlarged markedly due to the accumulation of glue-like substances before moth emergence. The Ng (a gene symbol for No-glue mutation located on chromosome linkage 12-21.8) mutant was one of the more than 400 mutations recorded in silkworm gene bank. The female moth of Ng mutant silkworm secreted only little glue-like substance and laid loose eggs naturally. The observation of anatomy showed that the typical shape of the colleterial gland had already formed in 7 or 8 days after pupal ecdysis in certain silkworm variety. However, the most interesting phenomenon is that the relatively small secretory region of colleterial gland can secrete a large amount of glue-like substance only in two or three days in silkworm. Interestingly, the female moth of Ng mutant secreted only little glue-like substance. Whereas some achievements in this field had been acquired, the molecular mechanisms of the secretion in a large quantity of glue-like proteins during a short period and the Ng mutant formation were still not understood well. Proteomics is a large-scale study of the gene expression at the protein level, whichultimately provides direct measurement of protein expression levels and insight into the activity state of all relevant proteins. Key elements of classical proteomics are the separation of proteins in a sample using immobilized pH gradient two-dimensional gel electrophoresis (2-DE) and their subsequent identification by biological mass spectrometry. The purpose of choosing colleterial gland in the present research material w as n ot o nly t hat t he c olleterial g land m orphologically enlarged m arkedly and secreted glue-like substance in one or two days, and that its mechanisms had not been clear. During this development, different functional genes might be activated and some s pecial p roteins i nvolved w ith c olleterial g land d evelopment and s ecretion o f glue-like proteins might be expressed. Moreover, there was a very good experiment material available in our silkworm genebank that was the silkworm strain E981 and its Ng mutant line. Therefore, we investigated the utility of a proteomic approach to improve the understanding of this complex bioprocess.1. In this research, the main methods of the total proteins extraction and preparation of different organs of insect (Bombyx mori) including the colleterial gland, mudgut, silkgland and hemolymph and the conditions of isoelectric focusing and SDS-PAGE were discussed and optimized. The methods and conditions of protein preparation and electrophoresis of different organs were different. The proteins extracted from colleterial gland, midgut and middle silk gland then separated by two-dimensional electrophoresis, the results showed that higher protein extraction rate, resolution (more than 1,000 spots detected by software), better reproducibility and more sample volume were acquired in the present experiment.2. In order to establish the 2D PAGE profiles database during different development stages, all the steps from protein sample preparation to the two-dimensional electrophoresis were operated more than two times, and the conditions were optimized. The image analysis software (Image master) typically detected more than 800 spots on each gel following sliver staining, and the protein spots distribution pattern were nearly same during different stages between normal and Ng mutantcolleterial gland. Furthermore, most of the protein spots arranged from 30 kD to 70kD and pH 4 to 8. Under the help of the software, it was found that most protein's expression volume in different development days were no significant difference, however, interestingly, the volume variation of 11 protein spots that changed more than 1.5 folds in expression level compared with moth emergence day, and 3 spots (spots 1, 2and 3) only expressed in the last one or two days of pupal stages and in the day of moth emergence and no expression in Ng mutant. All these differential proteins were excised for identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Spot No. 1 was unknown protein in the silkworm and its highest identity protein was found in fruit fly. Spots No. 2 and 3 were the same protein, cytosolic actin A3; Spot No. 9 was identified as eukaryotic translation initiation factor 5 A (eIF-5A). Actin is a highly c onserved protein of the contractile system, present in the cytoskeleton of all eucaryotic cells. Actins are involved in a variety of cellular events such as motion, chromosome segregation, transport of macromolecules and endo-and exocytosis. Interestingly, inBombyx m ori silk gland cells, cytoplasmic actin is an abundant component of apical bundles of micro filaments, which participates in exocytosis of silk proteins. The actins only expressed in the two days when the glue-like proteins are secreted markedly. Furthermore, there were no actins expressed in the Ng mutant silkworm that secreted little glue-like substance during the whole development stage. The main content of secretions from both colleterial gland and silk gland are proteins, it indicates that actins are important functional proteins, which might participate or regulate the secretion of colleterial gland, and probably play similar function in silk gland. The eIF-5A is important for the protein translation of eukaryotic cell, and its expression volume was increased gradually during the stage of glue-like protein secretion, therefore, it is important for protein synthesis. Except that, these differential expressed proteins might be related to the Ng mutant form and glue proteins secretion.3. For further study the difference between the normal and Ng mutant colleterial gland at proteomics level, the Fluorescence two-dimensional difference gel electrophoresis(2-DIGE) coupled with mass spectrometry were used to investigate mutant specific changes in the proteome of the secretory region of colleterial gland (normal and Ng mutant) in the moth emergence day. It was used Cy3 labeled proteins isolated from the normal colleterial gland and Cy5 labeled the proteins from the Ng mutant colleterial gland, and separated on the same 2-D gel along with a Cy2 labeled mixture of both 2 normal and mutant samples as an internal standard. Over 1000 protein spot-features were analyzed in the paired normal and mutant comparison. DeCyder software was used for simultaneous comparison of abundance of abundance changes and detected 20 spots which changed more than two-fold in expression level, and the expressed volume variation of some proteins even got almost 10 times. Using peptide mass fingerprint matrix-assisted laser desorption/ionization-time of flight mass spectrometry and public protein database, it was identified 4 proteins including PI09 protem-Bombyx mori. (spot 5), 50S ribosomal protein L5 (spot 9), Replication initiation protein (spot 13), Actin Al - silkworm (spot 17). According to the function of these differential expressed proteins, it indicated that these differential expressed proteins which connected to the protein synthesis and secretion might be have the relationship with glue-like protein secretion and Ng mutant form.4. The secreted protein mixture was separated by immobilized pH gradient two-dimensional gel electrophoresis. The results showed that more than 70 protein or polypeptide were detected in the gel (24 cm length), and the expressed volume of about 30 spots were abundance. According the distribution of these spots in gel, most of them arranged from pH 4-7, and the molecular weight of most protein and polypeptide more than 45 kD. It was identified 33 spots by mass spectrometry to understand the composition of the complex mixture. Peptide mass fingerprint of those protein spots were obtained and compared in the public database, the results suggested that most of the protein were the family of ribosomal protein occupied 30.4% among the identified proteins including the spot 3(60S ribosomal protein L4) ^ 5(60S ribosomal protein L14) .. 11 (40S ribosomal protein S6K 17(ribosomal proteinS27a) , 18 (60S ribosomal protein L7a) -. 27 (60S ribosomal protein L4) > 32 (60S ribosomal protein L6). Furthermore, two spots, 23 (Heat shock 70 kDa protein cognate 4) and 33 (Heat shock-related 70 kDa protein 2) , were identified as heat shock proteins that might be act as molecular chaperones to regulate protein folding and transfer the glue-like proteins out of special cells. Accepted that, some spots were identified as RNA-binding protein, elongation factor and so on, all the proteins composed the complex protein mixture secreted by colleterial gland ofBombyx mori. |
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