Studies On Silk Gland, Hemolymph, Fatbody Of Silkworm (Bombyx Mori) Using Two-dimensional Electrophoresis And Mass Spectrometry

The completion of the genome project, as a milestone, has provided a broader platform for life science. However, bio-diversity and complexity suggests that staying on information of genome is still far away from explanation of the mechanism of life. As the final product of most genes, proteins are the functional units in the hierarchical regulation cascade of genes. Intensive study on proteins is the requirement of further understanding of the mechanisms of lives, accordingly. With the completion of the Silkworm Genome Project, we step in the era of functional genomic research. The proteomics research of silkworm has gradually become one important post-genome research focus. In establishing the proteomic research platform of silkwormin this study, we investigate protein components of the silk gland, blood, fat body and other tissues y by proteomic tools, as will help people understand silkworm on protein level.1 The establishment of platform for high-quality silkworm proteomics research.The experimental conditions of two-dimensional gel electrophoresis in this study was optimized by different experimental conditions, including different sample processing, different time for isoelectric focusing, different types of equipment, different methods for gel staining, et al. The optimization are utilized to analyze silkworm organizations. The results show that high-speed centrifugal method is more appropriate than TCA precipitation method for silkworm organs. The better map of two-dimensional gel electrophoresis can be obtained and more protein spots can be separated from high-speed centrifugal method. Compared with previous studies, two-dimensional electrophoresis from this work have higher quality and the 2D map have better resolution and repeatability. In the experiment of in-gel digestion, trypsin digestion, size of excised gel, the parameters for matrix-assisted laser desorption time-of-flight mass spectrometry are analyzed and the optimum parameters are choosed for the experiments. In the experiment of analysis of silkworm peptide mass fingerprint (PMF), GPMAW software are utilized to construct the database of peptide mass fingerprint. Compare with the method by MASCOT on line, identification of proteins in local database by GPMAW could not only obtain the right result for reported protein, but also find some new proteins which have never been reported before, but have been predicted from the genome sequence of silkworm.2 Proteome analysis of silk gland of silkwormWith the establishment of platform of silkworm proteome, silk glands are firstly studied by proteomics tools. Using pH 3-10 strip and 12.5% SDS-PAGE, almost 700 spots can be obtained from two-dimensional electrophoresis map of silk gland, which are distributed in the area from 15kD to 100kD with pH 4-7. Then, total proteins from the middle silk gland (MSG) and posterior silk gland (PSG) are separated by 2-DE using pH 3-10, 18 cm Ready Strip IPG strips. After silver stained, 598±28 spots are detected in the MSG, while 612±12 spots are identified in the PSG The spot distribution patterns are similar between the MSG and PSG maps, but marked differences in high molecular weigh area are found in the acid region. The differentiated spots are excised, then are analyzed by MALDI-TOF MS. Some proteins related to chaperones, DNA transcription and protease inhibitor are identified. To affirm the result of identification, mass 1124.9 from spot 1 and mass 1451.3 from spot 3 are choosed to produce the fragment-ion segments with post source decay (PSD) method. When the result of fragment-ion segments are analyzed by software of Data explore, the precursor masses could be resolved completely, which support the result of identification of PMF. Through the analysis of their structure and expression patterns, the two protease inhibitors are presumed to play a role in protection silk proteins from degradation. In the map of posterior silk gland of silkworm, 98 spots are selected to be analyzed by MALDI-TOF MS, the results show that 88 spots have been identified successfully, which are involved in diverse functions, including chaperones, structural proteins, and metabolic enzymes in biochemical pathways. We compare our maps to previous results by Zhang et al. The result shows that light chain and P25, the important component of silk fibroin protein, differed greatly in different experiments. Through analysis of method and material of experiments, we speculate that different strains of silkworm might have different forms of silk protein in silk glands.3 Proteome analysis of hemolymph of silkwormIn different stages of development and metamorphosis, protein components in silkworm blood diversify greatly. We investigate blood protein from 1^st day of the fifth instar to the day of eclosion by SDS-PAGE. Then, two-dimensional electrophoresis tool are utilized to studying hemolymph of 3^rd day of the fifth instar of larvae, 6^th day of the fifth instar of larvae, 3^rd day of the pupa, 6^th day of the pupa and the day of eclosion. The result shows that some proteins are expressed up-regulatedly, while some proteins are expressed down-regulatedly during the development of silkworm. When the differentiated spots are identified by MAIDI-TOF MS, they contain not only SP storage protein, 30K protein, yolk protein, but also a number of novel proteins which have not been reported, such as Aldose reductase homolog, Apolipoprotein D homolog, and so on. They may be involved in important physiological activities during developmental stages of silkworm, such as food digestion, metabolism, and flights. In the experiment of identification of 30 K protein from silkworm blood, we analyze the spots around the 27-31kD area by two-dimensional electrophoresis and mass spectrometry, and identify five members of 30K family , of which the almost identical high expression levels, isoelectric points and molecular weights result in the clustering presentation on the gel. When examing blood protein of 3^rd day of the fifth instar, 47 spots are identified successfully, which contain a large number of enzymes related to metabolism, ion transport proteins, protease inhibitors and even to immune-related proteins. When silkworm is attacked by different microbes, the amount of many hemolymph proteins change significantly, , which contained 30K. proteins of silkworm, transferrin, Apolipophorin III proteins, and so on. Although most of them are not related to insect immune signal transduction pathway, the result suggests that they may be involved in immune system of silkworm.4 Proteome analysis of fat body of silkwormUsing proteomic tool, we investigate fat body from day 5 of the fifth instar larvae. Almost 700 spots can be obtained from two-dimensional electrophoresis map of fat body, distributing in the area from 15kD to 100kD with pH 4-7. When 41 spots are subjected to MALDI-TOF MS, 34 proteins have been successfully identified, which are related to immune response, development and metamorphosis, ribosomal protein, cytoskeletal proteins, and so on. In order to investigate the varieties of proteins during silkworm metamorphosis, 2^nd day fat body of spin period and 3rd day of the pupa are choosed to be analyzed by two-dimensional electrophoresis. The result shows that some proteins are expressed up- or down regulatedly during this stage. They are identified as silkworm 30K protein, actin cytoskeleton protein, tubulin, heat shock protein, et al. In addition, DIGE (two-dimensional differential in-gel electrophoresis) are applied to investigate the changing expression amount during metamorphosis. The result show that the silkworm fat body protein changes significantly during pupation, suggesting some important role of fat body protein involved in the process of metamorphosis.5 Preliminary analysis of midgut, testicle and embryos of silkwormUsing pH 3-10 strip and 12.5% SDS-PAGE, midgut of silkworm are examined by two-dimensional electrophoresis. After sliver stained, almost 600 spots can be obtained in the map of midgut, ranging from 15kD to 100kD with pH 4-7. And 34 proteins have been successfully identified from midgut, which are related to muscle protein, metabolism protein, ion transport protein, apoptosis factor, et al. In the experiment of testicle analysis by two-dimensional electrophoresis, about 350 spots can be detected in the area from 20kD to 80kD with pH 4-9. Additionally, eggs of different developmental stages are investigated by SDS-PAGE and the resulting significant diverse bands are subjected to MALDI-TOF MS. The cytoskeletal proteins Actin 3, an important reference for studying egg development has also been identified besides the ESP (specific protein egg) and VitellinThe research of silkworm proteomics has been gradually attracting people, but this research field is currently stills at the preliminary stage on the whole. In establishing a high-throughput and relatively complete research platform, we undergo a relatively intensive research on proteomics of various tissues of silkworm, such as silk gland, blood, fat body and so on, and the results will lay a good foundation for the future large-scale silkworm proteomics research.