【作者】 靳远祥；

【导师】 徐孟奎；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2004， 博士

【摘要】 同许多昆虫一样,家蚕雌性附腺由分泌部和贮存部组成,分泌部分泌的胶状蛋白贮存于贮存部中。胶状粘液蛋白主要作用是将产出的卵粘附于外界的物质上,以保证昆虫在有大风大雨的自然环境中也能正常继代。从形态学和解剖学上的研究发现,家蚕雌性附腺是逐步发育的,但非常有趣的是雌性附腺在羽化前的2～3天开始突然大量分泌粘液蛋白,并在贮存部中积蓄,导致贮存部迅速的膨大。而家蚕欧洲种的一些品种产下的卵为天然散卵,这些品种的雌体不能正常分泌胶状粘性物质,经研究确认为遗传性状,命名为Ng突变。家蚕雌性附腺是一个特殊器官,合成和分泌特异蛋白质在短时间内完成,对雌性附腺的研究有助于搞清楚细胞、组织或器官发育分化的调节机制。 蛋白质组学是在蛋白的水平大规模研究基因表达的产物,能够直接的研究蛋白质的表达水平和相关蛋白的活性状态。家蚕的雌性附腺无论是在形态上还是在功能上在发育过程上都发生了显著的变化。因此,本研究利用双向电泳和质谱相结合的蛋白质组学的研究方法对不同发育阶段的雌性附腺分泌部表达的蛋白进行比较研究,以进一步研究这个复杂的生物学过程。 1.探讨了家蚕不同组织包括雌性附腺、丝腺、中肠和血液等的总蛋白提取和样品制备方法,优化不同组织来源蛋白的一向等电聚焦电泳(固相胶条)和二向SDS电泳的实验条件。由于组织的特异性,对不同的组织采用不同的蛋白提取方法和电泳条件。研究结果表明,对雌性附腺、中肠和丝腺用不同的裂解缓冲液提取蛋白和用不同的电泳参数进行双向电泳分离,能获得较好的蛋白质提取率、较高的分辨率(>1000蛋白点)、良好的重复性。 2.为了获得理想的家蚕雌性附腺分泌部组织在不同发育阶段的蛋白质表达图谱,对不同发育阶段的正常和Ng突变体的雌性附腺的分泌组织蛋白质样品制备和电泳进行了多次重复实验,确立了实验条件,取得良好的实验结果,建立了蛋白质参考图谱。利用分析软件(Image master)可以检测到800以上个蛋白点,而且不同发育时期的蛋白分布图基本相似,蛋白点主要分部在分子量30kD到70kD和等电点pH4～8的范围内。进一步对凝胶进行对比和软件匹配以及表达量的分析,发现有11个蛋白点的表达量在不同的发育时期表达量差异达到1.5更多还原

【Abstract】 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.更多还原