【作者】 叶键；

【导师】 钟伯雄；

【作者基本信息】 浙江大学 ， 特种经济动物饲养， 2007， 硕士

【摘要】 家蚕是产丝的昆虫,体内具有1对丝腺,由下唇腺特化而成,位于幼虫消化管的腹面两侧,是1对半透明多屈曲的大型管状器官。家蚕后部丝腺的细胞,专门分泌丝素,幼虫进入第5龄以后,为了适应丝素大量合成的需要,整个后部丝腺细胞内的代谢系统以及亚细胞结构都发生巨大的变化。以丝素合成与分泌为中心,第5龄的后部丝腺发育可以分为3个生理时期：腺细胞生长期、丝素大量合成与分泌期和吐丝与腺细胞分解期。对后部丝腺的研究有助于搞清楚其分泌丝素的调节机制。蛋白质组学是在蛋白的水平上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。家蚕的后部丝腺无论是在形态上还是在功能上在发育过程中都发生了显著的变化。因此,本研究利用了双向电泳的方法对不同发育阶段的后部丝腺以及不同品种家蚕五龄后部丝腺细胞表达的蛋白质进行比较研究：1.建立了家蚕后部丝腺组织细胞的总蛋白提取和样品制备方法,优化了蛋白质双向电泳的实验条件。研究结果表明,采用本方法能获得较好的蛋白质提取率、较高的分辨率(>900蛋白点)、良好的重复性。2.研究了P50家蚕幼虫从四眠至五龄第十天后部丝腺细胞的蛋白质组成,共检测到个571个特异蛋白质斑点,其中五龄第1天和第2天出现的特异斑点最多,分别达到了109和168个,前者均匀分布于凝胶上,而后者的特异蛋白斑点主要分布于pH4-7的酸性区间内。推测后部丝腺细胞在5龄第1天和第2天处于代谢功能相异的两个重要阶段。5龄第8天检测到62个特异蛋白斑点,达到自5龄第2天后的第2个高峰,推测与后部丝腺细胞开始消亡相关。3.研究了13个品种五龄混合样后部丝腺细胞的蛋白质组成,检测到218个特异蛋白斑点,差异蛋白斑点可以明显的分为2大类,一类是只有在少数品种没有检测到,在其他品种中都可以检测到的蛋白,例如蛋白编号为1-71的蛋白；另一类为只在少数品种中检测到的蛋白,如编号为72-218的蛋白,从特异斑点的数量上来看,P50中检测到77个,罗尼7号检测到64个,秋丰检测到71个,白玉检测到76个,丰1检测到56个,除去涉及品种本身区别的差异蛋白斑点外,似乎区别不是很大。并且发现P50X秋丰表达的差异蛋白要明显多于其他品种,但其茧层量却比其他品种明显低,推测其低茧层量不是由于缺少了某些关键蛋白导致遏制了丝素的分泌,而是多分泌的蛋白遏制了丝素的分泌,推测蛋白斑点94-127可能与丝素分泌的阻遏过程相关并且这个阻遏过程涉及的蛋白较多,可能涉及了一个多蛋白协同作用的过程。B1共检测到特异蛋白斑点85个,R1共检测大特异蛋白斑点56个,J1共检测到特异蛋白斑点78个三个品种均各自表达了些特异蛋白,这些蛋白均无法在其他品种中检测到,其中B1表达了标号为187-204的蛋白,R1表达了标号为196、198、1 99、200、202以及标号为204-212的蛋白,J1表达了标号为213-218的特异蛋白斑点,推测出标号为196、198、199、200、202以及标号为204-212可能与R1(裸蛹)蚕H链的抑制有关。更多还原

【Abstract】 silkworm Bombyx mori is a insect that secrete fibroin, it has a pair of silk gland, which were developed from ectolabium gland, located on the side of the digest canal. The posterior silk gland of silkworm is a very important organ where fibroin is synthesized and secreted. when larva comes into the 5th instar, for the sake of adapting large amounts of fibroin’s synthesizing, the metabolic system and sub-cellular structure of posterior silk gland changed a lot.3 periods can be plotted out based on its capability of secreting fibroin cell growth period, massive fibroin secreting period, and cell languishing period.proteomics is a large-scale study of the gene expression at the protein level,which ultimately 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 and their subsequent identification by biological mass spectrometry. Using two-dimensional gel electrophoresis(2-DE)and image analysis system, we researched the proteins extracted form posterior silk gland of different period of development and different breeds of silkworm, we find out that:1.in this research, the main methods of the total proteins extraction and preparation of posterior silk gland protein, as well as the conditions of isoelectric focusing and SDS-PAGE were discussed and optimized.2.the proteins extracted from posterior silk gland of p50 silkworm in 4th molting and 5th instar were researched by two-dimensional polyacryamide gel electrophoresis(2D-PAGE)and image analysis system. A total of 571 differential protein spots were detected. Among them, the differential protein spots expressed on 1est and 2nd day in 5th instar contributed the largest quantity, namely 109 and 168 respectively, for the former, it distributing on the gel equably, while for the later, it mainly distributing between pH4-7. The results suggested that the 1est and 2nd day in 5th instar may locate on two different and important period of time by means of metabolism.62 protein spots were detected on 8th day in 5th instar, it become the second pinnacle of differential protein spots secretion after 2nd day in 5th instar, suggested that it may respected to the digestion of posterior silk gland cells.3.the mixed proteins extracted from posterior silk gland of different breeds of silkworm in 5th instar were researched by two-dimensional polyacryamide gel electrophoresis(2D-PAGE)and image analysis system. A total of 218 differential protein spots were detected, in which p50 takes 77, Luoni7 takes 64, Qiufeng takes 71, Baiyu takes 76, Fengltakes 56,this 5 breeds looks as if it makes without large difference in differential protein spots number. While, in p50 x Qiufeng, we find out that it secret notably much more differential protein spots than other breeds with its cocoon fiber weight notably lower than other breeds, we speculate that protein spots 94-127 may related with the stem of secreting fibroin protein, and a cooperational action may involved in this process. we detected 85 differential protein spots in B1, 56 differential protein spots in R1,78 differential protein spots in J1,and with their own differential protein spots respectively, which cannot be detected in other breeds. for B1,it secret protein 187-204,for R1,it secret protein 196、198、199、200、202 and protein 204-212,for J1,it secret protein 213-218.we speculated that protein spots 196、198、199、200、202 and protein spots 204-212 may related with the stem of R1 silkworm H chain.更多还原