家蚕蜕皮液蛋白质双向电泳及质谱分析

Analysis of molting fluid in silkworm (Bombyx mori) by two-dimensional electrophoresis and mass spectrometry

摘要:

蜕皮液是存在于新旧表皮之间的一层液体，在昆虫蜕皮和变态发育的过程中发挥了重要的作用。为进一步探究家蚕蜕皮液的功能，利用双向电泳技术对家蚕预蛹期及羽化前期的蜕皮液的蛋白质进行了分析，结果表明，预蛹期及羽化前期的蜕皮液中分别可以检测出超过200个蛋白点，它们主要分布在等电点4?9、分子量10?180 kDa之间。利用MALDI TOF/TOF对羽化前期蜕皮液的42个蛋白点进行了鉴定分析，结果表明34个蛋白点成功得到了鉴定，它们主要包括载脂蛋白类、蛋白酶与蛋白酶抑制剂、免疫相关蛋白、几丁质结合蛋白等，部分蛋白在预蛹期的蜕皮液和羽化前的蜕皮液之间存在明显的差异表达。为了进一步验证蛋白质组分析的结果，对其中1个差异表达明显的蛋白质Apolipoprotein D进行了进一步的分析，Q-PCR的结果表明，该蛋白主要在化蛹第1–4天存在高表达，其在羽化前蜕皮液中的高度累积暗示了它可能参与了家蚕羽化变态的过程。以上研究结果进一步丰富了人们对蜕皮液蛋白质的认识，为深入研究蜕皮液蛋白质的功能提供了一些参考。

Abstract:

Molting fluid, a liquid between the old epidermis and new epidermis, plays an important role in the process of ecdysis and metamorphosis for insect. In order to explore the function of molting fluid, we used two-dimensional electrophoresis to study the molting fluid during the prepupal stage and pre-eclosion stage. More than 200 protein spots were found in the molting fluid of the 2 stages, which distributed in the 4?9 of pI and 10?180 kDa of molecular weight. We selected 42 spots to be analyzed by the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/TOF) from the molting fluid of pre-eclosion stage, of which 34 proteins were identified successfully, including apolipoprotein, protease and protease inhibitors, chitin-binding protein and protein involved in immunity. Some of the proteins demonstrated differential expression between the two stages of metamorphosis. In order to validate the result from proteomics analysis, we studied expression of the apolipoprotein D by Q-PCR from the developmental stages. The results showed that the gene encoding apolipoprotein D had the high expression from the 1st day to the 4th day of the pupa stage, which indicated they could be involved in eclosion due to the abundant accumulation in the late pupa. Our results offered more clues for understanding the mechanism of ecdysis and metamorphosis in insect and could give reference for further study of molting fluid.