›› 2010, Vol. 53 ›› Issue (4): 369-378.doi:

• 研究论文 • 上一篇    下一篇

家蚕精巢蛋白质的双向电泳及质谱分析

聂红毅,钟晓武,邹勇,衣启营,赵萍,夏庆友   

  • 出版日期:2010-07-16 发布日期:2010-04-20
  • 通讯作者: 赵萍

Identification of testis proteins of silkworm Bombyx mori using two-dimensional electrophoresis and mass spectrometry

NIE Hong-Yi, ZHONG Xiao-Wu, ZOU Yong, YI Qi-Ying, ZHAO Ping, XIA Qing-You   

  • Online:2010-07-16 Published:2010-04-20

摘要: 精巢是雄性家蚕Bombyx mori的生殖腺,它的主要功能是产生精子,全面检测和鉴定精巢器官的蛋白分布将为分析家蚕雄性个体的发育和繁殖奠定基础。本研究利用双向聚丙烯酰胺凝胶电泳和蛋白硝酸银染色技术对家蚕5龄第5天幼虫的精巢组织进行了蛋白检测,利用基质辅助激光解析质量飞行时间质谱(MALDI-TOF-MS)对表达量较高的蛋白点进行了肽质量指纹图谱鉴定。结果表明:家蚕精巢蛋白质可以检测出1 000个以上的蛋白点,这些蛋白点主要集中在分子量为15~90 kD区域,等电点3.5~9之间,其中60个蛋白点得到了成功鉴定,按照已知或推测的蛋白功能,将其分为8类,包括:细胞骨架和细胞结构蛋白,膜蛋白或信号相关蛋白,大量应激反应蛋白(伴侣蛋白),线粒体和能量产生相关蛋白,转录调控和翻译及DNA/RNA结合相关蛋白,酶和少量血液组成蛋白。其中很多蛋白可能在鞭毛形成、能量代谢及减数分裂过程中有重要作用。这些结果为进一步认识家蚕精子形成过程提供了重要的生物学信息。

关键词: 家蚕;精巢;精子形成, 蛋白质;双向电泳;肽质量指纹图谱鉴定;质谱分析;生物信息学分析

Abstract: Testis is the generative gland in silkworm Bombyx mori that produces spermatozoa. A complete understanding of proteins expressed in the testis will provide biological information on development and reproduction in males of the silkworm. We investigated the testis proteins of 5th instar day 5 larvae of the silkworm using two-dimensional polyacrylamide gel electrophoresis and silver staining, followed by computer-assisted analysis. Then, the matrix-assisted laser desorption ionization time of flight mass spectrometry was applied to identify the major protein spots of the 2D map with peptide mass fingerprinting data. The results showed that over 1 000 spots were obtained by silver staining. The profiles of silkworm tissue proteins were established in a pI range of 3.5-9 and a molecular mass range of 15 kD to 90 kD. Among them, 60 proteins were identified successfully, which were divided into 8 groups according to their known or putative functions, including cytoskeleton and cell structure (cell differentiation), membrane protein receptors or cell signaling related, stress response (chaperones), mitochondrial and energy production related, translational and transcriptional regulation or DNA/RNA binding related, enzymes and blood components. Many proteins play important roles in flagellar formation, energy metabolism and meiosis. These results may provide valuable biological information for investigating spermiogenesis in the silkworm.

Key words: Bombyx mori, testis, spermiogenesis, proteins, 2D-electrophoresis, peptide mass fingerprinting, mass spectrometry, bioinformatical analysis