柞蚕微孢子虫部分孢壁蛋白的分离鉴定及孢壁蛋白8的序列分析

研究柞蚕微孢子虫的孢壁蛋白组成和结构特点,有助于解明柞蚕微孢子虫在侵染过程中与蚕体细胞的互作机制。分别采用煮沸法和Laemmli法分离提取柞蚕微孢子虫总蛋白和孢壁蛋白,回收30kD左右的蛋白条带进行液质联用离子阱电喷雾质谱分析,获得的短肽序列经Mascot在线检索工具进行蛋白同源序列比对,共鉴定出27种具有功能注释的蛋白,有3种注释为孢壁蛋白。其中获得了与家蚕微孢子虫孢壁蛋白8(NbSWP8)高度同源的柞蚕微孢子虫孢壁蛋白8(NaSWP8)的基因全长序列。序列分析表明NaSWP8与NbSWP8的氨基酸序列相似性达到90%,且均具有特征性的肝素结合基序,二者的编码基因核苷酸序列的非同义替换率和同义替换率比值(dN/dS)明显小于1,表明孢壁蛋白8基因在2种蚕类微孢子虫中受到纯化选择压力的作用,基因的功能相对稳定。

Extraction and Identification of Partial Spore Wall Proteins of Nosema antheraeae and Sequence Analysis of Spore Wall Protein 8

Researches on constitutional and structural features of spore wall proteins of Nosema antheraeae would facilitate the elucidation of interactive mechanism between N.antheraeae and silkworm cells in the infection process.Boiling and Laemmli methods were utilized to extract the total spore wall proteins of N.antheraeae.Protein bands of about 30 kD were recovered to conduct liquid chromatography and electrospray ionization mass spectrometry.The obtained peptide sequences were aligned with homologous protein sequences with Mascot online search tool.As a result,27 proteins were identified to have functional annotation,3 of which were annotated as spore wall proteins.Among them,one full-length gene sequence was found to code for the homologous protein of N.bombycis spore wall protein 8 (NbSWP8) and was thus named as N.antheraeae spore wall protein 8 (NaSWP8).Sequence analysis revealed that amino acid sequences of NaSWP8 and NbSWP8 were 90% identical and both of them had the typical heparin-binding motif.The ratio of the rate of non-synonymous substitutions (dN) to the rate of synonymous substitutions (dS) between NaSWP8 and NbSWP8 was far less than 1,indicating that purification selection pressure had been exerted on spore wall protein 8 gene in these two silkworm microsporidian species and the gene's function was relatively stable.