关键词: 东方蜜蜂微孢子虫, 孢壁蛋白, 亚细胞定位, 几丁质孢子壳, 极管蛋白, 互作

Abstract: 【Aim】 Nosema ceranae is known as the main pathogen of honeybee microsporidiosis and the work focusing on the infection mechanism of N. ceranae was rarely reported. The objective of this study is to prokaryotically express NcER_100148, a candidate novel spore wall protein of N. ceranae obtained by mass spectrometry identification in our previous work, clarify its subcellular localization and preliminarily explore its biological functions. 【Methods】Bioinformatic analysis on the sequence of the previously identified candidate spore wall protein NcER_100148 of N. ceranae was performed by online software. The expression level of NcER_100148 in the midgut of the A. mellifera adults infected with N. ceranae was detected by RT-PCR. The gene fragment of NcER_100148 was cloned into the prokaryotic expression vector pET30a, and the recombinant protein was induced by IPTG for preparing polyclonal antibody. Western blot was employed to detect the expression level of NcER_100148 in mature spores of N. ceranae. Subcellular localization of NcER_100148 in mature spores of N. ceranae was analyzed by indirect immunofluorescence and immunoelectron microscopy. Western blot and co-immunoprecipitation (Co-IP) were then employed to analyze the interaction of the recombinant NcER_100148 with chitin spore coats (CSCs) and the polar tube proteins NcPTP2 and NcPTP3 of N. ceranae, respectively. 【Results】 Sequence analysis result indicated that NcER_100148 of N. cerana has the predicted molecular weight of 12.169 kD, containing an O-glycosylation site, an N-glycosylation site and a glycosylphosphatidylinositol (GPI) anchor site. RT-PCR result showed that NcER_100148 began to be expressed in the midgut of A. mellifera adults at 4 d post infection by N. ceranae. Western blot result revealed that NcER_100148 could be expressed on the surface of mature spores and could interact with CSCs of N. ceranae. The subcellular localization result showed that NcER_100148 was localized on the spore wall of mature spores of N. ceranae. The results of Western blot and Co-IP demonstrated that the recombinant NcER_100148 could interact with the polar tube proteins NcPTP2 and NcPTP3. 【Conclusion】NcER_100148 is a novel spore wall protein located on the spore wall of mature spores of N. ceranae. NcER_100148 may play important roles in the construction of endospore, coil and fixation of polar tubes and regulation of infection to the host cells.

Key words: Nosema ceranae, spore wall protein, subcellular localization, chitin spore coats, polar tube protein, interaction