关键词: 家蚕微孢子虫, 海藻糖酶, 原核表达, RNA干扰, qRT-PCR

Abstract: 【Aim】 The purpose of this study is to preliminarily clarify the function of trehalase 3 (NbTre3) in Nosema bombycis, so as to provide theoretical basis and clues for the prevention and treatment of pebrine disease of Bombyx mori. 【Methods】 NbTre3 was amplified by PCR, and the prokaryotic expression vector pET28a-NbTre3 was constructed. The recombinant protein NbTre3 was expressed in Escherichia coli with IPTG induction and identified by Western blot. The recombinant protein NbTre3 was purified via Ni column affinity chromatography, and the polyclonal antibody was prepared by immunizing New Zealand rabbits with the obtained NbTre3. The localization of NbTre3 in mature spore of N. bombycis was investigated by indirect immunofluorescence assay. After N. bombycis infected the 5th instar larvae of B. mori, the transcription levels of NbTre3 in the midgut at different post-infection time were detected by qRT-PCR. The RNAi was carried out by injection of siRNA-1, siRNA-2 and siRNA-3, respectively, and the transcription levels of NbTre3 and 16S rRNA in the midgut of the 5th instar larvae of B.mori infected by N. bombycis at different time post RNAi were detected by qRT-PCR. 【Results】 The recombinant target protein NbTre3 was successfully expressed in E. coli and purified, with a size of about 34 kD. After immunizing the New Zealand rabbits, the serum was collected, and the polyclonal antibody of NbTre3 was obtained by purification and verified by Western blot. Indirect immunofluorescence result showed that NbTre3 was mainly distributed in the sporoplasm in mature spores of N. bombycis. The qRT-PCR results showed that the expression level of NbTre3 in the midgut of the 5th instar larvae of B. mori was the highest at 6 h post infection of N. bombycis. After the expression of NbTre3 was inhibited by siRNA, there was no significant change in the transcription level of 16S rRNA of N. bombycis. 【Conclusion】 The results suggest that NbTre3 may play an important role in the germinating process of the initial infection of N. bombycis.

Key words:  Nosema bombycis, trehalase, prokaryotic expression, RNA interference, qRT-PCR