家蚕双重氧化酶BmDUOX的序列特征、表达模式及病原诱导表达

Sequence characteristics,expression pattern and pathogen-induced expression of BmDUOX from the silkworm,Bombyx mori(Lepidoptera:Bombycidae)

【目的】研究双重氧化酶(dual oxidase,DUOX)在家蚕Bombyx mori中的表达模式,探析其在家蚕肠道免疫机制中的作用。【方法】通过氨基酸多重序列比对和系统进化分析对Bm DUOX蛋白氨基酸序列特征进行研究,并采用RT-PCR方法扩增获得家蚕Bm DUOX膜外部分Bm DUOX_OM基因序列。在大肠杆菌Eescherichia coli(DE3)中诱导表达并通过亲和层析法纯化获得重组表达蛋白,以其为抗原免疫昆明鼠,获得对应的多克隆抗体;利用所得的抗体检测Bm DUOX的表达和细胞定位。通过半定量RT-PCR方法分析Bm DUOX在家蚕不同发育时期和组织中的表达模式及病原诱导表达谱。另外,利用活性氧检测试剂盒分析家蚕微孢子虫Nosema bombycis诱导后家蚕Bm E细胞的活性氧(ROS)的含量。【结果】生物信息学分析表明,Bm DUOX内有保守的Peroxidase,Ferric_reduct,EF-hand,FAD-binding和NAD-binding结构域,且具有6个跨膜区,跨膜形式与人类Homo sapiens、果蝇Drosophila melanogaster等的DUOX蛋白的跨膜形式一致。多重序列比对分析表明,Bm DUOX的过氧化酶区域内具有过氧化物酶的保守活性位点。克隆获得家蚕Bm DUOX_OM基因,并纯化获得重组表达蛋白,制备的鼠多抗具有较好的特异性。间接免疫荧光实验(IFA)表明,Bm DUOX位于家蚕Bm E细胞的细胞膜上。表达模式分析表明,Bm DUOX在家蚕5龄第3日幼虫和成虫中表达量较高;且在幼虫表皮、精巢、卵巢和头内高量表达,在成虫的卵巢、精巢、表皮和脂肪体内也有较高的表达量。病原诱导分析表明,通过肠道起始感染的家蚕微孢子虫能够诱导家蚕幼虫中肠Bm DUOX基因持续上调表达,且其诱导后的Bm E细胞内活性氧含量也明显增加,提示Bm DUOX调控的肠上皮ROS应答参与抵抗家蚕微孢子虫的侵染。【结论】Bm DUOX含有典型的结构域及保守的活性位点,表明其在家蚕中具有保守的生物学功能。Bm DUOX在家蚕不同发育时期、不同组织及病原诱导下的表达谱提示其可能参与宿主肠上皮对家蚕微孢子虫的免疫反应。

[ Aim ] To explore the expression pattern and roles of dual oxidase （DUOX） in gut immunity in the silkworm, Bombyx mori. [ Methods ] The amino acid sequence of BmDUOX was analyzed by bioinformatic methods, and the outside region encoding gene, BmDUOX_OM, was cloned from B. mori and transferred into Eescherichia coli Transetta （DE3） to obtain the fusion protein. After purification by Ni affinity chromatography, the purified protein was treated as antigen to immunize mouse to prepare polyclonal antibodies, which were utilized for analyzing the expression and localization of BmDUOX in BmE cells. Semi-quantitative RT-PCR was employed to assay the expression patterns of BmDUOX in different developmental stages and different tissues and the pathogen-induced expression profiles. Additionally, the content of reactive oxygen species （ROS） in BmE cells after induction by Nosema bombycis was analyzed by using ROS detection kit. [ Results ] Bioinformatics analysis showed that BmDUOX has conserved peroxidase, ferric_reduct, EF-hand, FAD-binding and NAD-binding domains. There are also six transmembrane regions in BmDUOX, and their transmembrane form is consistent with that of hDUOX2 and DmDUOX. Multiple sequence alignment analysis showed that the peroxidase domain of BmDUOX has conserved activity sites. BmDUOX_OM gene was cloned, the recombinant protein was purified, and mouse-origin polyclonal antibody with high specificity was prepared. Indirect immunofluorescence assay （IFA） showed that BmDUOX was located on the plasma membrane of BmE cells. Expression pattern analysis showed that BmDUOX was highly expressed in the 5th instar day-3 larvae and adults of the silkworm, and in the tissues of integument, testis, ovary, head of larvae, and the ovary, testis, integument, fat body of its adults. Expression of BmDUOX in the larval midgut of the silkworm could be continuously induced by microsporidia Nosema bombycis, and the content of ROS in BmE cells was obviously increased after induction by N. bombycis, suggesting that DUOX-regulated intestinal epithelial ROS response may play important roles in the resistance against the infection of N. bombycis. [ Conclusion ] BmDUOX has the typical functional domains and active sites, showing its conservative biological function in B. mori. Expression profiles of BmDUOX in B. mori in different developmental stages, different tissues, and under induction by diverse pathogenic microorganisms suggest that BmDUOX may be involved in the immune response of host intestinal epithelium to the invasion of pathogenic microbes.