脂多糖对家蚕幼虫Toll样受体基因BmToll9-2表达的影响

摘要

[目的]Toll样受体是昆虫Toll信号传导途径的一个重要组成成员,对激活昆虫机体对入侵病原微生物的先天免疫应答起着至关重要的作用.本研究旨在探究脂多糖对家蚕Bombyx mori幼虫中Toll样受体基因BmToll9-2基因表达的影响.[方法]利用RT-PCR分析BmToll9-2基因在家蚕大造品系4龄和5龄幼虫表皮、脂肪体、马氏管、中肠、神经、丝腺、胸部肌肉共7种组织中的表达特征;将脂多糖和大肠杆菌Escherichia coli注射到5龄幼虫体腔内,诱导其免疫系统响应,采用实时荧光定量PCR分析注射后不同时间BmToll9-2基因在中肠中的表达水平.[结果]RT-PCR结果表明,BmToll9-2基因在家蚕4龄幼虫的脂肪体、马氏管、神经和丝腺中高度表达,在5龄幼虫脂肪体、中肠、马氏管和神经中均有较高表达.实时荧光定量PCR结果显示,注射脂多糖能诱导5龄幼虫中肠中BmToll9-2基因转录,在注射后6h时的诱导效果最好;注射大肠杆菌也能诱导BmToll9-2基因转录,在注射后3和6h时的诱导效果最好.[结论]家蚕幼虫BmToll9-2基因在脂多糖和大肠杆菌处理后上调表达,提示BmToll9-2受体可能参与昆虫Toll样受体对脂多糖和细菌的识别过程.%[Aim] Toll-like receptors are important members in Toll signaling pathway in insects and play an important role in activating the innate immune response to invading pathogenic microorganisms.This study aims to explore the influence of lipopolysaccharide (LPS) on the expression of the Toll-like receptor gene BmToll9-2 in larvae of the silkworm,Bombyx mori.[Methods] The expression profiles of BmToll9-2 in epidermis,fat body,Malpighian tubules,midgut,brain,silk glands and thoracic muscles of the 4th and 5th instar larvae of B.mori strain Dazao were detected by RT-PCR.LPS and Escherichia coli were respectively injected into the body cavity of the 5th instar larvae to induce their immune response,and the expression levels of BmToll9-2 in the midgut at different time after injection were detected by real-time quantitative PCR.[Results] RT-PCR results revealed that BmToll9-2 was highly expressed not only in the fat body,Malpighian tubules,brain and silk glands of the 4th instar larvae of B.mori,but also in the fat body,midgut,Malpighian tubules and brain of the 5th instar larvae.The real-time quantitative PCR results showed that injection of LPS could induce the transcription of BmToll9-2 in the midgut of the 5th instar larvae,and the best induction effect was at 6 h after injection.Injection of E.coli could also induce the transcription of BmToll9-2 in the midgut of the 5th instar larvae,and the best induction effect was at 3 and 6 h after injection.[Conclusion] Expression of BmToll9-2 in B.mori larvae is up-regulated after injection of LPS and E.coli,suggesting that BmToll9-2 receptor may be involved in the recognition process of Toll-like receptors to LPS and E.coli in insects.