烟草甲攻击素基因LsAttacin2的表达及其在抗细菌胁迫中的作用

Expression of attacin gene LsAttacin2 of Lasioderma serricorne(Coleoptera:Anobiidae)and its role in resisting bacterial stress

【目的】本研究旨在阐明烟草甲Lasioderma serricorne幼虫体内攻击素(attacin)基因LsAttacin2在响应细菌侵染过程中的功能。【方法】利用RT-PCR扩增烟草甲LsAttacin2的cDNA全长序列;利用RT-qPCR检测LsAttacin2在烟草甲不同发育阶段(卵、低龄幼虫、高龄幼虫、蛹、低龄成虫和高龄成虫)、4龄幼虫不同组织(头、表皮、前肠、中肠、后肠、脂肪体和马氏管)、大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus侵染及其肽聚糖处理4龄幼虫后的相对表达量;通过注射法利用RNAi沉默烟草甲4龄幼虫LsAttacin2的表达后,检测大肠杆菌和金黄色葡萄球菌侵染烟草甲4龄幼虫后的死亡率。【结果】克隆获得烟草甲LsAttacin2(GenBank登录号:MT635176)的cDNA全长序列,其开放阅读框长504 bp,编码167个氨基酸残基,LsAttacin2 N端存在信号肽,C端具有Attacin_C超家族的保守结构域。RT-qPCR结果表明,LsAttacin2在烟草甲测试的各发育阶段均有表达,且在高龄幼虫和蛹中高表达;LsAttacin2主要在4龄幼虫中肠、表皮和脂肪体等免疫组织中表达;大肠杆菌和金黄色葡萄球菌及其肽聚糖均诱导了烟草甲4龄幼虫体内LsAttacin2的上调表达。RNAi结合细菌生物测定结果表明,ds LsAttacin2注射组4龄幼虫中LsAttacin2的表达量在注射24和48 h时较对照组(注射ds GFP)分别显著下降了72.8%和80.4%;RNAi后48 h,与注射ds GFP的对照组相比,ds LsAttacin2注射组大肠杆菌和金黄色葡萄球菌侵染24 h时4龄幼虫的死亡率分别提高了21.1%和24.4%。【结论】本研究结果表明LsAttacin2可能在烟草甲抗细菌的免疫应答过程中起着重要作用。

【Aim】This study aims to clarify the function of an attacin gene(LsAttacin2)in Lasioderma serricorne larvae in response to bacterial infection.【Methods】The full-length cDNA sequence of LsAttacin2 from L.serricorne was amplified by RT-PCR.The relative expression levels of LsAttacin2 in different developmental stages(egg,early larva,late larva,pupa,callow adult and mature adult)and various tissues(head,cuticle,foregut,midgut,hindgut,fat body and Malpighian tubules)of the 4th instar larvae of L.serricorne,and in the 4th instar larvae after infection with Escherichia coli and Staphylococcus aureus and treatments with their peptidoglycans were examined by RT-qPCR.After silencing the expression of LsAttacin2 by RNAi through injection,the mortalities of the 4th instar larvae of L.serricorne post infection with E.coli and S.aureus were detected.【Results】The full-length cDNA sequence of LsAttacin2(GenBank accession number:MT635176)in L.serricorne was obtained.The open reading frame of LsAttacin2 is 504 bp in length encoding 167 amino acid residues.LsAttacin2 has a signal peptide at the N-terminus and a conserved domain of Attacin_C superfamily at the C-terminus.The RT-qPCR results showed that LsAttacin2 was expressed in the tested various developmental stages and highly expressed in the late larva and pupa.LsAttacin2 was mainly expressed in immune tissues,such as midgut,cuticle and fat body of the 4th instar larva.Both E.coli and S.aureus and their peptidoglycan induced the up-regulated expression of LsAttacin2 in the 4th instar larvae of L.serricorne.RNAi combined with bacterial bioassay results showed that the expression levels of LsAttacin2 in the 4th instar larvae in the ds LsAttacin2 injection groups were significantly reduced by 72.8%and 80.4%at 24 and 48 h,respectively,as compared to those in the control groups(injected with ds GFP).The mortalities of the 4th instar larvae at 24 h after infection with E.coli and S.aureus in the ds LsAttacin2 injection groups at 48 h after RNAi increased by 21.1%and 24.4%,respectively,as compared to those in the control groups injected with ds GFP.【Conclusion】The results of this study suggest that LsAttacin2 plays an important role in the immune response of L.serricorne against the bacterial infections.