克氏原螯虾duox1基因抵御金黄色葡萄球菌侵染的先天免疫机制

Innate immune mechanism of duox1 gene in Procambarus clarkii against Staphylococcus aureus infection

【目的】探究双氧化酶1基因(duox1)在克氏原螯虾抵抗金黄色葡萄球菌(Staphylococcus aureus)侵染先天免疫应答中的作用机制,为确保克氏原螯虾产业的持续健康发展提供技术支撑。【方法】采用实时荧光定量PCR检测金黄色葡萄球菌刺激后,duox1基因在克氏原螯虾血细胞、肝胰腺、肠道及鳃组织中的表达情况;通过RNA干扰(RNAi)敲低duox1基因表达再进行金黄色葡萄球菌刺激,统计克氏原螯虾存活率,采用H_(2)O_(2)含量检测试剂盒检测肝胰腺H_(2)O_(2)含量,电子显微镜下观察血淋巴黑化现象,并以实时荧光定量PCR检测肝胰腺中抗菌肽基因(toll1、dorsal、crustin3和crustin4)的表达情况。【结果】经金黄色葡萄球菌刺激后,克氏原螯虾duox1基因在血细胞、肝胰腺、肠道及鳃组织中的相对表达量较PBS组整体上呈上升趋势,故推测duox1基因参与克氏原螯虾的抗菌先天免疫应答,具有潜在的抵抗细菌侵染作用。与dsGFP+金黄色葡萄球菌组相比,经RNA干扰及金黄色葡萄球菌刺激后,克氏原螯虾存活率呈明显下降趋势,肝胰腺H_(2)O_(2)含量呈先降低后回升的变化趋势(在刺激后24 h达最低值),且克氏原螯虾血淋巴黑化反应程度明显减弱。干扰duox1基因表达并感染金黄色葡萄球菌后,克氏原螯虾肝胰腺Toll信号通路上的抗菌肽基因(toll1、dorsal、crustin3和crustin4)表达被抑制,导致参与抗菌反应的先天免疫能力下降,最终引起克氏原螯虾存活率下降。【结论】克氏原螯虾duox1基因通过调控H_(2)O_(2)产生、影响血淋巴黑化现象及调控toll1、dorsal、crustin3和crustin4等抗菌肽基因的表达,参与机体的先天免疫应答,进而协助机体抵御金黄色葡萄球菌的侵染。

【Objective】The study aimed to elucidate the role and mechanism of double oxidase 1 gene(duox1)in the innate immune response of Procambarus clarkii against Staphylococcus aureus infection,providing technical support for the sustainable and healthy development of P.clarkii industry.【Method】The real-time fluorescence quantitative PCR was used to detect the relative expression of duox 1 gene in hemocytes,hepatopancreas,intestine and gill tissues of P.clarkii after stimulation of S.aureus.By using RNA interference(RNAi)to down-regulate the expression of the duox1 gene and subsequently stimulating with S.aureus,the survival rate of the P.clarkii was determined.The contents of H_(2)O_(2)in the hepatopancreas were measured with an H_(2)O_(2)detection kit.Melanization in the hemolymph was observed under an electron microscope.The expression of antimicrobial peptide genes(toll1,dorsal,crustin3 and crustin4)in the hepatopancreas was assessed using real-time fluorescence quantitative PCR.【Result】After stimulation by S.aureus,the relative expression of the duox1 gene in hemocytes,hepatopancreas,intestine and gill tissues of P.clarkii showed an overall upward trend compared to the PBS group.This suggested that the duox1 gene may be involved in the innate immune response of P.clarkii against bacterial infection,with a potential role in combating bacterial invasion.Compared to the dsGFP+S.aureus group,after RNA interference and S.aureus stimulation,the survival rate of P.clarkii greatly decreased.The H₂O₂content in the hepatopancreas showed a trend of initial reduction followed by recovery(reaching its lowest point 24 h after stimulation),and the degree of hemolymph melanization was markedly weakened.After duox1 gene expression was interfered and S.aureus infection,the expression of antimicrobial peptide genes(toll1,dorsal,crustin3 and crustin4)in the Toll signaling pathway of the hepatopancreas was suppressed,leading to a reduction in innate immune capacity against bacterial infection,ultimately resulting in a decreased survival rate of P.clarkii.【Conclusion】The duox1 gene of P.clarkii participates in the innate immune response of the body by regulating H_(2)O_(2)production,affecting the melanization of hemolymph,and regulating the expression of antimicrobial peptide genes such as toll1,dorsal,crustin3 and crustin4,thereby assisting the body in resisting infection by S.aureus.