家蝇Mdsirt1基因在细菌、高温和重金属胁迫下的表达分析

Expression profiles of Mdsirt1 in Musca domestica ( Diptera: Muscidae) under stresses of bacteria,high temperature and heavy metal

【目的】探究家蝇Musca domestica sirt1基因(Mdsirt1)在各种胁迫条件下的表达模式。【方法】以家蝇2龄幼虫cDNA为模板,PCR扩增Mdsirt1基因序列并进行生物信息学分析;实时定量PCR(qRT-PCR)检测Mdsirt1在家蝇不同发育阶段(卵、1龄幼虫、2龄幼虫、3龄幼虫、蛹和成虫)的表达变化,2龄幼虫不同组织(表皮、肠道、脂肪体和血细胞)中的表达分布,以及胁迫条件(细菌刺激、热激及CdCl_2刺激)下的转录水平变化;通过RNAi干扰Mdsirt1基因表达,观察敲低Mdsirt1表达后家蝇幼虫抗病能力变化,并检测机体氧化应激水平。【结果】家蝇Mdsirt1基因编码蛋白含有SIR2结构域,与厩螫蝇Stomoxys calcitrans SIR2氨基酸序列一致性为66%。qRT-PCR结果显示,Mdsirt1基因主要在家蝇蛹期表达,在家蝇2龄幼虫脂肪体中表达量较高。家蝇幼虫Mdsirt1分别在大肠杆菌Escherichia coli刺激3 h,金黄色葡萄球菌Staphylococcus aureus刺激6 h,42℃热激30min以及30 mmol/L CdCl_2刺激48 h时表达量达到最高峰。敲低Mdsirt1表达的家蝇幼虫受到细菌感染(大肠杆菌和金黄色葡萄球菌1∶1混合感染)后存活率较对照组显著降低1.47倍,且敲低Mdsirt1后机体处于氧化应激状态,活性氧自由基水平和丙二醛含量较对照组分别升高1.58和1.59倍。【结论】Mdsirt1参与家蝇幼虫的抗菌免疫应答和抗逆反应。

[ Aim ] This study aims to explore the expression pattern of Musca doraestica sirtl gene （Mdsirtl） under various stress conditions. [ Methods] The sequence of Mdsirtl gene was cloned by PCR from the 2nd instar larvae of Musca domestica and subjected to bioinformatics analysis. The expression profiles of Mdsirtl in different developmental stages （egg, 1st instar larva, 2nd instar larva, 3rd instar larva, pupa and adult）, various tissues of the 2nd instar larvae （cuticle, gut, fat body and hemocyte）, and the 2nd instar larvae under stresses （challenged by bacteria, heat shock and CdC12 stimulation） were investigated via quantitative real-time PCR （qRT-PCR）. RNAi was employed to knock down the expression of Mdsirtl in M. domestica larvae by mieroinjeetion with dsRNA, and then the resistance of larvae to bacteria and oxidative stress was detected. [ Results] The deduced protein encoded by Mdsirtl contains a predicted SIR2 domain, and shows 66% sequence identity with SIR2 from Stomoxys calcitrans.qRT-PCR results showed that Mdsirtl was mainly transcribed in the pupal stage and fat body of the 2nd instar larvae of M. domestica. Enhanced expression of Mdsirtl was observed in response to Escherichia coli and Staphylococcus aureus challenge for 3 and 6 h, respectively. The expression of Mdsirtl was significantly induced by heat shock of 42℃ for 30 min, and cadmium stimulation （exposure to 30 mmol/L CdC12 ） for 48 h. The survival rate observed in larvae treated with dsRNA of Mdsirtl under bacterial challenge （E. coli： S. aureus = 1 ： 1 ） was 1.47-fold lower than that treated with dsRNA of GFP. RNAi- mediated knockdown of Mdsirtl led to oxidative stress, and caused rapid increase of 1.58- and 1.59-fold in the reactive oxygen species （ROS） level and malondialdehyde （MDA） content, respectively. [ Conclusion ] Mdsirtl is involved in immunity and stress resistance in M. domestica larvae.