摘要
【目的】本研究旨在克隆鉴定家蝇Musca domestica中8-羟基鸟嘌呤糖苷酶1(8-hydroxyguanine glycosylase 1,OGG1)编码基因Mdogg 1,明确其是否参与家蝇氧化应激调控。【方法】根据家蝇转录组数据,利用RT-PCR克隆Mdogg 1基因cDNA序列,并进行生物信息学分析;利用qRT-PCR检测Mdogg 1在家蝇不同发育阶段(卵、1-3龄幼虫、蛹和成虫)的表达变化、3龄幼虫不同组织(血细胞、肌肉、肠道和脂肪体)中的表达分布以及不同胁迫处理[2.5~100 mmol/L CdCl 2胁迫24 h,0.1 g/L盐酸阿霉素(DOX)浸泡30 min并恢复培养6,12和24 h,以及280-315 nm紫外线(UV)(强度5 J/cm 2)处理5~30 min]后2龄幼虫中的转录水平变化;通过RNAi技术敲低家蝇2龄幼虫Mdogg 1表达,并检测其丙二醛(malondialdehyde,MDA)含量、活性氧(reactive oxygen species,ROS)水平、超氧化物歧化酶(superoxide dismutase,SOD)活性和8-羟基鸟嘌呤(8-oxoguanine,8-oxoG)含量的变化。【结果】Mdogg 1(GenBank登录号:AYK27449.1)cDNA序列全长1062 bp,编码353个氨基酸,该蛋白的理论分子量和等电点分别为41.08 kD和9.02。MdOGG1氨基酸序列中含有螺旋-发夹-螺旋(HhH)结构域,并包含核酸内切酶Ⅲ保守结构域ENDO3c。qRT-PCR结果显示,Mdogg 1主要在家蝇卵和3龄幼虫脂肪体中呈高水平表达。2龄幼虫暴露于2.5~100 mmol/L CdCl 2下,Mdogg 1表达量呈现先上升再下降趋势,并伴有8-oxoG含量的持续升高。2龄幼虫浸泡于0.1 g/L DOX 30 min并恢复培养12和24 h以及UV照射30 min后,Mdogg 1表达量较未处理对照均显著上调。利用RNAi技术敲低家蝇2龄幼虫体内Mdogg 1的表达后,家蝇幼虫体内的ROS水平、MDA含量和8-oxoG含量较注射ds GFP的对照组显著增高,而SOD活性下降。【结论】Mdogg 1参与家蝇氧化还原平衡的维持,具有保护机体抵抗氧化损伤的生物学效应。
【Aim】The objective of this study is to clone and identify the 8-hydroxyguanine glycosylase 1 gene from Musca domestica(Mdogg 1),so as to verify its function in regulating redox balance.【Methods】Based on the previous transcriptome data of M.domestica,the cDNA sequence of Mdogg 1 was cloned by RT-PCR and then subjected to bioinformatics analysis.The expression levels of Mdogg 1 at different developmental stages(egg,1st-3rd instar larva,pupa and adult),in different tissues(hemocyte,muscle,gut,and fat body)of the 3rd instar larvae and in the 2nd instar larvae of M.domestica after different stresses[exposed to 2.5-100 mmol/L CdCl 2 for 24 h,soaked in 0.1 g/L doxorubicin hydrochloride(DOX)solution for 30 min and then recovered for 6,12 and 24 h,and exposed to 280-315 nm ultraviolet(UV)at the dose of 5 J/cm 2 for 5-30 min,respectively]were detected by qRT-PCR.The expression of Mdogg 1 in the 2nd instar larvae of M.domestica was knocked down by RNAi,and the changes in the malondialdehyde(MDA)content,reactive oxygen species(ROS)level,superoxide dismutase(SOD)activity,and 8-oxoguanine(8-oxoG)content were determined.【Results】The full-length cDNA sequence of Mdogg 1(GenBank accession number:AYK27449.1)was obtained.It is 1062 bp in length,encoding 353 amino acid residues,with the theoretical molecular weight of 41.08 kD and the isoelectric point of 9.02.The amino acid sequence of MdOGG1 includes a HhH(Helix-hairpin-Helix)motif and the conserved endonuclease III domain ENDO3c domain.The qRT-PCR results showed that Mdogg 1 was highly expressed in the egg stage and the fat body of the 3rd instar larvae of M.domestica.When the 2nd instar larvae of M.domestica were exposed to 2.5-100 mmol/L CdCl 2,the expression level of Mdogg 1 increased first and then decreased,and the 8-oxoG content increased.The expression levels of Mdogg 1 in the 2nd instar larvae soaked in 0.1 g/L of DOX solution for 30 min and then recovered for 12 and 24 h,and those exposed to UV for 30 min,were significantly up-regulated as compared to that in
作者
张玉明
邵梦华
李亚静
冯琴
魏丽亚
柳峰松
ZHANG Yu-Ming;SHAO Meng-Hua;LI Ya-Jing;FENG Qin;WEI Li-Ya;LIU Feng-Song(College of Life Sciences,Hebei University,Baoding,Hebei 071002,China;Institute of Life Science and Green Development,Hebei University,Baoding,Hebei 071002,China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2021年第1期51-60,共10页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31702064)
河北省自然科学青年基金项目(C2018201135)
河北省教育厅青年基金项目(QN2019117)。
关键词
家蝇
8-羟基鸟嘌呤糖苷酶
基因克隆
RNA干扰
氧化应激
Musca domestica
8-hydroxyguanine glycosylase
gene cloning
RNA interference
oxidative stress
