摘要
【目的】从家蝇Musca domestica中克隆一种C-型凝集素(C-type lectin)基因,并分析其在家蝇免疫过程中的功能。【方法】根据家蝇转录组信息,克隆了一条C型凝集素基因,将其命名为Mdlectin-C1。构建Mdlectin-C1的原核表达载体,在大肠杆菌Escherichia coli中表达并纯化r Mdlectin-C1蛋白,制备多克隆抗体。利用实时定量PCR(quantitative real-time PCR,qRT-PCR)及Western blot技术对家蝇不同发育阶段和细菌感染前后Mdlectin-C1的表达水平进行检测。运用RNAi技术敲低家蝇1龄幼虫Mdlectin-C1基因表达,然后进行细菌刺激并观察幼虫存活率变化。【结果】Mdlectin-C1 c DNA包含一个546 bp完整开放阅读框,编码181个氨基酸残基,所推导多肽N端包含由21个氨基酸残基组成的信号肽,成熟肽中含有一个保守的碳水化合物识别结构域(carbohydrate-recognition domain,CRD)。qRT-PCR及Western blot结果显示,家蝇从1龄幼虫到蛹的发育过程中,Mdlectin-C1表达量逐步上升,蛹期达到最大值。在革兰氏阴性菌大肠杆菌E.coli和革兰氏阳性菌金黄色葡萄球菌Staphylococcus aureus刺激后,家蝇2龄幼虫Mdlectin-C1均上调表达,并分别在36和3 h时达到最高值。利用RNAi技术成功敲低Mdlectin-C1表达。对Mdlectin-C1基因敲低的1龄幼虫进行细菌刺激,敲低实验组幼虫存活率显著低于正常对照组。【结论】Mdlectin-C1可能参与家蝇抗菌免疫应答,并在此过程中具有重要作用。
[ Aim ] This study aims to identify and characterize a new C-type lectin gene from the housefly Musca domestica, and to analyze its immune function. [ Methods ] Based on the transcriptome database, a new lectin gene, designated as Mdlectin-C1, was cloned from M. domestica. Recombinant Mdlectin-C1 was expressed in Escherichia coli, and purified rMdlectin-C1 was used to prepare polyclonal antibody. Quantitative real-time PCR （qRT-PCR） and Western blot techniques were employed to investigate the gene expression profiles in different developmental stages and larvae post bacterial infection. The survival rate of the 1st instar larvae subjected to Mdlectin-C1 RNAi was detected after bacterial challenge. [ Results] The cDNA sequence of Mdlectin-C1 contains a 546 bp open reading frame encoding 181- amino-acid residues. A putative signal peptide of 21-amino-acid residues is present at the N-terminus. A conservative carbohydrate recognition domain （CRD） is observed in the mature peptide. The results of qRT-PCR and Western blot revealed that the expression level of Mdlectin-C1 was gradually enhanced fromthe 2nd instar larval stage to the pupal stage and reached the maximum value at the pupal stage. The expression level of Mdlectin-C1 was up-regulated significantly in larvae at 36 and 3 h after challenge with Gram-negative bacterium E. coli and Gram-positive bacterium Staphylococcus aureus, respectively, qRT- PCR analysis showed that the expression level of Mdlectin-C1 was knocked down successfully by RNAi. The survival rate of the 1st instar larvae subjected to Mdlectin-C1 RNAi was significantly lower than the normal control post bacterial challenge. [ Conclusion ] Mdlectin-C1 is involved in antimicrobial immune response in the housefly and may play important roles in this process.
出处
《昆虫学报》
CAS
CSCD
北大核心
2016年第1期16-24,共9页
Acta Entomologica Sinica
基金
河北省自然科学基金项目(C2015201013)
河北省教育厅项目(QN20131065)
河北大学自然科学基金项目(2015308)
关键词
家蝇
C-型凝集素
RNAI
细菌刺激
先天免疫
模式识别受体
Musca domestica
C-type lectin
RNAi
bacterial challenge
innate immune
pattern recognition receptor
