亚洲玉米螟C型凝集素CTL6的克隆和功能分析  

Molecular cloning and functional analysis of C-type lectin 6( CTL6 ) in Ostrinia furnacalis( Lepidoptera: Crambidae)

作　　者：沈东旭[1] 初源[1] 洪芳[1] 刘杨[2] 王桂荣[2] 安春菊[1] 

机构地区：[1]中国农业大学农学与生物技术学院,北京100193 [2]中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京100193

出　　处：《昆虫学报》2015年第11期1177-1185,共9页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31201578;31172090)

摘　　要：【目的】鉴定出一种新的亚洲玉米螟Ostrinia furnacalis(Guenée)C型凝集素(C-type lectin),并对其功能进行初步研究。【方法】通过生物信息学分析,从亚洲玉米螟转录组中筛选得到一个可能的C型凝集素基因,命名为CTL6。利用RT-PCR技术分析该基因在亚洲玉米螟不同龄期、不同组织及不同病原物诱导下的表达模式。借助原核及杆状病毒真核表达系统产生重组CTL6蛋白,并利用细菌凝集实验对其功能进行初步研究。【结果】CTL6基因c DNA全长序列为1 034 nt,其中完整开放阅读框为945 nt。推导的CTL6多肽序列包括314个氨基酸残基,N端含有由22个氨基酸残基组成的信号肽。CTL6成熟肽中含有两个串联的糖识别结构域,与烟草天蛾Manduca sexta的IML-2(Immulectin-2)同源性最高。RT-PCR结果显示,CTL6在亚洲玉米螟5龄幼虫期转录水平最高,卵期其次,不同组织中则是在脂肪体中转录水平最高,呈诱导性表达。纯化的CTL6重组蛋白对大肠杆菌Escherichia coli具有一定的凝集作用。【结论】鉴定到的亚洲玉米螟CTL6是一种典型的C型凝集素,重组CTL6蛋白可能参与了亚洲玉米螟对病原菌的凝集作用。[Aim]This study aims to identify a C-type lectin in Ostrinia furnacalis（ Guenée） and to investigate its function. [Methods ] A potential C-type lectin gene was identified from O. furnacalis transcriptome with bioinformatics analysis and designated as CTL6. Its expression profiles in different developmental stages and tissues and after different pathogen induction in this insect were analyzed with RT-PCR. The recombinant CTL6 protein was produced in the prokaryotic or baculovirus eukaryotic expression system,and its function was investigated preliminarily with bacterial agglutination assay.[Results]The full-length c DNA of CTL6 is 1 034 nt,with an open reading frame of 945 nt. The deduced protein consists of 314 amino acid residues,including a predicted 22-residue secretion signal peptide.The mature CTL6 peptide contains two carbohydrate recognition domains. Database search indicated that O. furnacalis CTL6 has the highest amino acid sequence identity to IML-2（ Immulectin-2） from Manduca sexta. RT-PCR results showed that CTL6 was transcribed at the highest level in the 5th instar larval stage,followed by the egg stage,and its expression level in the fat body was the highest among the tested tissues. CTL6 mRNA level obviously increased after bacterial or fungal injection. Agglutination assay revealed that the purified CTL6 recombinant protein played a role in the agglutination of Escherichia coli.[Conclusion]The identified O. furnacalis CTL6 is a typical C-type lectin,which may be involved in the agglutination response of O. furnacalis against pathogens.

关 键 词：亚洲玉米螟 C型凝集素 CTL6 天然免疫反应 凝集作用 

分 类 号：Q966[生物学—昆虫学]