褐飞虱基因BphMIF1克隆及表达分析

Gene cloning and expression analysis of the Nilaparvata lugens(Hemiptera:Delphacidae)BphMIF1 gene

【目的】克隆水稻重要害虫褐飞虱Nilaparvata lugens唾液蛋白基因BphMIF1,探析BphMIF1在响应褐飞虱取食过程中的表达谱。【方法】采用RT-PCR法克隆了褐飞虱唾液蛋白基因BphMIF1,测序后对序列进行生物信息学分析,使用实时荧光定量PCR(qPCR)对褐飞虱1-5龄若虫及成虫以及不同成虫虫体部位BphMIF1的表达量进行分析,同时对BphMIF1进行了原核表达分析。【结果】克隆得到BphMIF1基因,编码119个氨基酸。qPCR结果表明BphMIF1基因在若虫4龄期表达会迅速上调,在成虫的头、胸、腹均有表达,以腹部表达量最高。原核表达结果显示褐飞虱BphMIF1基因以0.5 mmol∙L^-1的IPTG作为诱导浓度,诱导时间为4 h表达效果较好。【结论】BphMIF1基因在褐飞虱成虫头、胸、腹部均有表达,在若虫不同发育历期其表达量有所差异且在4龄若虫时期表达量有显著上调。该结果可为BphMIF1在褐飞虱取食过程中的功能研究奠定基础。

[Objectives]To investigate the expression profile of the saliva protein BphMIF1 gene of the brown planthopper,Nilaparvata lugens,an important insect pest of rice,in response to the feeding activity of this species.[Methods]BphMIF1 was cloned by RT-PCR and sequenced for bioinformatic analysis.The expression levels of BphMIF1 in nymphs(1st-5th instars),adults and different adult body parts,were analyzed with real-time fluorescent quantitative PCR(qPCR),and the prokaryotic expression of BphMIF1 was also quantified.[Results]The BphMIF1 gene was cloned and found to encode 119 amino acids.qPCR results showed that BphMIF1 was rapidly up-regulated in 4th instar nymphs.BphMIF1 was expressed in the head,thorax and abdomen of adults,but was most highly expressed in the abdomen.Prokaryotic expression analysis showed that the BphMIF1 gene was expressed at an induced concentration of 0.5 mmol/L IPTG for 4 h.[Conclusion]The BphMIF1 gene is differentially expressed in different age groups and body parts of N.lugens.These results provide a theoretical basis for the functional study of BphMIF1 in response to the feeding process of N.lugens.