摘要
【目的】鉴定一种新的家蝇Musca domestica防御素基因,并分析其功能。【方法】从家蝇转录组数据库中鉴定了1条新的防御素基因cDNA序列,并将其命名为家蝇防御素1(Md-defensin-1)基因Mdde-1。利用生物信息学网站、软件预测其结构等信息。以实时荧光定量PCR技术研究该基因的表达模式,并且利用基因步移技术获得了启动子序列,同时采取细胞转染技术验证Mdde-1启动子活性。【结果】该序列包含一个276 bp的开放阅读框,编码91个氨基酸残基。推导的氨基酸序列N端包括1个23个氨基酸残基的信号肽和1个28个氨基酸残基的前肽。成熟肽由40个氨基酸残基组成,含有1个典型的CSαβ基序。实时荧光定量PCR结果显示,家蝇2龄幼虫受金黄色葡萄球菌Staphylococcus aureus(G+)刺激后Mdde-1表达明显上调,而大肠杆菌Escherichia coli(G-)刺激后表达下调;Mdde-1在家蝇幼虫受到热激时呈上调表达。为进一步研究其调控机制,克隆了Mdde-1启动子,并证明了该启动子具有活性。【结论】据此认为Mdde-1是一种新的家蝇防御素,并且在免疫革兰氏阳性菌方面发挥重要作用;同时我们首先证明了Mdde-1的启动子具有活性。本研究为进一步研究家蝇防御素的作用机制奠定了基础。
[ Aim] Identify a new defensin gene from the housefly, Musca domestica, and analyze its function. [ Methods ] We identified the cDNA sequence of a new defensin gene from M. domestica transcriptome database and named Md-defensin-1 gene （Mdde-1）. Bioinformatics web site and software were used to predict the structure information and real-time fluorescent quantitative PCR technique to study the gene expression profiles. Meanwhile, Mdde-1 promoter sequence was obtained using genome walking method and its activity was validated using cell transfection technology. [ Results] The Mdde-1 sequence contains a 276 bp open reading frame, encoding 91 amino acid residues. A putative signal peptide of 23 amino acid residues and a potential propeptide of 28 amino acid residues are present at the N-terminus followed by a mature peptide of 40 amino acid residues, which contains a typical CSmotif. The results of real-time quantitative PCR indicated that the expression level of Mdde-1 was up-regulated significantly after the 2nd instar larvae of M. domestica were challenged by the gram positive bacterium Staphylococcus aureus while down-regulated after they were challenged by the gram negative bacterium Escherichia coll. The expression of Mdde-1 was up-regulated after the housefly larvae suffered heat shock. In order to further study its regulatory mechanism, we cloned Mdde-1 prompter and verified its activity. [ Conclusion] The results suggest that Mdde-1 is a new defensin from housefly, and it plays an important role in immune challenge by gram-positive bacteria. Mdde-1 promoter activity has been first proved. This study lays the foundation for further research on antibacterial mechanism of housefly dcfensin.
出处
《昆虫学报》
CAS
CSCD
北大核心
2013年第8期854-863,共10页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31101669)
教育部高等学校博士学科点专项科研基金项目(20101301120005)
河北省自然科学基金项目(C2011201027)
关键词
家蝇
先天免疫
抗菌肽
防御素
基因克隆
启动子
Musca domestica
innate immunity
antimicrobial peptide
defensin
genecloning
promoter
