›› 2013, Vol. 56 ›› Issue (8): 854-863.

• 研究论文 • 上一篇    下一篇

家蝇Denfensin-1基因的克隆、诱导表达及启动子活性分析

卢丹1,郑立2,王欣欣2, 王凡2, 唐婷2, 柳峰松2,*   

  1. (1. 河北大学学生处, 河北保定 071002;  2. 河北大学生命科学学院, 河北保定 071002)
  • 出版日期:2013-08-20 发布日期:2013-08-20

Cloning, induced expression and promoter activity of defensin-1 gene from the housefly (Musca domestica)

LU Dan1, ZHENG Li2, WANG Xin-Xin2, WANG Fan2, TANG Ting2, LIU Feng-Song2,*   

  1. (1. Students’ Affairs Division, Hebei University, Baoding, Hebei 071002, China; 2. College of Life Sciences, Hebei University, Baoding, Hebei 071002, China)
  • Online:2013-08-20 Published:2013-08-20

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。

关键词: 家蝇, 先天免疫, 抗菌肽, 防御素, 基因克隆, 启动子

Abstract: 【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 CSαβ motif. 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 coli. 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 defensin.

Key words: Musca domestica, innate immunity, antimicrobial peptide, defensin, gene cloning, promoter