家蝇抗菌肽Attacin-2基因的克隆、序列分析和诱导表达

Cloning,sequence analysis and induced expression of attacin-2 gene in housefly (Musca domestica)

攻击素(attacin)作为昆虫抗菌肽之一,在昆虫的先天免疫中起着重要作用。本研究通过家蝇Musca domesticaEST序列筛选并结合RACE技术克隆了家蝇的Attacin-2基因(Mdatta2)cDNA序列。其全长819 bp,包含一个726 bp的完整开放阅读框(open reading frame,ORF),以及42 bp的5'末端非翻译区(5'UTR)和51 bp的3'末端非翻译区(3'UTR),编码241个氨基酸残基,推导的多肽N端22个氨基酸残基为信号肽序列。同源性分析表明,其氨基酸序列与嗜凤梨果蝇Drosophila ananassae的Attacin一致性最高(46%)。以邻接法(Neighbor-Joining,NJ)构建的系统关系表明,家蝇的Attacin-2与其他双翅目昆虫的Attacin起源于共同的祖先,属于Attain_C超家族。应用实时荧光定量PCR的方法研究家蝇幼虫在受到外源细菌刺激时Mdatta2基因的表达,结果显示,在大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus分别刺激后3 h和6 h,家蝇幼虫Mdatta2表达量出现显著上调。Mdatta2基因在家蝇幼虫体内呈诱导型表达,表达水平随诱导时间的不同而变化,提示Mdatta2基因可能在家蝇免疫防御过程中起着重要作用。

As a member of the antimicrobial peptide family,attacin plays a key role in insect innate immune system.In this study,a cDNA of 819 bp encoding attacin-2 was cloned from housefly（Musca domestica） by RACE based on the EST information,and named Mdatta2.The cDNA sequence contains a 726 bp open reading frame（ORF） encoding 241 amino acid residues and is flanked by a 42 bp 5＇ UTR and a 51 bp 3＇ UTR.The deduced peptide contains a putative signal peptide of 22 amino acids.The amino acid sequence of Mdatta2 showed the highest identity（46%） with that of Drosophila ananassae by Blastp analysis.The phylogenetic tree indicates that the attacin-2 from housefly and those from other dipterans are descended from a single common ancestor and belong to Attain_C superfamily.The expression of Mdatta2 transcript was measured by real-time PCR.The results demonstrated that after housefly larvae were challenged with Escherichia coli and Staphylococcus aureus,respectively,the mRNA level of Mdatta2 at 3 h and 6 h post-challenge was up-regulated and significantly higher than that of the blank control.The expression of Mdatta2 is inducible,and its expression level varies with the induction time,suggesting that Mdatta2 may play an important role in housefly defensive system.