家蝇幼虫消减文库的构建及差异表达基因的鉴定

为了鉴定家蝇Musca domestica免疫相关基因，应用抑制性消减杂交技术，构建刺激家蝇幼虫差异表达cDNA消减文库。以大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus诱导12 h的家蝇幼虫与未诱导的家蝇幼虫为消减杂交对象，获得了差异表达基因的cDNA片段，将其与T/A载体连接并转化大肠杆菌DH5α，构建了刺激家蝇幼虫cDNA消减文库。PCR检测发现，文库的阳性克隆中插入的cDNA片段大小在200~1 000 bp之间，随机挑选了161个含大小不等差异片段的克隆进行测序和同源性分析，鉴定了36种蛋白的基因片段，包括抗菌肽、酶、核糖体蛋白、其他功能蛋白以及功能不明的蛋白。用半定量RT-PCR分析了其中6种蛋白基因的表达，结果显示：防御素和攻击素基因在细菌刺激后24 h内明显上调表达，而溶菌酶、酚氧化酶原活化因子、糜蛋白酶和蛋白质合成起始因子基因在细菌刺激后0-4 h内表达受抑制，12 h后上调表达。该研究结果为家蝇免疫相关基因的克隆和家蝇免疫防御机制的探讨奠定了良好的基础。

Construction of a suppression subtractive hybridization cDNA library to identify differentially expressed genes from Musca domestica (Diptera:Muscidae) larvae

To identify immune-related genes in housefly (Musca domestica) larvae, suppression subtractive hybridization (SSH) was performed to generate a subtracted cDNA library between bacteria-challenged (Escherichia coli and Staphylococcus aureus mixture) housefly larvae (Tester) and control housefly larvae (Driver) using the PCR Select^TM cDNA Subtraction Kit according to the manufacturer’s protocol. The subtracted target cDNAs were ligated into the pGEM-T-Easy vector using T4 DNA ligase and transformed into E.coli DH5α competent cells. Positive white clones were randomly selected and sequenced after PCR detection. PCR analysis showed that the white bacteria clones contained inserts of 200-1 000 bp. The sequences inserted were used to search GenBank with BLASTX. A series of ESTs of 36 kinds of proteins including antibacterial peptides, enzymes, ribosome proteins and other functional proteins as well as unknown proteins, were isolated from 161 clones sequenced randomly. RT-PCR analysis revealed that two differentially expressed genes, defensin gene and attacin gene, were up-regulated in housefly larvae challenged for 24 h, while the genes encoding other proteins like lysozyme 1, prophenoloxidase activating factor, chymotrypsin and eukaryotic translation initiation factor were first down-regulated in housefly larvae challenged for 0-4 h and then up-regulated at 12 h post treatment. These results have established a solid foundation for cloning immune-relevant genes from M. domestica and further studying immune mechanism in housefly.