关键词: 家蝇, 抑制性消减杂交, cDNA消减文库, 差异表达基因, 半定量RT-PCR

Abstract: 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.

Key words: Musca domestica, suppression subtractive hybridization, cDNA subtractive library, differentially expressed genes, semi-quantitative RT-PCR