关键词: 德国小蠊, 大肠杆菌, 酚氧化酶, 同源克隆, 免疫应答

Abstract: 【Aim】 In this study, we aim to identify the function of phenoloxidase in the immune response in Blattella germanica induced by Escherichia coli. 【Methods】 A full-length cDNA of B. germanica phenoleoxidase gene (BgPO) was cloned using homology-based cloning and rapid amplification of cDNA ends (RACE). The phylogenetic tree of BgPO and PO proteins from other insects was constructed by the software MEGA 5.1. The tissue expression patterns of BgPO and its transcription levels in hemocytes at different time points post E. coli infection were detected by RT-PCR. The antibacterial activity of hemolymph was measured by Hultmark’s method and the BgPO enzyme activity was determined by catechol method. 【Results】The full-length cDNA sequence of BgPO is 2 252 bp (GenBank accession number: KJ789157), which contains an ORF of 2 085 bp encoding 695 amino acid- residues. The deduced protein has a calculated molecular weight of 79.7 kDa and the theoretical pI of 6.19. The sequence alignment analysis showed that BgPO has high sequence identities with the homologies of other insect species, and shares an 80% amino acid sequence identity with the PO from Coptotermes formosanus. Phylogenetic tree analysis indicated that BgPO has the closest genetic relationship with the PO of C. formosanus. Gene expression profiling showed that BgPO was expressed mainly in the hemocytes and epidermis. The transcription level of BgPO mRNA increased from 24 h post E. coli infection, and reached a peak at 36 h p.i. The antibacterial activity and phenoloxidase activity increased after bacterial induction during 6-36 h p.i. The antibacterial activity showed significant differences 〖JP4〗between the bacteria-infected and the PBS-induced samples during 6-48 h p.i. (P<0.05), and the phenoloxidase activity also showed significant differences between the bacteria-induced and the PBS-induced samples during 24-36 h p.i. (P<0.05). 【Conclusion】Our findings revealed that BgPO is mainly expressed in the hemocytes and epidermis. BgPO may participate in the immune response against E. coli invasion. The results provide a good foundation for the further research on immune response of BgPO in B. germanica.

Key words: Blattella germanica, Escherichia coli, phenoloxidase, homology-based cloning, immune response