Cloning, Identification And Expression Analysis Of Immune Factor Apdorsal From Antheraea Pernyi

Antheraea pernyi is a kind of important economic insect. Every year, there is huge economic losses due to the disease of Antheraea pernyi. Therefore, there is growing concern with the immunity of Antheraea pernyi. Studies had suggested that insect, when are infected by different microorganism, can induce the expression of different antimicrobial peptides which are mainly regulated by two different signaling pathways:Toll pathway and Imd pathway. The two pathways control the expression of different antimicrobial peptides through Rel/NF-KB. Rel/NF-KB family protein is an important transcription factor, which plays a central role in immune response, inflammation, cell apoptosis, growth and development.Firstly, degenerate primers were designed according to conserved domain of the sequence of the Rel/NF-KB from other species which have already reported by GeneBank. The full length of two cDNAs encoding NF-κB genes were cloned from Antheraea pernyi using RACE technology, and named ApdorsalA and ApdorsalB respectively. ApDorsalA and ApDorsalB contain classic features of Rel family protein-RHD after BLAST. Comparison and phylogenic analysis between the amino acid sequence of ApDorsalA and other Rel proteins showed that ApDorsal have a high similarity to those of other animal Rel proteins. ApDorsal have a higher evolutionary relationship with the Dorsal of Bombyx mori and maduca sexta which belong to the Dorsal class of Rel family proteins, suggesting that they have been derived from a common ancestral gene.Secondly, the quantitative real-time RT-PCR was applied to analyze the distribution of Apdorsal in different tissues and different pathogens infecting. The results showed that expression of Apdorsal exists in all tissues and is the highest in the epidermis and fat body before immunity. The expression of Apdorsal was increased after different pathogens challenged in epidermis and fat body, but there were no changed in trachea, hemocyte, testicle and brain. After injected with NPV, B.subtilis and Yeast, Apdorsal expression of fat body had significantly increased. However, expression of Apdorsal has no obvious changed after injected with E.coli, indicating that Apdorsal is probably involved in fat body immune response against virus, fungi, gram-positive bacterium in Antheraea pernyi, but not in Gram-negative bacteria.At last, the antimicrobial activity of antimicrobial peptides against Gram-negative bacteria, which were produced by Antheraea pernyi in different pathogens, was detected by using of inhibition zone. The results show that E.coli and B.subtilis can induce the expression of anti Gram-negative bacteria peptides, and antibacterial activity of E.coli is the better, and the B.subtilis'is poorer. There is not obvious inhibition zone by induced with Yeast and the NPV. Those results suggest that the immune signaling pathway to gram-negative bacteria is different from the other immune signaling pathways to gram-positive bacteria, fungi and viruses. This also further proved that Apdorsal is involved in the immune response against virus, fungi, gram-positive bacterium in Antheraea pernyi, but not in Gram-negative bacteria.