Role Of Ha-DFP1Gene In Innate Immunity Of The Cotton Bollworm(Helicoverpa Armigera)

Hemocytes play important roles in insect innate immune responses.The main celllular immune responses mediated by hemocytes are phagocytosis, encapsulation and nodulation. However, until now, molecular mechanisms for the regulation of celllular immune response are largely unknown. Recent transcriptome analysis of the cotton bollworm (Helicoverpa armigera) hemocytes identified a potential immune-related gene and named defense protein1(Ha-DFP1). In this paper, we used RNAi and other methods to investigate the function of Ha-DFP1gene in the innate immunity of H.armigera. The results are as follows.(1) Cloning and sequence analysis of Ha-DFP1gene.A Ha-DFP1cDNA was identified from the transcriptome of H. armigera hemocytes. The full-length cDNA sequence of Ha-DFP1is410bp long, containing a complete open reading frame (ORF) of204bp. The predicted protein encoded by Ha-DFP1was67-aa long. Multiple sequence alignment showed that the amino acid sequence of Ha-DFP1have a significant similarities with that of Danaus plexippus and Galleria mellonella JH-inducible protein (JHIP).(2) Tissue specific expression.RT-PCR was used to compare transcript abundance of Ha-DFPl in different tissues of H. armigera. The result showed that Ha-DFP1was expressed in all tested tissues (epidermis, midguts, fat bodies and hemocytes).(3) The expression of Ha-DFP1in the hemocytes of H. armigera after the injection of hormone.qRT-PCR was used to examine the expression levels of Ha-DFP1mRNAs in the hemocytes of H. armigera larvae after larvae were injected with hormone. The results showed that Ha-DFP1transcript levels in the hemocytes were up-regulated by methoprene, whereas the injection of20E had no influence on the expression levels of Ha-DFP1. This result suggested that Ha-DFP1might also be a kind of juvenile hormone induced expression gene.(4) The expression of Ha-DFP1in the hemocytes of H. armigera induced by beads challenge. qRT-PCR was used to examine the expression levels of Ha-DFP1mRNAs in the hemocytes of H. armigera larvae after larvae were injected with beads. The result show that the expression levels of Ha-DFP1in the hemocytes was up-regulated after the injection of beads. This result suggested that Ha-DFP1expression is inducible upon beads challenge.(5) The function assays of rHa-DFP1in vitro.Prokaryotic express system was used to obtain the recombinant protein rHa-DFP1.The result of the function assays showed that rHa-DFP1could promote hemocyte encapsulation in vitro. Agglutination test showed that rHa-DFP1had a strong agglutination activity against Escherichia coli in the presence of Zn2+. Moreover, rHa-DFP1could enhance the PO activity of plasma in vitro. The function assays in vitro suggested that Ha-DFP1may be involved in the celluar immunity and humoral immunity of H. armigera.(6) RNAi assays.RNAi was used to further analyze the roles of Ha-DFP1gene in H. armigera immunity. The results showed that the spreading ability of plasmatocytes and the encapsulation ability of hemocytes as well as the prophenoloxidase (PPO) activation in hemolymph were impaired when the expression of Ha-DFP1in H. armigera hemocytes was inhibited by dsHa-DFP1. These results further confirmed that Ha-DFP1gene may be involved in the encapsulation response and the PPO activation of H. armigera.In summary, Ha-DFP1gene was identified from H. armigera. In vitro and in vivo function assays indicated that Ha-DFP1gene plays important roles in H. armigera innate immunity, possibly by mediating encapsulation response and PPO activation system.