[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

A secreted phospholipase A2 (BmsPLA2 ) regulates melanization of immunity through BmDDC in the silkworm Bombyx mori.

Insect immune-associated phospholipase A2 (PLA2 ) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA2 on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA2 gene (BmsPLA2 ), and its protein had characteristic features of secreted PLA2 (sPLA2 ). After injection of bacteria, BmsPLA2 expression and sPLA2 activity in hemolymph significantly increased. BmsPLA2 fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA2 was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA2 by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA2 overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA2 inhibitor dosage. These results demonstrated that BmsPLA2 plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA2 after injection of bacteria, implying that BmsPLA2 was activated prior to BmDDC. Moreover, the alteration of BmsPLA2 by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA2 regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects.