PBX and Dfd cooperatively regulate stage‐specific expression of 30 K protein BmLP1 in <i>Bombyx mori</i>

Nutrient accumulation is essential for insect metamorphosis. As a group of important nutrient-storage proteins, forty-six 30 K proteins (30KPs), including BmLP1-BmLP46, have been identified in the silkworm, Bombyx mori. Most 30KPs are synthesised in the last instar larvae, and the stage-specific expression of 30KPs is believed to be regulated by juvenile hormone (JH)-dependent pathways; however, the specific regulatory mechanism remains unclear. In this study, we found that a 30KP gene Bmlp1 was expressed after Day 3 of the fifth instar, and its expression was down-regulated by JH analogue. We also identified a cis-response element (CRE) on the promoter of Bmlp1. Dfd was determined to bind to this CRE adjacent to another CRE that serves as a binding site for PBX. Dfd is a HOX transcription factor found to exhibit an expression pattern similar to that of PBX. The interaction between PBX and Dfd was confirmed using bimolecular fluorescence complementation and GST pull-down experiments. The expression of Bmlp1 was down-regulated when PBX and Dfd were overexpressed in BmN cells, whereas it was up-regulated when PBX and Dfd were knocked down in BmN cells. Our data show that the transcription factor Dfd, and the cofactor PBX, synergistically regulate the transcription of Bmlp1 in B. mori. This study provides a reference for an in-depth understanding of the regulation of insect development mediated by JH.