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PBX and Dfd cooperatively regulate stage-specific expression of 30 K protein BmLP1 in Bombyx mori
Qingqing Linghu1, Haoyun Li1, Jiahui Wan1
1Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Biological Science Research Center, Southwest University, Chongqing, China.
Abstract:
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.
Insights
Juvenile hormone (JH) regulates insect development. In silkworms, transcription factors Dfd and PBX bind to the Bmlp1 gene promoter, controlling nutrient storage protein synthesis during metamorphosis.
Area of Science:
- Entomology
- Molecular Biology
- Developmental Biology
Background:
- Nutrient accumulation is critical for insect metamorphosis.
- Forty-six 30K proteins (30KPs) are involved in nutrient storage in silkworms (Bombyx mori).
- Juvenile hormone (JH) is thought to regulate stage-specific 30KP expression, but mechanisms are unclear.
Purpose of the Study:
- Investigate the regulation of the 30KP gene Bmlp1 during silkworm development.
- Elucidate the role of JH and specific transcription factors in Bmlp1 expression.
Main Methods:
- Gene expression analysis of Bmlp1 in response to JH analogue.
- Identification of cis-response elements (CREs) in the Bmlp1 promoter.
- Chromatin immunoprecipitation and protein-protein interaction assays (bimolecular fluorescence complementation, GST pull-down) to study Dfd and PBX binding and interaction.
- Overexpression and knockdown experiments in BmN cells to assess the functional impact of Dfd and PBX on Bmlp1 expression.
Main Results:
- Bmlp1 expression occurs after Day 3 of the fifth instar and is repressed by a JH analogue.
- A CRE in the Bmlp1 promoter binds Dfd, with PBX binding to an adjacent CRE.
- Dfd and PBX interact and synergistically regulate Bmlp1 transcription.
- Overexpression of Dfd and PBX down-regulates Bmlp1, while knockdown up-regulates it.
Conclusions:
- The transcription factor Dfd, along with cofactor PBX, synergistically controls Bmlp1 expression in Bombyx mori.
- This provides insight into JH-mediated regulation of insect development and nutrient storage protein synthesis.
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