Biochemical characterization and functional analysis of the POU transcription factor POU-M2 of Bombyx mori.

POU-M2 is a homeodomain transcription factor which plays important roles in the development and silk synthesis of Bombyx mori. In this study, we expressed, purified and characterized POU-M2 and studied its transcription regulation on fibroin heavy chain gene of Bombyx mori. Gel filtration showed POU-M2 existed as a dimer in solution. Far-UV circular dichroism spectra indicated POU-M2 had a well-defined α-helix structure and the α-helix content was about 26.4%. The thermal unfolding transition of POU-M2 was a cooperative process. Tm, ΔH and ΔS were 45.15±0.2°C, 138.4±0.5KJ/mol and 0.4349±0.04KJ/(mol·K), respectively. Western blotting analysis indicated the expression level of POU-M2 increased slightly from day 3 to day 7 of the fifth instar larvae in the posterior silk gland. POU-M2 was positioned in the nucleus of cells. The luciferase reporter assay demonstrated POU-M2 could stimulate the promoter activity of fibroin heavy chain gene, and the activation effect was dependent on the amount of POU-M2. Our study suggested POU-M2 may be involved in the transcriptional regulation of fibroin heavy chain gene. These findings expand toward a better understanding of the structure of POU-M2 and its function in silk synthesis of Bombyx mori.

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