RESUMO
Vitellogenin receptors (VgRs) transport vitellogenin (Vg) into oocytes, thereby promoting egg growth and embryonic development. VgRs recognize and transport multiple ligands in oviparous animals, but their role in insects is rarely reported. In this study, we investigated whether Bombyx mori VgR (BmVgR) binds and transports lipoprotein-1 (BmLP1) and lipoprotein-7 (BmLP7) of the 30 kDa lipoproteins (30 K proteins), which are essential for egg formation and embryonic development in B. mori. Protein sequence analysis showed BmLP7, similar to reported lipoprotein-3 (BmLP3), contains the cell-penetrating peptides and Cysteine position, while BmLP1 has not. Assays using Spodoptera frugiperda ovary cells (sf9) indicated the direct entry of BmLP7 into the cells, whereas BmLP1 failed to enter. However, co-immunoprecipitation (Co-IP) assays indicated that BmVgR could bind BmLP1. Western blotting and immunofluorescence assays further revealed that over-expressed BmVgR could transport BmLP1 into sf9 cells. Co-IP assays showed that SE11C (comprising LBD1+EGF1+OTC domains of BmVgR) or SE22C (comprising LBD2+EGF2+OTC domains of BmVgR) could bind BmLP1. Over-expressed SE11C or SE22C could also transport BmLP1 into sf9 cells. Western blotting revealed that the ability of SE11C to transport BmLP1 might be stronger than that of SE22C. In the vit mutant with BmVgR gene mutation (vit/vit), SDS-PAGE and western blotting showed the content of BmLP1 in the ovary, like BmVg, was lower than that in the normal silkworm. When transgenic with hsp70 promoter over-expressed BmVgR in the vit mutant, we found that the phenotype of the vit mutant was partly rescued after heat treatment. And contents of BmLP1 and BmVg in vit mutant over-expressed BmVgR were higher than in the vit mutant. We conclude that BmVgR and its two repeat domains could bind and transport BmLP1 into the oocytes of the silkworm, besides BmVg. These results will provide a reference for studying the molecular mechanism of VgR transporting ligands in insects.
RESUMO
GATA transcription factors (GATAs) are widely expressed among various organisms and belong to the zinc finger protein family. GATA transcription factors play important roles in the proliferation, differentiation, and development of eukaryotes. Previous studies have shown that GATA participates in oogenesis by selective splicing in silkworms. In this study, we investigated the function of GATAs during vitellogenesis using female silkworms (Bombyx mori). Six types of GATA transcription factors were successfully cloned in the fat body of silkworms during the wandering stage and only BmGATAß4 induced the activity of the Bombyx mori vitellogenin (BmVg) promoter. Furthermore, BmVg and BmGATAß4 exhibited similar expression patterns in the fat body of female silkworms during the wandering stage. Electrophoretic mobility shift assays, cell transfection assays, and chromatin immunoprecipitation showed that BmGATAß4 was involved in regulating the transcription of BmVg by directly binding to the GATA cis-response element 1 (CRE1) and GATA cis-response element 2 (CRE2) in the promoter of the BmVg gene. RNA interference of BmGATAß4 in female silkworms downregulated BmVg transcription, resulting in a decrease in egg size and shortening of the length of egg tubes relative to the control. In summary, our results indicated that BmGATAß4 bound to the GATA CRE1 and CRE2 motifs in the BmVg promoter to upregulate BmVg expression in the fat body of female silkworms.