Figure 1.
The Genotypes and Colors of Silk Glands and Cocoons in Bombyx mori.
The silk glands from Qiubai, Dazao, Jianpuzhai and 03-520, were obtained from last instar larvae stage at 5 days of age. The colors of silk glands are similar as the colors of cocoons from the same strains. The white bars indicate 1 cm.
Table 1.
PCR Primers for RT-PCR and the Construction of pcDNA3.1 B/pEGFP-N1/pBiFC Expression Vectors.
Figure 2.
Protein Sequence Comparison Between CBP and cbp.
There was an absence of 79 amino acids on the N-terminal of cbp compared to CBP. The boxed area represents the region of steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. The black highlight areas indicate mutation of amino acids residues in cbp compared to CBP.
Table 2.
Restriction Endonucleases for the Construction of pcDNA3.1 B, pEGFP-N1, pBiFC-VC155 and pBiFC-VC173 Vectors.
Figure 3.
The mRNA Expressions of Cameo1, Cameo2, CBP and cbp in Midguts and Silk Glands in Bombyx mori.
The midguts and silk glands were obtained from last instar larvae stage at 3, 4, 5, and 6 days of age. CBP mRNA was expressed in both midguts and silk glands from Jianpuzhai and 03-520; Cameo2 mRNA was expressed in midguts and silk glands from all strains except the silk glands from 03-520.
Figure 4.
Carotenoids Content in Tissues and Cocoons from Qiubai, Dazao, Jianpuzhai and 03-520 strains.
The midguts, hemolymph and silk glands were obtained from last instar larvae stage at 5 days of age. In these four Bombyx mori strains, the types of major carotenoids in silk glands were consistent with cocoons. Qiubai and Dazao hardly showed carotenoids in their hemolymph, silk glands and cocoons. The major coloring pigment in silk glands and cocoons from Jianpuzhai was lutein, but 03-520 was β-carotene.
Figure 5.
Immunoblots of Cameo1, Cameo2, CBP and cbp in the HEK293 Transfected with Various Combination.
Markers of protein molecular weight are indicated on the left.
Figure 6.
Lutein Concentrations in the HEK293 Cells Transfected with Various Combination of Cameo1, Cameo2, CBP and cbp.
The cells expressing CBP had slightly higher lutein concentration (P<0.05) than the cells transfected with empty vector, EGFP and cbp. The cells expressing Cameo2 had higher lutein concentration (P<0.05) only than the cells transfected with empty vector. The lutein concentration in the cells expressing Cameo2+CBP was 2 fold higher (P<0.01) than other transfected cells. Different letters represent significant difference.
Figure 7.
The Absorption Rate of Lutein Associated with Time of Incubation and Concentration Gradient of Lutein in Transfected HEK293 Cells.
(A) Time course of cellular uptake of lutein into the transfected HEK293 cells. The lutein absorption rate in the cells expressing Cameo2+CBP increased with the prolonged incubation time and achieved plateau after 8 h incubation. (B) Effect of lutein concentration on the cellular uptake of lutein. The absorption rate of lutein was positively related to the lutein concentration in cells expressing Cameo2+CBP, and reached plateau at higher lutein concentration (8–16 µM).
Figure 8.
Subcellular Localization of Cameo1, Cameo2, CBP and cbp in Transfected HEK293 Cells.
(A) Protein primary structures of the Cameo1 Cameo2, CBP and cbp. The grid boxes represent predicted transmembrane domains. Both Cameo1 and Cameo2 contain two transmembrane regions on each near C- and N-terminal and a putative large extracellular domain. The value represents the number of amino acid residues in protein. (B) The fluorescence of HEK293 cells expressing CBP-EGFP+cbp-His and Cameo1-His+Cameo2-EGFP. The labeling of recombinant expressed proteins with His tag was detected with an antibody raised against the His tag epitope of the proteins and Cy3 conjugated secondary antibody (red). Fusion proteins with EGFP tag glows green. Cell nuclei were stained with DAPI (blue). The white bars indicate 5 µm. The fluorescence of Cameo1-His and Cameo2-EGFP was detected only on the plasma and nuclear membranes in transfected HEK293. In contrast, the fluorescence of CBP-EGFP and cbp-His was mainly presented in the cytosol. (C) Immunoblots of CBP-EGFP, cbp-His, Cameo2-EGFP and Cameo1-His in membrane fractions and cytosol fractions from transfected HEK293 Cells. Cameo1 and Cameo2 were expressed only in the membrane-rich fractions, while CBP and cbp were expressed only in cytosol fractions.
Figure 9.
Protein-protein Interactions of Cameo1+CBP and Cameo2+CBP from Bimolecular Fluorescence Complementation Analysis.
Cell nuclei were stained by DAPI (blue). Yellow fluorescence indicated that two separated non-fluorescent fragments can interact with each other to form complete yellow fluorescent proteins. The yellow fluorescence was detected in the HEK293 cells expressing Cameo1+CBP or Cameo2+CBP. YFP represents yellow fluorescent protein. The white bars indicate 25 µm.