| In the silkworm, Bombyx mori (B. mori), pigments from the mulberry leaves are absorbed in the midgut lumen by the midgut epithelium and through the mi dgut into the hemolymph, then transfered into the silkgland, where the pigments are accumulated in the layers of the cocoon sericine, then the coulorful cocoons are shaped. The main pigments of yellow-red cocoon strains of silkworm include lutein,β-carotene, a-carotene and violaxanthin. The the best well studied mecha nism among the yellow-red cocoon strains is that how the yellow cocoon produc ed, in which the main carotenoids of cocoons are lutein, and thenβ-carotene, an d 3 genes involved:Y, I, C. The Y gene colors the hemolymph yellow and cont rol the uptake of carotenoids from the midgut lumen into the midgut epithelium.The I gene is a suppressor gene that down-regulates the yellow coloring of the hemolymph and controls carotenoid transfer from the midgut epithelium into the hemolymph. The C gene colors the cocoon golden-yellow by controlling the upt ake of carotenoids from the hemolymph to the silk gland. Only the combination of Y+IC can produce yellow cocoon. The Y gene encodes CBP, and C gene con trols expression of Cameo2.CBP have a START domain and binds lutein in a 1:1 molar ratio. In mam mals, the START domain involves in the absorption and metabolism of many lip ids, including cholesterol, phosphatidyl choline, ceramide and lutein. Traditional g enetic analysis suggests that Y gene controls the carotenoid transport in the mdig ut of silkworm, however, new evidences show that CBP, the product of Y gene, has protein expression in both midgut and silkgland, indicating the possibility of its carotenoid transport function in both of the two tissues. Besides, the Y gene plays a central role in the formation of several different color strains of yellow-red cocoon when the cocoon is being produced, indicating that it may involve in transport of different carotenoids, therefore, CBP may bind various carotenoids.Camoe2 is homologous to mammalian SR-B Iand ninaD of Drosophila, both of which are CD36 gene family members. SR-B I is HDL receptor in mammal s, and it involves in the absorption and transport of many lipids, including chole sterol, lutein, zeaxanthin,β-carotene, vitamin E. NinaD involves in absorption an d transport of lutein, zeaxanthin,β-carotene in Drosophila. The Camoe2 that enc oded by C gene, has mRNA transcription in both midgut and silkgland.Considering all these above, questions are raised that whether CBP or Camo e2 has the carotenoid transport function? If yes, do they have a cooperative fun ction on it? Do their proteins have physical interactions? Do the homologene of Cameo2, Cameo 1 has the carotenoid transport related function? Some studies we re accomplished following these questions and the results are as follows.1. The correlation of mRNA transcription of Cameol, Cameo2 and CBP with th-e lutein transport in silkwormRNA was abstracted from the 3-6 days of midgut and silkgland, the last lar va instar of silkworm, both of which are the key tissues that control the caroten oid transport, reverse transcription to cDNA was accomplished.RT-PCR shows that in the strains(03-210、Jianpuzhai and 02-320) of silkwor m that cocoon mainly pigmented with lutein, Cameo1, Cameo2 and CBP have a co-existence mRNA transcription in the midgut and silkgland. However, in the sil kworm strains(03-520、Dazao and Qiubai) that cocoon mainly pigmented with ot her carotenoids rather than lutein, Cameo1, Cameo2 and CBP are not transcripte d in both midgut and silkgland at the same time, suggestting that Cameo1, Cam eo2 and CBP may participate in the transport of lutein in both midgut and silkg land at the same time.2. The cellular carotenoid transport function of carotenoid transport related protei ns of silkwormCameo1, Cameo2, CBP, cbp (deletion form of CBP) were cloned into euk aryotic expression plasmids, and transfected into HEK293 cells. The result indicat es that Cameol and Cameo2 are located on the plasma membrane, while CBP a nd cbp are located in the whole cell region. Lutein and (3-carotene were added t o the medium and incubated with the transfected cells, cells were collected and sonicated, carotenoids were extracted and measured with HPLC. Data was analyz ed with SPSS 17.0. The amount of lutein in Cameo2+CBP expressing cells is 2. 71 times as much as the EGFP expressing cells, showing significant difference (P =0<0.01). The saturation and concentration-dependence of the amount of accumu lated lutein in the cells indicates that it is a carrier conducted transport process of lutein transport by Cameo2 and CBP. However, these proteins have no such f unction on theβ-carotene(P>0.05).3. Preliminary investigation of physical interactions of silkworm carotenoid trans port related proteinsCameo1 and Cameo2 were cloned into eukaryotic expression plasmid pBiFC-VC155(HA-CMV backbone), CBP and cbp were cloned into pBiFC-VN173(FLA G-CMV backbone), plasmid combinations of CBP with Cameo1, CBP with Came o2, cbp with Cameo1, cbp with Cameo2, were co-transfected into the HEK293 c ells. Fluorescences shows that there may be protein-protein interactions or inter-a pproachings between CBP and Cameo1, CBP and Cameo2 at the plasma region.In summary, our results show that Cameo2 and CBP may have a protein-pr otein interaction or inter-approaching and they have a cooperation on the transpor t of lutein from extracellularly to intracellularly, while the function of Cameol r emains elusive. Based on the results, we speculate that extracelluler luteins are c aptured by Cameo2 on the cell membrane, then transfered to the intracellular lut ein binding protein CBP, and then perform its physiological functions or accumul ate in the cell or are transported away; The protein-protein interaction or inter-ap proaching of Cameo2 and CBP at the plasma region may play a role of lipid(lut ein) transfering; The transport of lutein in process of yellow cocoon formation m ay need the coexistence and cooperation of both Cameo2 and CBP in the midgu t and silkgland of silkworm. |
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