Research Of Color Formation Mechanism In Yellow-red Cocoon From Silkworm,Bombyx Mori

Silkworms with colored cocoons are divided into two categories:yellow-red cocoon and greed cocoon varieties, according to the types and contents of main colored pigments. Carotenoids are the main coloring pigments in yellow-red cocoons. Silkworms can’t synthesis carotenoids by themselves, but they can be absorbed from mulberry leaves. This process of carotenoids from the midgut into the silk gland via hemolymph and combining with sericin is controlled by many genes, such as yellow blood gene (Y), yellow blood inhibitor gene (I), yellow cocoon gene (C), and fresh cocoon gene (F) and so on.CBP, Cameo2and SCRB15are the product of Y, C and F, respectively. Cameo2controls the formation of yellow cocoon with CBP; SCRB15controls the formation of fresh cocoon with CBP. However, yellow-red cocoons have many other beautiful colors besides yellow and fresh color. Meanwhile, the depth of cocoon color could be different. What is the relationship between the type and content of carotenoids and the color difference of cocoons? How does the carotenoids accumulation in silkworm larvae affect the color formation of yellow-red cocoons? Whether the genes involving in the color formation of yellow cocoon and fresh cocoon are related to the formation of other cocoon colors? How do these genes adjust the transport of carotenoids in silkworm larva? Whether are there any other factors involving the formation of cocoon color? Answering these questions will help to understand the formation mechanism of yellow-red cocoon. Not only it provides a basic theory for the development and utilization of natural colored cocoons, but also contributes significant information to understand the transport mechanism of carotenoids in mammals and humans.In current research, we studied the relationship between the accumulation of carotenoids in larvae of different silkworm varieties and the color formation of yellow-red cocoon, analyzed the expression of the related regulatory genes in different developmental stage of silkworms larvae, preliminarily studied the roles of those regulatory genes encoding proteins during the uptake and transport of carotenoids in vitro cell culture system, and isolated and identified the carotenoid-binding proteins from the silk glands of rust cocoon varieties, The main results are:1. The relationship between accumulation of carotenoids in silkworm larva and the color formation of yellow-red cocoon.Qualitative and quantitative analysis of carotenoids from mulberry leaves, cocoons and tissues from last instar larvae stage at day5of different cocoon color silkworm varieties was done using RP-HPLC-DAD methods. Results are as follows:(1) Types of major colored carotenoids in mulberry leaves, silkworm larvae and cocoons are lutein,(3-carotene and violaxanthin.(2) The ratio of lutein and β-carotene in cocoons is associated with the depth of cocoon color, the higher the ratio of lutein and β-carotene, the more depth the yellow color of cocoon.(3)The composition and content of carotenoid in silk glands is basically the same as cocoons.(4) The carotenoid absorption of silk glands show differences in varieties, time and absorption site。 Lutein is the major absorption pigment of the silk glands from yellow cocoon varieties; β-carotene is the major absorption pigment of the silk glands from rust cocoon varieties. The silk glands of yellow cocoon varieties can absorb lutein from last instar larvae stage at day3; the silk glands of red cocoon and rust cocoon varieties can absorb β-carotene from last instar larvae stage at day3and the silk glands of red cocoon varieties can absorb lutein from last instar larvae stage at day5after. Posterior portion of the middle silk glands from red cocoon and rust cocoon varieties mainly absorbs β-carotene, and middle section portion of the middle silk glands from red cocoon varieties mainly absorbs lutein. It can be seen silk glands selectively absorb carotenoids from the blood that is main reason showing the color differences in yellow-red cocoons.2. The interrelation analysis between the accumulation of carotenoids and the expression patterns of CBP, Cameo2and SCRB15in different yellow-red cocoon varietiesThe expression of CBP, Cameo2and SCRB15were analyzed by RT-PCR. The result showed that CBP was expressed in midguts and silk glands from yellow cocoon, red cocoon and rust cocoon varieties. Cameo2was expressed in midguts and silk glands from yellow cocoon and red cocoon varieties besides midguts from rust cocoon varieties. SCRB15was expressed in silk glands from yellow cocoon, red cocoon and rust cocoon varieties. The expression patterns of CBP, Cameo2and SCRB15were analyzed by real-time PCR in the midguts and silk glands of last instar larvae stage at1-7days of age from different cocoon color varieties. The result showed that the expression patterns of Cameo2and SCRB15are consistent with lutein and β-carotene, respectively. The results above suggest that CBP, Cameo2and SCRB15could be involving in the formation of red and rust cocoon. Furthermore, CBP was always expresses along with Cameo2and/or SCRB15in silk glands from yellow-red cocoon varieties, indicating CBP has the potential combined effect with Cameo2and/or SCRB15to regulate carotenoid accumulation in silk glands.3. Preliminary study on the roles of CBP, Cameo2and SCRB15during the uptake and transport of carotenoidsThe structure prediction on line showed that CBP belongs to StAR protein superfamily and has no transmembrane area. Both Cameo2and SCRB15belong to CD36protein superfamily, had two transmembrane regions on each near end of C-and N-terminal. Subcellular localization and co-localization showed that SCRB15and Cameo2were located at cell plasma membrane, vesicle membrane and nuclear membrane, but CBP was expressed in the whole cell. CBP and SCRB15or Cameo2can overlap in the same place. The results above suggest that SCRB15and Cameo2are membrane proteins, CBP might be cytoplasmic protein.Carotenoid absorption experiment of transfection cells showed that β-carotene concentration in the cells expressed SCRB15was1.82fold higher (P<0.05) than control; the cells co-expressed SCRB15+CBP could absorb higher β-carotene (P<0.05) than other transfected cells. The results above suggest that SCRB15can improve the cellular uptake of β-carotene; CBP has the combined effect with SCRB15on facilitating the cellular uptake of β-carotene. The transfection cells were incubated in culture medium containing different carotenoids micelle. The results show that the cells expressing SCRB15+CBP specifically absorbed β-carotene, while the cells expressing Cameo2+CBP specifically absorbed lutein. The absorption rate of β-carotene in transfected cells expressing SCRB15+CBP was time-dependent, concentration-dependent and reached saturation. The absorption rate of lutein in transfected cells expressing Cameo2+CBP was also ime-dependent, concentration-dependent and reached saturation. The results above suggest that SCRB15and Cameo2, as membrane proteins, specifically identified and mediated the cellular uptake of β-carotene and lutein, respectively. BiFC results showed that Cameo2or SCRB15has directly interacted with CBP at cellular level.From protein structure prediction, subcellular localization and co-localization, carotenoid absorption experiment and BiFC experiment, we conclude that in vitro cell culture system, SCRB15and Cameo2, as membrane proteins, specifically absorbed β-carotene and lutein, respectively. It is possible that they transported carotenoids into transfected cells through plasma membrane invagination forming vesicles. CBP, as a cytosol protein, and SCRB15or Cameo2could combined effect to facilitate vesicle transport of carotenoids.4. Purification and identification of carotenoid binding protein from the silk glands of from rust cocoon03-520.In order to looking for new carotenoid transport adjustment factors, Carotenoid binding proteins of the silk glands from rust cocoon03-520larvae were purified and identified. CBP, ecdysone oxidase and retinoic acid binding protein were obtained.CBP was mainly combined with lutein (71.14%) and p-carotene (22.75%). The spectrum of Carotenoid-CBP purified sample has carotenoid typical characteristic, its absorption wavelengths were432nm,462nm and492nm. The purified sample spectrum of retinoic acid binding protein also has carotenoid typical characteristic, its absorption wavelengths were434nm,456nm and482nm. Although the purified sample spectrum of ecdysone oxidase has not carotenoid spectrum characteristics, there are two absorption peaks in the visible area and the absorption wavelengths of these two peaks are399nm and466nm, respectively. It is the first time that β-carotene is detected in the purified samples of retinoic acid binding protein and ecdysone oxidase. The two proteins might be involved the transportation and metabolism of β-carotene in the silk gland cells.