The Determination Of Flavonoid Contents In Silkworm,bombyx Mori With Different Cocoon Colors And Analysis On Expression Of UGT86 Gene Associated With Green Cocoon Color

Silkworm colored cocoon varieties can mainly be classified into two families: yellow-red cocoon series and green cocoon series. Yellow and pinkish cocoons are derived from carotenoids, while those green are from ?avonoids. Mechanism for the carotenoid cocoon has been well established, while the formation of green cocoon, the mechanisms for metabolism, uptake and transport of dietary ?avonoids, mechanism of gene regulation and the inheritance pattern for the ?avonoid cocoon are much more complicated and largely unknown.In this paper, we detected and analyzed the flavonoid contents in the midgut, hemolymph, silkgland of the 5th instar larvae and their cocoon layer with different cocoon colors; investigaed the cocoon color segregation of hybrid progeny from varieties with different cocoon colors; we also analyzed quantitative expression of UGT86 gene which is responsible for Gb by Real-Time fluorescence quantitative PCR. Above all can guide us to reveal the differences in mechanism of pigment, the formation mechanism of green cocoon, benefit strain breeding of natural colored-cocoons, and promote the industrial development of natural colored-cocoon. The main result of this text is as follows:1. Analysis on flavonoids content in tissues of 5th instar of varieties with different cocoon color.G₁ and other green-cocoon varieties have strong capabilities of combining flavonoids in the midgut, transferring flavonoids from the midgut epithelium to the silkgland via hemolymph, their silk fiber have strong abilities to combine flavonoids as well, resulting in many flavonoids accumulated in silk gland and the layers of the cocoon, and green cocoons.While Y12-1B and other yellow-cocoon varieties have strong capabilities of combining flavonoids in the midgut, and the flavonoids contents in hemolymph and silkgland are also at a high level, the cocoons do not show green. Therefore, we supposed that this can be deduced in two reasons, the first one is the hemolymph of Y12-1B and other yellow-cocoon varieties have high level of oxidation to flavonoids, resulting in colorless hemolymph; second, the flavonoids in hemolymph and silkgland of yellow-cocoon varieties are not visible at natural light, resulting in colorless middle silkgland.2. Analysis on flavonoids content in cocoon layer of varieties with different cocoon color.The differences of flavonoids content in cocoon layer of different cocoon color varieties are significantly.The content of flavonoids of green cocoon varieties are higher that in yellow-red cocoon varieties. The content of flavonoids of white cocoon varieties is significantly lower than that in natural colored cocoon series.3. Analysis on cocoon color separation and inheritance pattern of filial generation F₁.The cocoon color of hybrids from green cocoon variety G₁ and white cocoon variety C108 was light green, lighter than G₁. The segregation ratio on cocoon color segregation of selfcross F2 and backcross generations goes together harmoniously with Mendel's regulavity of segregation, indicated that green cocoon trait of silkworm variety G₁ was incompletely dominant to white cocoon trait.The cocoon colors of hybrids F₁ from yellowish-pink cocoon variety MⅡand green cocoon variety Daizo are golden yellow, indicated that interaction happens between cocoon-color genes locate on chromosome.4. Analysis on flavonoids content in cocoon layer of aboriginal species and their hybrids F₁.The flavonoids content of hybrids F₁ from different cocoon color varieties are between their parents.The cocoon colors and flavonoids content of hybrids F₁ from green cocoon variety G₁ and white cocoon variety C108 are between variety G₁ and C108, as well as hybrids F₁ from yellow cocoon variety Y12-1B and variety C108, indicated that green cocoon trait of silkworm variety G₁ and yellow cocoon trait of Y12-1B are incompletely dominant to white cocoon trait. In green cocoon series, the cocoon colour depth is positive correlation with the contents of flavonoids, sugesting that the contribution major green-cocoon genes show different effect on combining and transporting of flavonoids in silkgland tissues.The cocoon colors of hybrids F₁ from yellowish-pink cocoon variety MⅡand green cocoon variety Daizo are golden yellow, the flavonoids contents of cocoon layer of them are at a high level, higher than the parent MⅡ, slightly lower than Daizo, indicating that genes in Daizo controlling the combination flavonoids are incompletely dominant.5. The expression of UGT86 varied with tissues and varieties of different cocoon colors.The expression of UGT86 gene varied with tissues, the expression level of UGT86 in midgut is obviously higher than that in silkgland.The expression of UGT86 varied with varieties of different cocoon colors: it has the high expression level in varieties of green cocoon such as G₁,resulting in amounts of flavonoids absorbed from mulberry leaves glucosylated at the 5-O position in the midgut, 5-O-glucosylation of flavonoids dramatically increases the solubility, target tissue uptake, or disposition of flavonoids, this promote the absorption of flavonoids from midgut to silkgland, resulting in a marked increase in the amount of ?avonoids in silkglands, green cocoon.We just deceted miner expressin in varieties of yellow cocoon such as Y12-1B, resulting in a mounts of flavonoids absorbed from mulberry leaves can not be glucosylated at the 5-O position in the midgut, and the capabilities of transferring flavonoids without glucosylation from midgut to silkgland is low, resulting in just a little flavonoids accumulated in silkgland.Our study shows that 5-O-glucosylation of flavonoids may be the major pigment of green cocoon, because the formation of green cocoon can be influenced by many factors, such as the permeability of midgut epithelium and silkgland of flavonoids, hemlymph's oxidation abilities to flavonoid pigment and so on, the expression level of UGT86 gene in tissues is not positive correlation with the contents of flavonoids in tissues, indicated that UGT86 gene has the role in the formation of green cocoon, But it is not the only green cocoon gene.