The Research For Mechanism Of Sex Identification By The Fluorescent Cocoon Color Of Silkworm Bombyx Mori

Silkworm (Bombyx mori) cocoon layer showed various fluorescent color under uv irradiation.It ofen can be divided into two kinds of different shades of yellow and blue purple. Quality and mechanical properties male and female silkworm cocoon are obvious different, the cocoon silk fineness and economic characters of male silkworm are excellent, its raw silk reeling silk are higher than female silkworm cocoon 1 ~ 2 levels. In recent years, suzhou university institute YuXiaoHua sericultural inventions the method of cultivating silkworm breed that cannot be convicted sex by cocoon fluorescent color into being convicted sex by cocoon fluorescent color, he had bred cultivars of sex identification by the fluorescent cocoon color of silkworm Bombyx mori the "male dawn" in 2006, " and nurtured the hatch out together, fast development and good raised sex identification by the fluorescent cocoon color of silkworm Bombyx mori varieties Sue. the male×Ying xiao in 2008, Which male cocoon is yellow fluorescent and female cocoon is purple fluorescence, and the accuracy of sex identification by the fluorescent cocoon color may achieve 100%, and started to popularize in the production, male and female silkworm cocoon are filature respectively, produced 6 level of high quality male silk, and the quality of female silkworm cocoon silk are also improved. Although we had produced the variety of sex identification by the fluorescent cocoon color, but the mechanism to sex identification is not clear.Clarifing the mechanism of sex identification by cocoon color fluorescent is great significance for riching silkworm physiology, genetics theory and fluorescence convicted sex breeding practice.In our studies, by controling the pigment varieties and contents in artificial feed to observe the change of fluorescent pigments in the cocoon layer. Through the fluorescence illuminate to observe the conditions of fluorescent pigments accumulation and distribution in organs such as midgut, blood, silk gland and cocoon layer between male and female silkworm (bombyx mori). Appling real-time fluorescence quantitative polymerase chain reaction (PCR), to analyse the level of transcription of the gene of pigment binding protein in midgut, the level of transcription of the gene of pigment oxidation and metabolic enzymes in midgut and silk gland, to understand the role of the gene of pigment binding protein in permeance and the gene of pigment oxidation and metabolic enzymes in forming of the fluorescent pigments in silkworm (bombyx mori). The main results as follows.1、From 3rd day of fifth star the convicted sex by cocoon color fluorescent silkworm were fed with normal atificial diet,alcohol bleaching atificial diet and adding quercetin after alcohol bleaching atificial diet. Then measured cocoon fluorescence.T?he results show that the cocon of silkworm fed with untreated artificial diet can be distinguished male and female by cocoon color fluorescent under ultraviolet light; however, the cocon of male and female silkworm fed with alcohol decolorization artificial diet both showed purple fluorescent color, we were unable to distinguish male and female by cocoon fluorescent color; the cocon of female silkworm fed with feed adding quercetin after ethanol bleaching, showed purple fluorescent, and male showed yellow fluorescent color. The rate of sex-identified fluorescent cocoon color was more than 80 percent. This shows the material of fluorescent pigment-based in male and female cocoon was alcohol-soluble substances derived from mulberry leaves. One important is quercetin or similar things of quercetin. Also prove the fluorescence yellow element decided to the differences of cocoon fluorescent color between the male and female silkworm.2、By the fluorescent microscope observation of freeze-dried fifth instar silkworm’s blood, organs and tissues , we discovered that all or part of the yellow fluorescent substance were accumulated at the back of the female silkworm midgut(about 1 / 3 to 2 / 5) in the fluorescent silkworm cocoon species, and the males silkworm no this phenomenon. It is the difference of absorption of the fluorescent pigment in female silkworm’s midgut, resulting in sex-identified fluorescent cocoon color. This study found that a large number of yellow fluorescent pigment accumulated at the posterior portion of midgut is part of these cylindrical cells. It is speculated that there may be fluorescent pigment-binding protein.3、Observing changes in the fluorescent color in male and female blood and intestinal tissue of 5 year-old silkworm on a daily basis, the result show that blood fluorescent colors between male and female silkworm appear a significant difference in the 4th day. This difference was maintained until silking without any change. Under uv irradiation, 1 day before spinning fluorescent colors in blood of female silkworm was darker, but the blood of male silkworm showed bright yellow fluorescence. If we drops the blood on paper and dried it, we can find obvious difference in the middle of a fluorescent color spots, in central blood spots of female silkworm present deep fluorescent purple, that is the yellow fluorescent substance and purple fluorescent material mixed color, and the central blood spots of males silkworm present very bright yellow fluorescence, that is the yellow fluorescent substance and the pale blue purple fluorescent mixture color. Whitch show that fluorescent color difference of male and female silkworm’s blood is beginning at feed high stage of 5th star bombx mori.4、Using sex-identified fluorescent cocoon color silkworm as experimental material, we analyzed the fluorescence spectra of cocoon shell and cocoon shell extracts. The result showed that there are yellow and blue-violet emission peak in male and female cocoons. This shows that there was no difference of fluorescent pigments types in male and female cocoon.T?he difference in fluorescent color of male and female cocoon was caused by differences of yellow and blue-violet fluorescence intensity.5、Silica gel thin layer chromatography and fluorescence spectroscopy showed that blue-violet fluorescent material was composed of five kinds of blue-violet fluorescent pigment composition at least, and yellow fluorescent substance was composed of at least three kinds of yellow fluorescent pigment composition.Y?ellow fluorescent pigment extracted from male cocoons layer was identical to the pigment accumulated in the back of the female silkworm midgut.6、UV spectra and AlCl3 color reaction showed that three kinds of yellow fluorescent pigments are flavonoids or flavonoid glycosides, one of the yellow fluorescent pigment (Rf =0.82) was the main composition. The maximum emission peak of fluorescence emission spectra was 533nm, the excitation spectrum peak was 377nm.7、By measuring the transcription level of midgut 30Kc19, BmLP-C21, BmLP-C23, carotenoid-binding protein (CBP) in silkworm feed by Normal, alcohol extraction and adding quercetin diet after alcohol extraction artificial feed respectively. The results show that carotenoid-binding protein (CBP) was involved in the specific-accumulation of fluorescent pigment in the female silkworm midgut. And 30Kc19 is a specific important gene in the course of accumulating pigment in the silkworm midgut.8、By measuring the transcription level of BmUGTs P450s and BmGSTs gene in normal feed, feed alcohol extraction and adding quercetin after alcohol extraction artificial feed silkworm’s midgut and silk gland, BmUGT29 participate in the formation and metabolism of the pigment in the female midgut of sex identification by the fluorescent cocoon color of silkworm, BmUGT30 mainly involved in quercetin metabolism in the sex-identified fluorescent cocoon color silkworm midgut, BmUGT59 mainly involved in the form and metabolism of quercetin in the silkgland of the female silkworm. BmGSTe1 and BmGSTe5 mainly involved in the metabolism of quercetin in the silkgland of the male silkworm. CYP6au1 and CYP6ae8 involved in the quercetin formation and metabolites in midgut of silkworm.These results suggest that the base material that cause different fluorescent color of silkworm cocoon to male and female silkworm cocoon comes from mulberry leaf alcohol-soluble material. The difference of male and female silkworm fluorescent color began in feed high stage of 5th star bombx mori. Sex identification by cocoon color fluorescent is mainly due to the female silkworm bowel exist in fluorescent pigments protein to combinate specific yellow fluorescent pigments and accumulated it in midgut of silkworm, which lead to relative fluorescence intensity of the yellow and blue purple fluorescent color between male and female cocoon, eventually reflect different for the cocoon color.