The Flavonoids Of Bombyx Mori Cocoon And Pupa, And Related Gene Expression Analysis

The resources of colourful cocoon Bombyx mori’s variety are rich in our country,colorful cocoon silk with moisturizing, antibacterial, anti-oxidation, anti-UV properties;and the pupa is not only rich in protein, contain a variety of active substances and a lot of trace elements for human. Therefore, the development of Bombyx mori cocoon and pupa have attracted a lot of attention. Colourful cocoon contains abundant pigments,mainly from mulberry leaves, mainly from mulberry leaves, after Bombyx mori feeding,absorbed by the intestinal epithelial cells enter the haemolymph, finally into silk glands and accumulated in the sericin layer, thus make the cocoon silk color the Bombyx mori feed, Central intestinal epithelial cells after absorption into the hemolymph, and finally into the silk gland and silk accumulation layer, thereby enabling the colored silk.Studies showed that silkworm green cocoon main flavonoids pigment.This study detection of silkworm cocoon and content of flavonoids in silkworm chrysalis, and use high performance liquid chromatography(High Performance Liquid Chromatography, abbreviated HPLC) method for the analysis of rutin and quercetin in silkworm chrysalis, quercetin, milk vetch glycosides and the content of chlorogenic acid,which provides an important reference for the better development and utilization of natural color cocoon and pupa resources. Fluorescence quantitative PCR analysis of the midgut, fat body and green cocoon silk gland genes related UGT86 and UGT65 and related genes expression of CBP and yellow cocoon Cameo1 age five different varieties of silkworm, Bombyx mori to explore the differences between organizations pigment and the formation mechanism of green cocoon, provide new reference. The main results are as follows:1. The analysis of flavonoid contents in white, yellow and green cocoon.Green cocoon cocoon shell content of flavonoids was significantly higher than the yellow and white cocoon cocoon and cocoon color depth and content of flavonoids in green cocoon is directly correlated to the Department. There is no yellow cocoon shell between flavonoids content and white cocoon significant difference, even a small number of yellow cocoon cocoon shell content cultivars in flavonoids lower than white cocoon varieties phenomenon, indicating that not all color cocoon cocoon content of flavonoids than the high white cocoon.2. The analysis of flavonoid contents in the different colour cocoon’s pupa.Content of the white, yellow and green three kinds of color cocoon pupa flavonoids ranged between 2.00 mg/g ~ 5.00 mg/g. Green cocoon content of flavonoids relative to other color cocoon pupae though high, has not reached a significant level, but the content of some varieties among pupae flavonoids significantly different. The content of the same breed silkworm pupae flavonoids is much higher than the cocoon shell in an amount(ranging from 1 to 10 times).3. Provides a way to use HPLC method to measure the home of flavonoids content in silkworm chrysalis.Detection of four chromatographic conditions pupa flavonoids are: mobile phase consisted of acetonitrile and carboxylic acid groups(A: 0.05% H3PO4 aqueous solution,B: acetonitrile) using a gradient elution program is: 0 ~ 9min, B 20 % ~ 80%; 9 ~ 12 min,B 80%; 12 ~ 12.1min, B 80% ~ 20%; 12.1 ~ 16 min, B 20%; volume flow rate: 0.8m L /min; injection volume: 20 u L; detection wavelength: 254nm; Column temperature:room temperature. The test is simple, fast, high sensitivity, the pupa develop in terms of consumption provides a practical and reliable detection method.4. Different cocoon color pupa in the isolation and identification of flavonoids.Detected by HPLC home pupa rutin, quercetin, Isoquercitrin, Astragalin four kinds of flavonoids and chlorogenic acid content: pupa samples detected in this study do not contain chlorogenic acid; different the total content of flavonoids pupa cocoon of different color, same color cocoon type and content of flavonoids, there are significant differences between different species pupa, chrysalis cocoon green quercetin,isoquercitrin content than other color cocoon pupae higher of these two flavonoids can be used as a green cocoon marker; yellow cocoon pupae no or low rutin.5. Relative expression levels of Green Cocoon related gene(UGT65 and UGT86) and yellow cocoon related gene(CBP and Cameo1).Examined the relative expression levels of different varieties of color cocoon fifth instar larval midgut, silk gland and fat body of four-color cocoon related gene expression yellow cocoon variety CBP is relatively high, relatively high expression of green cocoon varieties UGT86. The Cameo1 and UGT65 expression level of color cocoon formed and showed no apparent direct relationship. Larval stage(5L3d),pupa body fat middle and late periods of three pupae have CBP expression, yellow and green cocoon cocoon significantly higher, especially after entering the pupal stage.UGT86 gene expression in different periods of yellow cocoon pupa body fat at first stabilized after rising phenomenon, and green cocoon is manifested after the first decline in stable trend.