【作者】 许乃霞；

【导师】 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2011， 博士

【摘要】 家蚕(Bombyx mori)茧层在紫外线照射下呈现多种荧光色,通常可以分为深浅不同的黄白和蓝紫两大类;家蚕雌雄蚕茧茧丝质和力学性能存在明显差异,雄蚕茧丝纤度细,经济性状好,其缫制的生丝高于雌蚕茧1～2个等级。近年苏州大学蚕桑研究所虞晓华等发明了将不具备荧光茧色判性特性的蚕品种转育成荧光茧色判性蚕品种的方法,2006年育成荧光判性品种“雄晓”,2008年又育出孵化齐一、发育快、好养的荧光判性品种苏.雄×荧晓,雄茧为黄荧光,雌茧为紫荧光,判性准确率均可达到100%,并开始在生产上推广,雌、雄蚕茧分开缫丝,生产出了6A级高品质雄蚕丝,而雌蚕茧生丝品质也同时提高。虽然通过一定的方法可以培育出判性品种,但是判性机理目前的研究并不深入,许多方面还不清楚,因此阐明家蚕荧光茧色判性机理,对丰富家蚕生理学、遗传学理论及荧光判性育种实践都有很大意义。在我们的研究中,以人工饲料控制饲料中的色素种类和含量,观察茧层的荧光色素变化情况;通过荧光照射观察雌雄家蚕体内中肠,血液,丝腺等器官以及茧层中的荧光色素的积累与分布情况;通过薄层色谱分析,荧光色素的荧光光谱分析和荧光色素的紫外吸收光谱等方法,分析雌雄家蚕荧光色素的种类与组成。应用实时荧光定量PCR,分析色素结合蛋白相关基因在中肠的转录水平、色素氧化及代谢酶类基因在中肠和丝腺内的转录水平,来了解荧光茧色判性家蚕中肠色素结合蛋白对荧光色素透过的作用以及色素氧化酶及代谢酶类对荧光色素形成的作用。主要结果如下:1、从5龄3天开始分别用正常饲料、酒精脱色饲料和酒精脱色后加入槲皮素的饲料饲养荧光判性品种家蚕,测定蚕茧荧光,结果表明,饲以未经处理的桑叶粉配制的人工饲料所营的蚕茧在紫外线下可判别雌雄;而饲以酒精脱色饲料所营蚕茧雌雄均为均匀的紫荧光色而无法分辨雌雄;饲以酒精脱色后加入槲皮素的饲料,所营蚕茧雌性绝大部分表现为紫荧光色,雄茧均表现为黄荧光色,荧光茧色判性率达到80%以上。这表明引起雌雄蚕茧荧光色差异的荧光色素基础物质来源于桑叶醇溶性物质,槲皮素或者是槲皮素的类似物是其中重要的一类物质;也证明了荧光黄色素决定雌雄蚕茧荧光色的差异。2、通过对冻干的五龄家蚕血液、器官和组织的荧光显微观察发现,家蚕荧光茧品种中,雌蚕中肠后部(约1/3～2/5)积累大量黄色荧光物质,而雄蚕无此现象,正是雌蚕中肠对黄色荧光色素的差异吸收,导致家蚕荧光茧色的判性。实验发现中肠后部大量积累黄色荧光色素的部分是圆筒形细胞部分,推测可能存在荧光色素结合蛋白。3、对5龄家蚕的雌雄蚕血液和中肠组织的荧光色变化进行逐日观察,结果表明,五龄第4日起,雌雄蚕血液荧光色出现明显的差异,这种差异一直保持到吐丝前。在紫外光照射下,吐丝前1天的雌蚕血液荧光色较暗,而雄蚕血液发出很明亮的黄色荧光;将血液滴在纸上风干后,其斑点中央的荧光色呈明显差异,雌蚕呈深紫色荧光斑,是黄色荧光物质与紫色荧光物质的混合色;而雄蚕血在斑点中央呈很亮的黄荧光和很淡的蓝紫荧光的混合色。说明雌雄蚕血液的荧光色差异是从家蚕5龄盛食期开始。4、以家蚕荧光茧色判性品种为实验材料,对蚕茧层表面和茧层抽提物的荧光光谱分析表明,雌雄蚕茧都出现黄色和蓝紫色二个荧光发射峰,茧层中不存在荧光色素种类的差异。雌雄茧之间的荧光色差异主要因黄色和蓝紫色相对荧光强度不同而引起的。5、应用硅胶薄层色谱和荧光光谱分析显示,蓝紫荧光色物质至少由5种蓝紫荧光色素成分组成,而黄色荧光物质至少由3种黄色荧光色素成分组成;从雄蚕茧层中提取到的黄色荧光色素与雌蚕后部中肠积累的色素完全相同。6、紫外光谱分析和AlCl3显色反应表明,3种黄色荧光色素属于黄酮类或黄酮苷类化合物,其中一种黄色荧光色素(Rf=0.82)为主要成分,其荧光发射光谱最大发射峰为533nm,激发光谱峰值为377nm。7、通过测定正常饲料、酒精脱色饲料和酒精脱色后加入槲皮素的饲料饲养的家蚕中肠的30Kc19、BmLP-C21、BmLP-C23、类胡萝卜素结合蛋白(CBP)基因的转录水平,表明类胡萝卜素结合蛋白(CBP)是参与荧光色素雌家蚕中肠内的特异性积累的主要基因,30Kc19也是家蚕中肠色素特异积累过程中的重要基因。8、通过测定正常饲料、酒精脱色饲料和酒精脱色后加入槲皮素的饲料饲养的家蚕BmUGTs、P450s、BmGSTs基因在家蚕中肠和丝腺中的转录水平,结果表明, BmUGT29参与荧光茧色判性家蚕雌性中肠内色素的形成与代谢,BmUGT30参与荧光茧色判性家蚕中肠对槲皮素的代谢。BmUGT59主要参与槲皮素在雌性丝腺内的形成与代谢。BmGSTe1和BmGSTe5参与了荧光茧色判性家蚕雄性丝腺的色素代谢。CYP6au1,CYP6ae8参与家蚕中肠内槲皮素的形成与代谢。综上所述,引起荧光茧色判性家蚕雌雄蚕茧荧光色差异的荧光色素的基础物质来源于桑叶醇溶性物质,雌雄蚕荧光色差异是从家蚕5龄盛食期开始;家蚕荧光茧色判性主要是由于雌蚕中肠存在荧光色素的结合蛋白对黄色荧光色素产生特异性积累,导致雌雄茧之间的黄色和蓝紫色相对荧光强度不同,最终反映为茧色的不同。更多还原

【Abstract】 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.更多还原