琥珀蚕茧色形成机理研究

Antheraea Assamensis collected in Yunnan Province in recent years is a precious wild silkworm resource, has the high economic value due to its silk with the native golden color，and it is a fine material for studying on the pigment metabolism and the formation mechanism of cocoon color, too. In this study, the nature and types of the key coloration pigments of cocoon color in Antheraea Assamensis will be identified by using UV spectrophotometer, high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance, the metabolic pathway and the variation rule of the pigments in vivo will be made clear, and the physiological and biochemical mechanism of cocoon color formation in Antheraea Assamensis will be ascertained. Moreover, the key genes controlling cocoon color in Antheraea Assamensis will be unearthed by bioinformatics combined together RNA-Seq sequencing technologies and protein purification techniques, and the relationship between the expression feature of target genes and the pigment changes will be studied and analyzed by using Southern blot, Northern blot, qPCR and Western blot. The function of target genes will be tested and verified in the cellular level and in vivo by using cell transfection and transgenic RNAi technologies, the molecular mechanism of cocoon color formation in Antheraea Assamensis will be elucidated. This study will analyze the key scientific question, that is the mechanism of cocoon color formation in Antheraea Assamensis, from the physiological and biochemical level and the molecular level. The results will have great significance for protecting this rare resource and developing and utilizing the natural color silk of the rare resource.

琥珀蚕是近些年在云南收集到的一个珍贵野蚕资源，其蚕丝具有天然的金黄色，有较高的经济利用价值，是研究色素代谢和茧色形成机理的良好材料。本研究拟采用紫外分光光度计、高效液相色谱、质谱和核磁共振技术鉴定琥珀蚕茧色主要呈色色素的种类与性质，弄清该类色素在生物体内的代谢途径和变化规律，明确琥珀蚕茧色形成的生理生化机理；同时，利用生物信息学方法结合RNA-Seq技术以及蛋白质纯化技术，发掘控制琥珀蚕茧色形成的关键基因，采用Southern blot、Northern blot、qPCR和Western blot技术研究靶基因表达特征与色素变化的关系，并利用细胞转染技术和转基因RNAi技术在细胞水平和生物活体上研究靶基因的功能，阐明琥珀蚕茧色形成的分子机制。本研究将从生理生化和分子水平上解析琥珀蚕茧色形成机理这一关键科学问题，研究结果对于保护这一珍稀资源和开发利用琥珀蚕天然彩色茧丝具有重要意义。

琥珀蚕是一种珍贵的野蚕资源，其蚕丝具有天然的金黄色，有较高的经济利用价值，研究其茧色形成、解开茧丝呈色的生命机理，对于保护这一资源和开发利用琥珀蚕天然彩色茧丝具有重要意义。本项目采用紫外可见光光度计法、高效液相色谱、质谱和核磁共振技术鉴定了琥珀蚕茧色色素的性质，结合对色素化学特性和琥珀蚕营茧特征研究，认为琥珀蚕茧呈色色素组成、茧色形成较为复杂，茧色形成既源于生物体内色素物质，也可能与吐丝结茧后的环境因素相关，胆色素、黑色素可能是呈色色素的组成成分；利用RNA-Seq测序技术进行了琥珀蚕中肠、丝腺转录组测序，获得了大量数据信息，分析转录组数据表明，两个胆色素类结合蛋白（胆绿素结合蛋白）基因在中部丝腺高量表达，在中肠、后部丝腺中小量表达，同时分析到胆色素代谢通道上的多个酶在转录组中均有表达；定量PCR研究表明，胆绿素结合蛋白基因在琥珀蚕丝腺组织中特异高量表达，在中部丝腺与后部丝腺中以及丝腺不同发育时期的表达存在差异，其可能与色素代谢和色素形成的组织特异性相关，克隆获得了该基因的全长cDNA。研究结果为进一步弄清琥珀蚕茧色色素种类、呈色机理和分子机制提供了线索、靶标、基因数据和分子证据。