不同品质鸡蛋壳中糖胺聚糖(GAGs)的结构特征及其分子机理

GAGs are key regulators for the qualities of biominerals, while the biological activities of GAGs are determined by their own fine structures, and the fine structures of GAGs are shaped by upstream synthetases or modification enzymes. However, it is unknown what the structural characters of eggshell GAGs are, how the structures affect eggshell qualities, and which genes contribute to the biosynthesis and modification of shell GAGs. Therefore, based on previous work, this project will furhter systemly perform the studies on biochemistry and moleclar biology of eggshell GAGs. On one side, with technique of FACE (fluorophore-assisted carbohydrate electrophoresis), we will characterise the constituent structures (content of total GAGs, types of GAGs, content of each type of GAGs) and fine structures (length of each type of GAGs, kinds of disaccharides in each type of GAGs, and content of each kind of disaccharides) of GAGs from various types of eggshells, such as pimple shell and normal shell, shells with various thickness, and shells with different strength. Through this work, it is in prospect to uncover special structures of shell GAGs which are significantly related with eggshell qualities. On the other side, using techniques of gene chip, gene overexpression, RNAi, and FACE, the candidate genes involved in biosynthesis and modifications of eggshell GAGs will be sreened, and which biological functions will be further probed into. This work will identify specific genes which contribute to fine structures of eggshell GAGs. Overall, it is the final aim of this project to found the molecular mechanisms, i.e. some specific genes function as major contributors to the qualities of eggshell through regulating the fine structures of shell GAGs.

糖胺聚糖(GAGs)对生物矿化物晶体的品质有重要作用，其生物活性受自身精细结构影响，而精细结构由加工修饰酶决定。但是鸡蛋壳中GAGs的精细结构对蛋壳品质的影响尚属未知，而修饰蛋壳GAGs精细结构的上游基因更属空白；因此，本项目计划在前期发现涉及GAGs合成、修饰的蛋壳强度候选基因的基础上，系统开展蛋壳GAGs的生化和分子生物学研究。一方面，利用荧光辅助糖电泳(FACE)技术研究砂壳蛋与正常蛋壳之间、厚度差异蛋壳之间、强度差异蛋壳之间GAGs的组成性结构(蛋壳中GAG的种类及含量)和精细结构(GAG所含二糖结构类型及含量、GAG糖链长度)的变化规律，以期发现与蛋壳品质密切相关的GAGs结构特征；另一方面，利用基因芯片、RNAi、FACE等技术筛选合成修饰蛋壳GAGs的候选基因并研究其功能，以期发现调控蛋壳GAGs精细结构的基因。本项目旨在构建特定基因调控GAGs精细结构而影响蛋壳品质的机理。

鸡蛋壳由壳膜、钙化壳和表面角质层构成；含95%的CaCO3以及4-5%的有机基质。有机基质最主要成分是蛋白质和糖胺聚糖；已知有机基质参与调控钙化壳形成，从而影响蛋壳品质，但是具体机制缺乏认知。本项目发现：1) 四类糖胺聚糖（透明质酸、硫酸软骨素/硫酸皮肤素、硫酸角质素、肝素/硫酸肝素）在蛋黄、浓蛋清、稀蛋清、壳膜、钙化壳中均有分布；但是，蛋黄中4类糖均极少，蛋清中有丰富的硫酸角质素，壳膜中有丰富的透明质酸，钙化壳中有丰富的硫酸软骨素以及肝素。2) 每枚鸡蛋的蛋清可以纯化3mg硫酸角质素，属于N-型糖，分子量约36.8 kDa 至40.6 kDa，并且1/10为帽子结构神经氨酸。3) 建立了鸡蛋壳膜和钙化壳中，四类糖胺聚糖精细结构的群体分布特征；并且壳膜中硫酸软骨素的二糖之间、以及肝素的二糖之间存在高度线性相关；但钙化壳中硫酸软骨素的二糖之间却很具异质性。4) 鸡蛋壳膜中，硫酸软骨素/硫酸皮肤素及其二糖结构4SCS-A和0SCS-O均显著影响蛋壳强度；透明质酸0SHA和角质素NSKS显著影响蛋形指数。但是钙化壳内，除肝素6SHS显著影响蛋形指数外，其它糖胺聚糖及其二糖结构均与蛋壳品质无显著关联。5) 克隆了鸡蛋壳中糖胺聚糖的主要核心蛋白OC-116的编码基因，该基因高度保守。6) 初步鉴定DSE、CHSY3、B4GALT7、B3GALT6、Xyl2-O-kinase、B3GALT3、CHPF等基因与OC-116肽链上糖胺聚糖的合成密切相关。7) 沙蛋壳中酸不溶性基质、水不溶性基质、酸和水兼溶基质以及总基质的百分含量均显著高于正常蛋壳；尽管沙蛋壳的壳重显著低于正常蛋壳，但是单枚沙蛋壳中有机基质却极显著多于正常蛋壳。正常蛋壳的破碎强度与上述4种基质的百分含量无明显相关。8) 鸭和鹅的钙化壳中各种基质的含量很相近；鸭蛋和鸡蛋一样，各种基质的含量均与蛋壳品质无显著相关；但是鹅蛋的基质含量却与蛋壳品质显著相关。9) 蛋白质组学研究鉴定了正常蛋和沙壳蛋钙化壳中42种主要蛋白，这些主要蛋白是三种基质的构成主体；同时发现了25个影响蛋壳品质的蛋白因子。上述成果有利于从糖胺聚糖和蛋白质角度理解蛋壳品质的影响机制。