【作者】 江洪波；

【导师】 陈立侨；

【作者基本信息】 华东师范大学 ， 动物学， 2003， 博士

【摘要】 中华绒螯蟹(Eriocheir sinensis H.Milne—Edwards)俗称河蟹，隶属于甲壳纲(Crustacea)，十足目(Decapoda)，方蟹科(Crapsida)，绒螯蟹属(Eriocheir)，是我国特有的水产养殖珍品。广泛分布于我国东南部沿海的咸淡水或淡水水域中，北起辽河、南至瓯江均有出产，而以长江流域所产品质为最佳。因其营养丰富、肉味鲜美、风味独特，倍受国内外市场青睐。 自上个世纪90年代以来，养蟹业已成为我国淡水养殖的重要产业。随着河蟹养殖的集约化和规模化发展，在实际生产过程中出现了一系列亟待解决的问题，如雌蟹亲体的性腺发育不良，抱卵率下降，亲蟹利用效率较低；苗种培育过程中，胚胎发育死亡率高，且幼体育成率低，质优量足的苗种供应不足；营养素的不平衡导致成蟹养殖过程中的性早熟等，都严重制约了河蟹养殖产业的健康、稳步发展。究其原因，这些问题均与河蟹本身的生理特性、营养需求，以及亲蟹培育期未能获取合理的营养素等有直接关系。而这些问题的解决则要求对中华绒螯蟹生殖过程中的摄食和消化生理机制，亲体性成熟期对各种必需营养素(如必需氨基酸、必需脂肪酸和维生素等)的需求，卵细胞发生过程中营养物质的合成、转运及贮存规律，以及在受精卵、胚胎和后续以内源性营养为主的溞状幼体发育阶段对卵黄磷蛋白的消耗和利用模式等环节进行较为深入的研究，在全面系统地了解中华绒螯蟹的生殖营养生理的基础上，探讨亲体生殖营养对卵质、孵化及后续幼体生长发育的影响。 蛋白质是动物体内维持正常结构和功能的物质基础，其生理作用主要有：为机体的生长和发育提供原料，用于构建有机体的器官、组织和细胞等结构；在代谢过程中调节并控制生命活动；可通过分解作用为机体多项生理过程提供能量。目前有关经济虾蟹不同生长阶段的蛋白质营养需要的研究较多。研究发现虾蟹对蛋白质的营养需求与多种内外因素密切相关，如生长发育的阶段、生理状况、饲料蛋白源及其生存环境等。对处于生殖季节的虾蟹而言，饵料蛋白质消化吸收后，一部分用于合成体内的生理调节物质，如酶和激素等，可诱导亲体的成熟，促进雌雄交配；另一部分则用于合成性细胞发育所需的营养物质，尤其是合成卵黄磷蛋白，有利于提高卵质和卵细胞数目，维持正常的受精作用和胚胎发育过程，从而增加幼体的存活率。然而迄今，有关蛋白质营养对虾蟹繁殖作用的影响却鲜见报道。华东师范大学2003届博卜学位论文 为此，本研究在弄清中华绒鳌蟹自然条件下卵巢发育过程中肝胰腺和卵巢的蛋白质含量及其氨基酸组成的基础上，探讨了饵料蛋白质水平对其卵巢发育过程中肝胰腺和卵巢的蛋白质含量及氨基酸组成的影响;在制备中华绒鳌蟹成熟卵巢卵黄磷蛋白抗体的前提下，研究了蛋白质营养对成熟期卵巢卵黄磷蛋白合成的影响:并对仔蟹期的部分必需氨基酸适宜需要量，蛋白质和维生素B6的相互作用进行了研究。此外，还研究了蛋白质营养对中华绒鳌蟹卵巢发育期和仔蟹生长期消化酶的影响，以期为中华绒鳌蟹的生殖营养生理研究积累基础资料，同时也为人工育苗生产过程中的规范亲体培育及全价配合饲料的研制提供理论依据。一、中华绒鳌蟹卵黄磷蛋白的分离、纯化与鉴定 目前在判定虾蟹卵巢成熟和卵质时，大多采用性腺指数、蛋白质和脂类的组成与含量等生化指标，尚不够全面。本研究试图建立一种有效的免疫检测手段来测定卵巢中卵黄磷蛋白含量，以较为直接地反映卵巢成熟程度及卵细胞质量的优劣，这要求先获得纯化的卵黄磷蛋白，供制备抗体之用。因此，运用SePhadexG一200凝胶层析法(gelchromatography)从中华绒鳌蟹的成熟卵巢中分离、纯化得到一种蛋白复合物，经聚丙烯酞胺梯度凝胶电泳(队GE)确定其纯度;该蛋白复合体可被苏丹黑(Sudan black)，Stain一all和Schiff试剂染色，表明其含有脂类、糖类和磷等组分，由此可以确定该蛋白复合物即是卵黄磷蛋白(li povitellin)。又经SDS一聚丙烯酞胺梯度凝胶电泳(S DS一PAGE)分析发现，卵黄磷蛋白分子量为520.85士19.02 kDa，并由分子量分别为96.92士2.83 kDa和73.86士1.89 kDa的两种亚基组成。对纯化卵黄磷蛋白的氨基酸组成和含量也进行了分析，发现必需氨基酸总量占51.30%，略高于非必需氨基酸;其中，谷氨酸(Glu)和天门冬氨酸(AsP)含量较高，分别达10.80%和14.31%;亮氨酸(Leu)、赖氨酸(切s)、精氨酸(Arg)和撷氨酸(val)次之，含量分别为9.43%、7 .83%、7.08%和6.88%;而牛磺酸(Tau)、半肤氨酸(cys)和蛋氨酸(Met)含量较低，百分含量分别为0.17%、1 .01%和1 .40%。分析中华绒鳌蟹卵黄磷蛋白的氨基酸组成及含量可为其亲体阶段的必需氨基酸营养需求提供标准参考。二、中华绒鳌蟹卵巢成熟期蛋白质和卵黄磷蛋白含t变化的研究 探讨卵巢成熟过程中卵黄磷蛋白合成和积累的规律，对于深入研究亲蟹的生殖营养至关重要。于2001年8一12月自上海市郊淀山湖取中华绒鳌蟹雌体亲蟹。在采用纯化的卵黄磷蛋白免疫兔而获得其抗体的基础上，运用酶联免疫吸附检测中文摘要中华绒鳌蟹蛋自质价养生理研究法(enzyme一linked immunos更多还原

【Abstract】 In crustaceans, vitellogenesis is associated with several complete processes that lead to the formation of mature oocytes within the ovary. Substantial quantities of yolk protein (mainly lipovitellin) accumulate within the developing oocytes, and serve to meet the basic requirements of embryonic and early larval development, dependent of the maternal organism. So crustacean broodstock nutrition and feeding have been shown to greatly improve not only egg and sperm quality but also seed production. The availability of an optimal diet is identified as a crucial factor for the sexual maturation and reproduction of shrimp and crab. Gonadal development and fecundity are affected by certain essential dietary nutrients, especially essential amino acids, essential fatty acids, and vitamins etc. Thus, during the last two decades, more attention has been paid to the optimal level of different nutrients in broodstock diets.It is well known that protein is a kind of indispensable nutrient because of its important physiological function on body structure and supplying energy to the growth and development of shrimp and crab. Protein and amino acids of broodstock diet have been identified as major dietary factors that determine successful reproduction and survival of offspring. Up to date there are no reports on optimal protein levels, or amino acid profiles for crustacean (especially shrimp and crab) broodstock diets.The Chinese mitten-handed crab, Eriocheir sinensis, is considered to be one of the most important species for commercial production in China. Nutritional requirement for protein at different grow stages has been studied by many researchers. But at present few attach importance to nutriology and digestive physiology of E. sinensis broodstock. So in this paper, effects of dietary protein levels on the content of protein and amino acid profile in hepatopancreas and ovaries during ovarian maturation are investigated on basis of analyzing protein content and amino acid composition and content in hepatopancreas and ovary of broodstock in nature. Influence of dietary protein on concentration of lipovitellin in ovary of broodstock after purification and preparation antibody of lipovitellin from mature ovaries of E. sinensis is evaluated. And nutritional requirement for arginine and lysine, and interaction between protein and vitamin B6 of juvenile E. sinensis are also studied. Finally digestive enzyme activities in hepatopancreas of E. sinensis fed on experimentdiets with different protein level during juvenile and ovarian maturation. The purpose is to accumulate basal information for reproductive nutrition, and provide reference for breeding broodstock in seed production and formulation of broodstock diet.1. Isolation, purification and characterization of lipovitellin from the ovary of female Eriocheir sinensis broodstockLipovitellin was extracted and purified using gel chromatography and electrophoresis procedures from ovaries of Chinese mitten-handed crab. One kind of lipovitellin was obtained. Lipovitellin could be stained by Sudan black, Stand-all, and Schiff reagent, which indicated that it comprised some component such as lipids, carbohydrate and phosphorus etc. The characterization of lipovitellin was carried out by SDS-PAGE. The results indicated that lipovitellin had a molecular weight of approximately 520.85?9.02kDa and was composed of two kinds of subunits, whose molecular weights were 96.92+2.83 kDa and 73.86?.89 kDa respectively. Major amino acids made up of lipovitellin are aspartic acid (asparagine), and glutamic acid (glutamine), their contents were 10.80% and 14.31%, some amino acids including leucine, lysine, arginine and valine were less, their contents were 9.43%, 7.83%, 7.08% and 6.88%, respectively, while contents of taurine, cysteine and histidine were very low, only 0.17%, 1.01% and 1.40%. The purpose to analyze amino acid profile of lipovitellin is to provide a kind of reference standard for broodstock amino acid nutritional requirement. The purified lipovitellin can be used更多还原