萍乡红鲫论文：萍乡红鲫ZP3蛋白组织差异性表达及其在卵母细胞中免疫荧光定位.doc

萍乡红鲫论文：萍乡红鲫ZP3蛋白组织差异性表达及其在卵母细胞中免疫荧光定位【中文摘要】萍乡红鲫(Pingxiang red-transparent crucian carp, Carassius auratus var. Pingxiangnensis)主要分布于江西省萍乡地区,是三倍体野生鲫鱼的一种突变种。因其通体呈肉红色、透明而得名肉红鲫,是一种集食用和观赏价值于一体的鲫鱼品种,在2008年被国家水产原良种委员会审定为新的养殖品种-萍乡红鲫。萍乡红鲫可以行雌核发育和两性生殖两种生殖方式。本论文主要包括以下三部分：一、利用RACE-PCR技术克隆得到萍乡红鲫ZP3基因cDNA全长序列,该序列全长1385bp,有一个1308bp的完整开放阅读框,编码435个氨基酸,经过氨基酸序列比对,121-376位氨基酸残基属于ZP家族domain超家族结构域。二、利用荧光定量PCR的方法检测了ZP3基因在萍乡红鲫卵巢、心脏、肝脏、脾脏、肾脏、脑等组织中的差异性表达,相对定量分析结果显示ZP3基因在肝脏中表达量最低,在卵巢组织中的表达量显著多于其他五个组织,约是肝脏中的26656倍,除卵巢组织外,ZP3基因在所检测的其他五个组织中都有微量的表达。三、将纯化好的ZP3蛋白作为抗原免疫白兔,制备得到了多克隆兔抗鱼ZP3蛋白血清。用Elisa间接法检测了抗血清的效价。将得到的抗血清用于卵母细胞冰冻切片免疫荧光定位实验。Elisa实验结果显示：抗血清效价约为25600且抗血清的特异性良好。免疫荧光实验结果显示：在早期发育的卵母细胞中,就有少量的ZP3蛋白存在,随着卵母细胞的发育,ZP3蛋白的合成累积量逐渐增加。在V时相的卵母细胞中,ZP3蛋白在卵母细胞细胞膜上和细胞质中的存在量达到了最大。ZP3蛋白在精卵识别的过程中起到了关键作用。本研究为进一步了解萍乡红鲫ZP3蛋白在受精时的具体功能,提供了一点分子生物学方面的数据。【英文摘要】Pingxiang red-transparent crucian carp, Carassius auratus var. Pingxiangnensis, mainly distributed in Pingxiang city of Jiang Xi Province, is a triploid wildness crucian carp mutant. For its whole transparent body, it was named Carassius auratus var. Pingxiangnensis. The fish is a good crucian carp specie for edible value and ornamental value, and it is approved by national aquatic original improved variety committee as a new cultural fish specie in 2008. Carassius auratus var. Pingxiangnensis has two modes of reproduction,which includes bi-sexual and gynogenetic reproductive mode.The content of this research includes three parts:First:The ZP3 cDNA sequence from Carassius auratus var. Pingxiangnensis is cloned by the method of RACE-PCR. Its full length is 1385bp, and an ORF is 1308bp for encoding a peptide of 435 aa. By blasting of amino acid sequence, amino acid residue from 121 to 376 aa belongs to ZP family domains.Second:The ZP3 mRNA expression levels of different tissues in Carassius auratus var. Pingxiangnensis are detected by the technology of real-time PCR. The tissues include heart, liver, spleen, kidney, brain and ovary. The results of relative quantitative analysis show that mRNA expression in the liver was the lowest, and the ovary posses more higher expression level than other tissues. The ZP3 mRNA expression in the ovary is about 26,656 times as much as in the liver. Except for the ovary, there is a slight expression in all the other tissues.Third:The purified ZP3 protein is used as antigen to immunize the rabbits. The polyclonal antibody of rabbit-anti-fish serum is prepared. The potency of antiserum is detected by Elisa. The antiserum is used for oocytes frozen section immunofluorescence localization experiments. The result of Elisa shows that the potency of antiserum is about 25,600 and the specificity of antiserum is good. Immunofluorescence results show that:ZP3 protein existed in the early developmental oocytes, and with the development of oocytes, ZP3 protein synthesis accumulation gradually increases.In the V growth period of oocyte,the cumulant of ZP3 protein on the cell membrane and in the cytoplasm reachs maximum.ZP3 protein plays a great important role in the period of sperm-egg recognition. In order to investigate the function of ZP3 protein in the period of fertilization, this research provide a few bio-molecular dates.【关键词】萍乡红鲫 ZP3 荧光定量PCR RACE-PCR 免疫荧光【英文关键词】Carassius auratus var. Pingxiangnensis ZP3 RACE-PCR real-time PCR Immunofluorescence【目录】萍乡红鲫ZP3蛋白组织差异性表达及其在卵母细胞中免疫荧光定位摘要3-4ABSTRACT4-5第一章 文献综述9-161.1 卵透明带(zona pellucida,ZP)研究进展91.2 关于ZP3的研究进展9-141.2.1 关于ZP3 cDNA序列克隆10-111.2.2 关于ZP3基因转录和蛋白的合成位点11-121.2.3 关于ZP3精子受体功能12-131.2.4 ZP3蛋白在不同卵母细胞发育时期中的存在量和表达量13-141.3 本论文的研究目的和意义14-16第二章 萍乡红鲫ZP3基因cDNA序列克隆及其组织差异性表达分析16-392.1 材料和方法16-232.1.1 实验材料162.1.2 RACE-PCR引物设计16-172.1.3 萍乡红鲫多个组织总RNA提取17-182.1.4 用SuperScriptTM RT合成ZP3 cDNA第一链18-192.1.5 扩增萍乡红鲫ZP3基因cDNA全长19-202.1.6 回收目的片段,与载体连接,感受态细胞制备以及转化测序20-222.1.7 荧光定量PCR检测ZP3基因在萍乡红鲫不同组织中的差异表达22-232.2 实验结果与分析23-362.2.1 多个组织RNA的提取23-252.2.2 萍乡红鲫ZP3基因全长克隆25-272.2.3 萍乡红鲫ZP3基因cDNA序列及其氨基酸序列分析27-312.2.4 萍乡红鲫ZP3基因的进化以及同源性分析31-322.2.5 荧光定量PCR检测萍乡红鲫ZP3基因组织差异性表达32-362.3 讨论36-39第三章 萍乡红鲫ZP3糖蛋白多克隆抗体制备及在卵母细胞中的免疫荧光定