【作者】 汪晓鸿；

【导师】 刘娣；

【作者基本信息】 东北农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 骨桥蛋白(OPN)是一种含精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp，RGD)的分泌型糖基化磷蛋白，归类于细胞外基质蛋白。有研究表明OPN在分泌期子宫内膜上皮细胞、侵袭的滋养细胞、蜕膜腺上皮、胎盘上都有表达。且OPN在哺乳动物的母-胚界面持续表达，人们推测OPN是影响子宫-胚胎微环境的重要因素。因此，应用现代分子生物学技术研究猪OPN基因的分子遗传结构和时空表达，对研究猪的繁殖性状的候选基因具有十分重要的理论意义。本研究以大白猪、野猪和野家杂种猪为研究对象，克隆了大白猪和野猪的OPN基因，对其结构功能及分子进化进行了分析，并对大白猪和野家杂种猪的OPN基因在心脏、肝脏、胃、脾脏、肾脏、肺脏、大肠、小肠、肌肉、子宫和卵巢的表达情况进行了比较分析。主要研究结果如下：通过克隆测序的方法克隆了大白猪和野猪OPN基因的全长cDNA序列，通过序列比对发现，野猪的变异位点较大白猪群体内的变异位点丰富。通过对OPN基因的结构功能域的分析发现，发现OPN蛋白仅有一个OPN特征性功能域，功能相对单一，对其进行系统发育分析表明猪与牛、水牛、羊有较高的同源性，而鸡的变异程度稍大，处于进化树的末端。半定量RT-PCR检测结果表明，大白猪OPN基因在不同生长发育阶段的心脏、肝脏、胃、脾脏、肾脏、肺脏、大肠、小肠、肌肉、子宫和卵巢共11个组织中均有表达，但不具有明显规律性，而在性腺组织中的表达表明OPN表达随着日龄的增加，逐步增高，270日龄达到高峰，以后有所下降。OPN基因在不同日龄的大白猪和野家杂种猪的不同组织的表达情况不同。而OPN基因在大白猪和野家杂种猪性腺组织表达来看都存在一个先高后低，以后达到高峰，大白猪出现在270日龄，而野家杂种猪出现在360日龄。采用Western blot技术检测了大白猪不同组织的骨桥蛋白表达情况，结果表明不同组织表达情况不同，心脏、肺脏、子宫表达70kDa和45kDa的蛋白，胃、肾脏、小肠表达70kDa、45kDa和25kDa的蛋白而肝脏和肌肉只表达70kDa的蛋白。利用诱导表达和RT-PCR相结合的方法，检测了在子宫内膜上皮细胞里添加不同的激素对OPN表达的影响，结果发现孕激素正调控OPN基因的表达而雌激素负调控OPN基因的表达。更多还原

【Abstract】 Osteopontin (OPN) is an secretion type glycosylation phosphoprotein, which contains anArg-Gly-Asp (RGD) sequence, and it is classified a component of the extracellular matrix (ECM).The subsequent research found that OPN was expressed in cellula epithelialis uterineendomembrane, the invaded trophoblastic cell, the glandular epithelium of decidua and placenta inthe secretion phase. Furthermore, OPN is expressed on the maternal-fetal interface continuouslyand inferred that OPN play a key role in the regulation of uterine-placenta microenvironment.Therefore, it has important significance for studying on pig’s candidate genes of reproductive traitthat molecular genetic construction and space-time expression of OPN gene is studied by modernmolecular biological technology.This research applied to Yorkshire, wild boar and Crossbred pig as the object of studying,OPN gene of Yorkshire and the wild boar was cloned, and its structure function and the molecularevolution had analyzed, at the same time, expression situation of OPN gene of Yorkshire andCrossbred was analyzed in the heart, the liver, the stomach, the spleen, the kidney, the lung, thelarge intestine, the small intestine, the muscle, the uterus and the ovary. Main research results asfollows:Getting completed OPN cds of Yorkshire and wild boar. From sequence comparation we foundthat there are five mutant sites in Yorkshire and eight in wild boar. The analysis on domin of OPNindicate that there is only one characteristic domin in OPN. Phyletic anlaysis found that OPN of pighas high homology with cattle, bufflo,and sheep but low with fowl.The results of RT-PCR indicate that OPN of Yorkshire expressed in eleven tissues at diffentdevelopmental stage, and did not have obviously regulatory. In the glandular tissue, OPN expressedincreasingly as time passed by. The results of semi-quantitative RT-PCR indicate that OPNexpressed differently in Yorkshire and wild family hybrid pig. But both in Yorkshire and wildfamily hybrid pig the expression trend is high to low to high and reach the peak. The peak ofYorkshire is in the 180 days and wild family hybrid pig is 360 days.The results of Western blot indicate that OPN expressed differently in different tissues. OPNmotified differently in different tissues, which lead to different hydrolization degree and diversityexpression format.Induced expressing and RT-PCR detecting OPN expreesion variation in endometrium endothelial cell with different hormones. The detected results found that pregnancy hormone increased OPNexpressing but hormones decreased.更多还原