【作者】 刘慧；

【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， 2008， 硕士

【摘要】 腺苷一磷酸脱氨酶(AMPD)是仅在真核生物中发现的一种酶,是嘌呤代谢中的关键酶之一,它的作用是催化AMP(一磷酸腺苷)脱氨生成IMP(肌苷酸)。AMPD基因由多基因家族编码,家族成员有AMPD1、AMPD2、AMPD3。AMPD1基因在所有的真核生物中都存在,但主要在骨骼肌中高水平表达。有研究发现AMPD1基因与NGFB基因紧密连锁,因NGFB基因定位区域与肉质胴体性状(如胴体重、肩肉重、肌肉重、肌肉面积等)有关,所以也将AMPD1基因作为胴体性状的候选基因来研究。本研究以人、鼠的AMPD1基因序列为基础,首次克隆了野猪和家猪AMPD1基因的cDNA序列;并且以野猪、民猪、大白、长白、杜洛克为研究对象,利用PCR-SSCP方法检测它们的多态性,探讨不同基因型在不同品种(系)间的分布规律;采用半定量RT-PCR方法研究了这个基因在不同组织及相同组织不同生长时期的表达特性。主要研究结果如下:1.采用同源克隆首次克隆了家猪和野猪AMPD1基因的cDNA序列,猪AMPD1基因编码区全长2244 bp,编码747个氨基酸;野猪AMPD1基因cDNA全长2240 bp,开放阅读框位于399~2240 bp,编码613个氨基酸。采用生物信息学方法对其编码的多肽链进行了结构和功能域的初步分析。2.采用PCR-SSCP方法在AMPD1基因编码区上共寻找到11个单核苷酸突变,其中有4个突变属于错义突变。群体遗传学分析表明,所检测的4个位点中,5个品种间基因型的分布均存在着极显著的差异(P<0.01);野猪和民猪之间除了S5位点基因型的分布存在显著差异外(0.01<P<0.05),其他3个位点上基因型分布差异均不显著(P>0.05)。3.半定量RT-PCR方法检测了野猪和纯种大白猪AMPD1基因在心、肝、胃、肾、脾、肺、肌肉、大肠、小肠、性腺共10种组织表达情况,结果表明猪的AMPD1基因仅在其肌肉组织中表达;同时检测了AMPD1基因肌肉组织不同生长时期的表达特性,结果显示AMPD1基因在1日龄大白猪肌肉组织中表达量最高,其次是7日、15日、21日龄,3月龄表达较少,6月、9月、12月龄几乎不表达,随着日龄的增加,表达量呈依次减弱的趋势。更多还原

【Abstract】 Adenosine monophosphate deaminase(AMPD)has only been found in all eukaryote. It is one of the key enzymes in the purine nucleotide cycle and catalyzes the deamination of AMP to IMP. AMPD is a complex allosteric enzyme encoded by a multigene family in mammals including AMPD1, AMPD2 and AMPD3. The AMPD1 gene is expressed predominantly in skeletal muscles. It is shown from research that AMPD1 was tightly linked with the NGFB gene. The porcine AMPD1 maps within known QTL with effects on carcass traits (carcass weight cold, shoulder meat weight, bacon meat weight, chops weight, head weight and muscle area). So AMPD1 may be regarded as a positional candidate for these traits. This study was designed to clone the cDNA of swine and wild boar AMPD1 gene based on the sequences of human, rat and mouse AMPD1 gene; to detect the polymorphisms by PCR-SSCP in five breeds of pigs; to investigate the expression characteristic of the gene by RT-PCR. The main results are as follows:1. The cDNA sequences of domestic pig and Wild boar AMPD1 gene were cloned in the first time by homologous cloning. The results showed that coding region of swine AMPD1 gene was composed of 2244 bp encoding 747 AA. The cDNA of AMPD1 gene in Wild boar was composed of 2240 bp and the open reading frame encodes 613 AA.2. 11 SNPs in exons of AMPD1 gene were found by PCR-SSCP method, and there were 4 missenses. Population genetics analyses showed that in each polymorphic locus, there was extremely significant difference among 5 breeds in the distribution of genotypes (P<0.01). While no significant difference was found between Wild boar and Min pig except the locus detected by primer S5.3. The expression characteristic of AMPD1 gene in heart, liver, stomach, kidney, spleen, lung, large intestine, small intestine, muscles and gonad tissue was investigated by RT-PCR in Wild boar and Yorkshire. The results showed that the pig AMPD1 gene was expressed only in skeletal muscle. And the expression of AMPD1 in muscle on different growing period in Yorkshire was analyzed. The expression level in muscle of Yorkshire was the most at the age of birth. There was barely expression at 6、9 and 12-month. Along with the increasing of age, the expression level showed a weakening trend.更多还原