【作者】 王训翠；

【导师】 陈宏权；

【作者基本信息】 安徽农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 摘 要本研究建立了PCR-SSCP和PCR-RFLP两种方法。以PTGS2基因和PRL基因的多态性为研究内容，旨在探索此两个候选基因与小尾寒羊高繁殖力之间的关系，为我国开展小尾寒羊高繁殖力的遗传学基础研究提供科学依据。应用PCR-SSCP和PCR-RFLP技术对两组样本进行PTGS2基因和PRL基因多态性分析。一组为有产羔记录的小尾寒羊血样；另一组为四个无产羔记录的绵羊品种血样（湖羊，特克塞尔羊、萨福克羊、多赛特羊）。得到如下结果：PTGS2基因两对引物扩增片段研究发现：引物1扩增片段即启动区经SSCP分析不存在多态性。引物2扩增片段即外显子3到外显子5区域经RFLP分析存在酶切位点的突变，其中936位为多态切点，当该位点发生变异而导致该酶切位点出现时，产生420bp和516bp的两个片段。具有两种基因型（AA、AB），两个等位基因（A、B）。A基因频率明显高于B基因频率，遗传特性（杂合度、多态信息含量（PIC）和有效等位基因数）在五个绵羊品种差异不明显，特克塞尔羊的有效等位基因数、杂合度和PIC最小，分别为1.0951、0.0869、0.0831；萨福克羊的有效等位基因数、杂合度和PIC最大，分别为1.4204、0.2960、0.2522。对于多态信息含量而言，除萨福克羊达到中度多态外，其余四个绵羊品种均属于低度多态。经X2适合性检验，五个绵羊品种此扩增片段均处于Hardy-Weinberg平衡状态。在有产羔记录的小尾寒羊中，引物2扩增片段与繁殖性状的最小二乘分析结果表明，对于小尾寒羊第一胎和第二胎产羔数，不同基因型对应的最小二乘平均值关系都是AB>AA，但影响均未达到显著水平。PRL基因两对引物扩增片段研究发现：引物2扩增片段即外显子5经SSCP分析不存在多态性。引物1扩增片段即5＇端调控区经SSCP分析存在位点突变。在有产羔记录的小尾寒羊中，出现两种基因型（AA、AB），两个等位基因（A、B），且A基因频率明显高于B基因频率，有效等位基因数、杂合度和PIC分别为1.0960、0.0878、0.0839，为低度多态。其余四个无产羔记录的绵羊品种均只发现了一个等位基因，达到了纯合。经X2适合性检验，五个绵羊品种此扩增片段均处于Hardy-Weinberg平衡状态。引物1扩增片段的突变序列进行克隆和测序分析，结果表明在5＇端调控区发生了一处碱基突变，即第63个碱基由G突变成T。此位点突变可能对PRL基因的转录调控有一定的影响，但尚需进一步确证。在有产羔记录的小尾寒羊中，引物1扩增片段与繁殖性状的最小二乘分析结果表明，对于第一胎产羔数，AB型小尾寒羊产羔数比AA型平均多0.50只，影响达到0.05<WP=8>显著水平（P<0.05）。对于第二胎产羔数，AB型小尾寒羊产羔数比AA型平均多0.57只，影响达到0.05显著水平（P<0.05）。本研究中发现的PTGS2基因引物2扩增片段多态性和PRL基因引物1扩增片段多态性可能与繁殖性状有关，但还需进一步研究。更多还原

【Abstract】 The methods of PCR-SSCP and PCR-RFLP were established in this study. Based on the genetic polymorphism of PTGS2 gene and PRL gene, we try to link these candidate genes with the high productivity of Small Tail Han sheep, to further provide scientific evidence for the basic genetic research on Small Tail Han sheep high productivity.Applying the powerful PCR-SSCP and PCR-RFLP technique to analyze the polymorphism of prostaglandin-endoperoxide synthase 2 gene and prolactin gene, we divided our samples into two groups, one group is the blood sample of Small Tail Han sheep; another group is the blood sample of four different sheep breeds without littering records (Hu sheep, Texel sheep, Suffolk, Dorset). The results were as follows: fragments amplified by two primers of PTGS2 gene showed that the fragment amplified by primer 1 namely promoter region didn’t exhibit polymorphism by SSCP analysis, that the fragment amplified primer 2 namely region of spanning from exon 3 to exon 5 exhibited base difference by RFLP analysis. Thereinto 936 locus was polymorphic locus, when this locus exhibited base mutation, 420bp and 516bp were digested. All five sheep breeds had two genotypes (AA and AB) and two alleles (A and B).A gene frequency was significantly higher than B gene frequency. The difference of the genetic characteristic (heterozygosity, polymorphic information content (PIC) and effective number of alleles) was not evident in five sheep breeds. The effective number of alleles, heterozygosity and PIC of Texel sheep were least at this locus. They were 1.0951、0.0869、0.0831 separately. The effective number of alleles, heterozygosity and PIC of Suffolk sheep were most. They were 1.4204、0.2960、0.2522 separately. As for polymorphic information content, except Suffolk sheep achieving moderate polymorphism, other four sheep breeds were all intermediate polymorphism.X2 fitness test showed that fragment amplified by primer 2 is in a state of Hardy-Weinberg Equilibrium.Among Small Tail Han sheep with littering records, least squares analysis showed that fragments amplified by primer 2 had no significant effects on the first littering records and the second littering records .Fragments amplified by two primers of PRL gene showed that the fragment amplified by primer 2 namely exon 5 region didn’t exhibit polymorphism by SSCP analysis, that the fragment amplified primer 1 namely 5＇flank region exhibited base difference by SSCP analysis. Olny Small Tail Han sheep with littering records had two genotypes (AA and AB) <WP=10>and two alleles (A and B). A gene frequency was significantly higher than B gene frequency. The effective number of alleles, heterozygosity and PIC of were 1.0960、0.0878、0.0839 separately, The PIC belonged to intermediate polymorphism. Only one allele was found in other four sheep breeds without littering records. X2 fitness test showed that fragment amplified by primer 1 was in a state of Hardy-Weinberg Equilibrium. We cloned and sequenced fragment amplified by primer 1，which included one mutation locus.Our sequences show that there was one mutation (C→A) in 63bp of 5＇flank of prolactin gene. Maybe the locus mutation had influence upon transcription and regulation, but need studies to conform it.Among Small Tail Han sheep with littering records, least squares analysis showed that fragments amplified by primer 1 had significant effects on the first littering records (P<0.05), AB genotype was much more 0.5 than AA genotype, and also had significant effects on the second littering records (P<0.05), AB genotype was much more 0.57 than AA genotype.According to all of our results, we conclude that the polymorphism of fragment amplified by primer 2 of prostaglandin-endoperoxide synthase 2 gene and fragment amplified primer 1 of prolactin gene probably associated with Small Tail Han sheep productive trait, but need further studies.更多还原