【作者】 余梅；

【导师】 李奎；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2003， 博士

【摘要】 猪是为人类提供动物蛋白的最主要畜种之一，孟德尔定律的重新发现和数量遗传学的诞生及其在猪育种中的应用，使得猪的常规育种取得了很大成效。而高密度猪基因图谱的建立，以及对猪基因的结构、功能及与性状表型关系等的充分了解，则将是应用分子育种技术对猪重要经济性状进行遗传改良的基础。 本论文根据比较基因定位信息，分离、鉴定和定位猪12号染色体上的新基因，取得了如下结果： 1．依据人17号、小鼠11号染色体上的同源基因保守序列设计CATS引物，从二花脸猪基因组中分离克隆了四个猪中新基因片段，并提交GenBank数据库，这四个基因分别是神经胶质细胞原纤维酸性蛋白(GFAP)基因，GenBank收录号AF250778；铁氧还蛋白还原酶(FDXR)基因，GenBank收录号AF317683；杆细胞磷酸二酯酶γ亚单位(PDEG)基因，GenBank收录号AF317684；蛋白酶体激活亚单位3(PSME3)基因，GenBank收录号AF317685。 2．用猪×啮齿类体细胞杂种板将四个新分离的猪基因进行染色体区域定位，GFAP基因区域定位结果为SSC12p11-(2/3)p13(P=0.8897，与该区域标记间的相关系数为0.9177，P＜0.1％)，FDXR基因被定位于SSC 12(1/3 p13)-p15(P=0.8989，与该区域标记间的相关系数为0.9250，P＜0.1％)，PDEG基因位于SSC12(1/3 p13)-p15(P=0.8181，与该区域标记间的相关系数为0.9220，P＜0.1％)，PSME3基因定位于SSC12p11-(2/3)p13(P=0.8901，与该区域标记间的相关系数为1.000，P＜0.1％)。 3．用猪辐射杂种板将四个新分离的猪基因进行染色体精细定位，结果这四个基因均与猪12号染色体上的基因或标记紧密连锁，GFAP基因与GH紧密连锁 (LOD=9.17)，FDXR基因与S0143紧密连锁(LOD=12.36)，PDEG基因与SW2490紧密连锁(LOD=7.78)，PSME3基因与GH紧密连锁(LOD=6.52)。 4．将四个新基因的定位结果与已公布的猪12号染色体RH图谱整合，获得一张猪12号和人17号染色体的同源基因比较定位图，发现这四个新基因在猪12号染色体上的排列顺序是PDEG-FDXR-PSME3-GFAP，而在人17号和小鼠11号染色体上，PSME3和GFAP这两个基因的排列次序是相反的。 5．采用PCR—RFLP分析技术，在不同群体中检测了FDXR的HhaⅠ-RFLP，PDEG的MspⅠ-RFLP多态性，计算了基因型频率和基因频率，分析了各等位基因在不同猪群中的分布差异。并发现了位于PSME3基因第6内含子的HhaⅠ-RFLP和第8外显子的AluⅠ-RFLP多态性位点。华中农业大学博士学位论文 6.在大白x通城，长白，x通城猪的上下代个体中分析了尸ay及、尸刀心G和尸夕团孙基因上述多态性位点的遗传方式，结果表明均呈共显性遗传方式。 7.在群体中检测到只引困孙基因的长度多态性，进一步分析该多态性产生的原因发现是由于第5内含子中存在一段147bp的缺失突变。 8.利用猪EST数据库的信息，以人尺饥伍3基因编码区序列为信息探针，采用电脑克隆策略并结合RT.PCR技术，获得猪只孙刃刃基因的完整编码区序列，推导出相应的氨基酸序列。将所得到的核昔酸序列和推导的氨基酸序列与其它物种相应序列进行同源性比较，结果表明猪尸及团叨基因在核昔酸水平上与人的同源性为%%，而推导的氨基酸序列与人和小鼠的同源性达100%。 9.以荷兰动物科学与卫生研究所Marinus te Pas博士赠送的大白猪群为实验材料，分析了尸凡阴百了基因与部分生产性状的关联，结果发现第8外显子中的Alul-RFLP位点两种基因型AA和AB的170日龄屠宰时的背膘厚有显著差异(P<0.05)。更多还原

【Abstract】 Pig is one of the predominant meat producing around the world and so the pig industry is of economic importance to both developed and developing countries. The rediscovery of the law of Mendel and the establishment of quantitative genetics caused a sea change in animal breeding. In order to make the genetic improvement in important economic traits of pigs a rapid progress by using the molecular breeding approach, further effort should be made to establish high-density gene map and have full understand about the gene structure, function and their association with the economic traits. The objective of this thesis was to isolate, characterize and physically map the novel genes on pig chromosome 12 by use of information derived from comparative mapping. The main results are as follows:1. The CATS primers were designed from well conserved-sequence regions of homologous genes between the human chromosome 17 and the mouse chromosome 11. Using the CATS primers, four novel porcine gene fragments were isolated and identified from Erhualian genomic DNA. The four novel porcine genes were as follows: glial fibrillary acidic protein (GFAP) gene ( GeneBank accession number: AF250778), ferredoxin reductase (FDXR) gene ( GeneBank accession number: AF317683), rod cGMP-phosphodiesterase gamma-subunit (PDEG) gene (GeneBank accession number: AF317684), and proteasome activator subunit 3 (PSME3) gene ( GeneBank accession number: AF317685).2. The pig ×rodent somatic cell hybrid panel (SCHP) was used for regional assignment of the four genes on the pig chromosome. SCHP analysis mapped all the four genes to porcine chromosome 12p (SSC12p). The GFAP and PSME3 were localized to SSC12pll- (2/3) p13 with error risk <0.1%. The probability of regional localization and concordance for the two genes was 0.8897, 0.9177 and 0.8901, 1.0, respectively. The FDXR and PDEG genes were physically mapped to SSC12 (1/3 p13)-p15 with error risk <0.1%. The probability of regional localization and concordance for the two genes was 0.8989, 0.9250 and 0.8181, 0.9220, respectively.3. The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of the four genes. Statistical analysis revealed that both the GFAP and PSME3 genes were located closely to GH already placed on chromosome 12, with LOD score threshold 9.17 and 6.52, respectively. The FDXR and PDEG genes, which were mapped on the other linkage group, were closely linked to SO 143 and SW2490, which both had been localized on chromosome 12, withLOD score threshold 12.36 and 7.78, respectively.4. An improved RH comparative map of human chromosome 17 (HSA17) and pig chromosome 12 (SSC12) was established by integrating the four genes with the recently published first-generation porcine whole-genome radiation hybrid map. The most likely order of the four genes on chromosome 12 given by the two-point distance was PDEG-FDXR-PSME3-GFAP.Comparing the SSC12 framework map with HSA17 GB4 map, an inverted gene order between PSME3 and GFAP was observed.5. A search for mutation in the amplified fragment of the four genes was performed by PCR-RFLP. The polymorphism of FDXR Hhal-RFLP and PDEG MspI-RFLP were detected and allele frequencies among different pig populations were also determined. RFLP screening with the restriction digestion enzymes Hhal and Alul revealed two polymorphic sites within PSME3 gene .The Hhal site is located in intron 6,whereas the’Alu! site is placed within exon 8.But the GFAP fragment obtained with GFAP CATS primer did not show any polymorphism.6. Codominant inheritance of the FDXR Hhal-RFLP, PDEG MypI-RFLP and PSME3 Hhal-RFLP were demonstrated in the Landrace x Tongcheng and Large White x Tongcheng two-generation pedigrees.7. Within intron 5 of PSME3 gene, a length variation caused by a 147bp fragment deletion was observed. Genotype and allele frequencies among several pig populations were identified.8. Using the information available in the EST database, th更多还原