【作者】 蓝贤勇；

【导师】 陈宏；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 本研究采用了PCR-RFLP、PCR-SSCP、RMAPD、DNA序列分析和微卫星等标记技术分析了西农萨能奶山羊CSN1S2基因、CSN3基因、IGFBP3基因、POU1F1基因、GH基因、FSHR基因的基因座多态及其与经济性状（产奶量、产羔数、初生重、成年体重和体尺指标）的相关性，为奶山羊的改良、开发和利用提供DNA水平的理论依据；同时，还利用PCR-RFLP和PCR-SSCP方法分别检测了西农萨能奶山羊、关中奶山羊、陕南白山羊、安哥拉山羊和布尔山羊5个山羊品种共计175个个体的10个基因座的遗传多态性，为山羊品种资源保护和畜牧业的发展提供资料。本论文通过上述研究首次取得了以下结果：西农萨能奶山羊的PCR-RFLP研究1.1.1 CSN1S2基因:①群体等位基因F/N的频率为0.087/0.913且处于Hardy－Wenberg平衡状态；②西农萨能羊CSN1S2基因座F等位基因与高产奶量呈负相关，如FF基因型个体在平均产奶量、第二胎产奶量上显著或极显著地低于NN型个体(P<0.05，P<0.01)；③作者首次发现CSN1S2基因对产羔数有显著影响(P <0.05)，该基因的产羔效应是由与之连锁FecB基因产生的；FF、NF基因型个体第一胎产羔数较NN型多（P <0.05）；在第四胎产羔数上，NN基因型个体产羔数较NF型和FF型多(P <0.01);④在初生重、体高指标上，NF、FF基因型和NN型间差异显著（P <0.05）。1.1.2 CSN3基因：①CSN3-HaeIII基因座未发现多态性。②CSN3-HindIII和CSN3-TaqI基因位点对产奶量没有显著影响（P >0.05）。③作者第一次证实CSN3、CSN1S2等酪蛋白基因与控制羊产羔数的主效基因FecB连锁，而且它们之间遗传距离很小；CSN3-TaqI位点与FecB位点一样对产羔数有显著影响：AB和BB基因型第一、二胎产羔数均值差异显著或极显著（P <0.05和P <0.01）；CSN3、CSN1S2等酪蛋白基因可作为产羔性状分子标记的有效侯选基因④CSN3基因对初生重和成年体重等指标上有显著差异（P<0.05,P<0.01）。1.1.3 IGFBP3基因：①利用HaeIII、Alw26I、TaqI分析西农萨能羊IGFBP3基因多态性，结果表明均为单态。作者认为山羊IGFBP3基因第299→302位序列没有丢失酶切位点是导致了山羊未出现HaeIII酶切多态性的根本原因。②IGFBP3<WP=8>基因经酶切后表现明显的山羊、绵羊、水牛和普通牛等的物种间多态性。1.1.4 POU1F1基因：69只西农萨能奶山羊和62只关中奶山羊POU1F1基因的HinfI酶切结果表明，在2个山羊品种种未发现多态性。2 西农萨能羊SNP位点多态的PCR-SSCP检测及与经济性状的相关分析①PCR-SSCP检测表明FSHRexon10基因座无基因多态性；GHexonII基因座处于Hardy－Weinberg平衡态状态，GHexonV基因座存在BB型、AC型、AB型和BC型，处于Hardy－Weinberg非平衡态状态。②GHexonII位点与产奶量不相关（P<0.05）；GHexonV位点与产奶量相关，如不同基因型在第三、四、五、六、七胎产奶量和平均产奶量上存在显著差异(P <0.1,P <0.05，P <0.01)。③GHexonII基因座AB型个体第五胎产羔数显著多于BB型个体(P<0.01)，BB型个体第七胎产羔数优于AB型(P <0.05)；GHexonV基因座BC型个体第三胎产羔数分别比AB、AC型个体多0.250只(P <0.1)。④GHexonII和GHexonV 基因位点对体尺指标没有影响(P >.05)。⑤与牛杂合子类型序列进行DNA序列分析，西农萨能奶山羊FSHRexon10基因座杂合子序列第54位（A→G）的转换导致了Gln→His的氨基酸的变化。⑥经DNA序列分析表明：GHexonV基因座存在SNP位点，其分子机理是该基因座位存在11个位点的转换或者颠换等遗传变化。微卫星标记利用2对微卫星引物对萨能羊进行微卫星分析，结果未发现多态性。4. RMAPD与西农萨能奶山羊经济性状的相关性研究①RMAPD（随机微卫星引物扩增多态DNA）标记是一种新型的分子标记。②RMAPD与西农萨能奶山羊经济性状存在一定的相关性：HEL1-2+F09标记、MFW20-1+F09、HEL1-1+F09标记对产奶、产羔性状和初生重、体高等体尺指标均有显著影响（P<0.05）。 5 5山羊品种遗传多态性的PCR-RFLP检测①CSN1S2、CSN3-HindIII、CSN3-TaqI、CSN3-HaeIII、IGFBP3-HaeIII和β-1g等6个基因座的PCR-RFLP检测结果表明：CSN3-HaeIII和IGFBP3-HaeIII基因座未表现多态性，而CSN3-HinfI基因座的A等位基因为稀有基因仅在关中奶山羊中低频率表现；②对6个乳蛋白基因座进行数据分析，得到结果：关中奶山羊的遗传多样性最高，其次是西农萨能奶山羊，陕南白山羊和布尔山羊的遗传多态性最低；6 5山羊品种遗传多态性的PCR-SSCP检测 <WP=9>GHexonII、GHexonV 和FSHRexon10以及CSN3exonIV等4个基因座位的PCR-SSCP检测结果表明：5个山羊品种PIC均值在0.1805-0.3281之间，其中西农萨能奶山羊遗传多态性最丰富，其PIC值最高达到0.3281，其次是关中奶山羊（PIC=0.3000），最低的是安哥拉山羊和布尔山羊（PIC分别为0.1805和0.1875）；更多还原

【Abstract】 DNA molecular marker technology , such as PCR-RFLP, PCR-SSCP, RMAPD (Random-microsatellite amplified polymorphic DNA), DNA sequence analysis, microsatellite marker, were applied to evaluate the effects of genotypes of six candidates genes (CSN1S2 gene, CSN3 gene, GH gene, IGFBP3 gene, POU1F1 gene, FSHR gene) in Xinong Saanen Dairy goat; And PCR-RFLP and PCR-SSCP was used to detect genetic diversity of five goat breeds (including Xinong Saanen dairy goat, Guanzhong dairy goat, Shaanan White goat, Angora goat and Boer goat) . The main goal were to construct the foundation of making good use of economic traits (milk performance, litter sizes and body sizes) in Xinong Saanen dairy goat and protecting breed resources, so that molecular theory about breeding in Xinong Saanen dairy goat and constructing useful database in five goat breeds were provided primary. The results were as follows:1. Research on PCR-RFLP in Xinong Saanen dairy goatCSN1S2 gene: (1) In 69 Xinong Saanen dairy goats population which was at Hardy-weinberg equilibrium, the frequencies of allele F/N for CSN1S2 gene loci were 0.087/0.913; (2) Allele F was always bound with lower milk yield, such as genotype FF individuals showed lower milk yield than that of genotype NN in average milk yield and 2nd milk lactation (P<0.05, P<0.01). (3) CSN1S2 gene locked with FecB gene affected litter sizes because different genotypes for CSN1S2 gene loci had significant difference in 1st litter sizes and 4th litter sizes (P<0.05,P<0.01). Besides, there was also significant difference among genotypes in body sizes (P<0.05). CSN3 gene: No polymorphisms for CSN3-HaeIII gene loci was detected; The locus of CSN3-HindIII and CSN3-TaqI had no significant effect on milk performance; It was verified firstly in this trial that CSN3, CSN1S2, CSN1S1 and CSN2 gene locks with FecB gene because litter sizes of CSN3-TaqI gene loci had significant difference among different genotypes in 1st litter sizes and 2nd litter sizes(P<0.05, P<0.01). Hence CSN3, CSN1S2, CSN1S1 and CSN2 genes can be regarded as candidate genes for effective molecular marker about litter sizes.By the way, the polymorphic loci also affect some body sizes (P<0.05).IGFBP3 gene: The results about restriction nucleauase HaeIII、Alw26 and TaqI digesting IGFBP3 gene demonstrated singular genotype. With DNA sequence analysis for DNA sequence FSHRexon10 of from bovine heterozygote and Xinong Saanen goat , no changes in HaeIII restiction cleavage site (form 299th to 302nd ) resulted in no polymorphism of IGFBP3 gene. While obvious <WP=11>polymorphisms of IGFBP3 gene digested with HaeIII appeared among goat, sheep, buffalo and bovine.POU1F1 gene: In Sixty-nine individuals of Xinong Saanen dairy goat and sixty-two individuals of Guanzhong dairy goat populations, there were no polymorphism at HinfI cleavage site in POU1F1 gene. 2. The polymorphisms of SNP loci detected by PCR-SSCP in Xinong Saanen dairy goat and its relationship with economic traits No polymorphism was discovered in FSHRexon10 gene loci; while GHexonII gene showed polymorphism in Xinong Saanen dairy goat population which was at Hardy-weinberg didn’t associated with milk yield (P<0.05), and GHexonV gene concluded genotype AB,AC, BB, BC which showed significantly difference in milk performance in 3rd milk yield , 4th milk yield , 5th milk yield , 6th milk yield and 7th milk yield and average milk yield . Moreover, the different genotypes in GHexonII and GHexonV gene loci affected significantly litter sizes in 3rd litters sizes, 5th litter sizes and 7th litter sizes. To analyze SNPs of GHexonV gene by DNA sequence analysis in this population, there was transition or transversion in 11 places. As far as difference between goat and bovine in FSHRexon10 was considered, the result of DNA sequence analysis showed amino acid change for Gln→His appears in 54th loci for goat.3. Microsatellite MarkerIn this trial, two anonymous microsatellite primer from fish were amplified in Xinong Saanen goat genomic DNA and no polymorphism was detected.4. RMAPD更多还原