【作者】 张佳兰；

【导师】 昝林森；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 本研究分别以中国荷斯坦、澳大利亚荷斯坦、加拿大荷斯坦及陇州奶牛的395份血样为材料提取基因组DNA，运用生物信息学方法和同源序列克隆技术结合电子PCR(ePCR)，对牛的磷酸甘油酰基转移酶ζ(AGPAT6)基因的基因组DNA序列进行了克隆与序列分析。运用PCR-SSCP、PCR-RFLP以及PCR-RFLP、SSCP结合的方法对奶牛泌乳性能两个候选基因的部分基因组DNA片段进行了单核苷酸多态(SNP)检测，运用一般线性模型研究了AGPAT6基因和PRLR基因与上述4个奶牛群体泌乳性能性状的相关性，在5个方面取得了研究进展。1．牛AGPAT6基因的克隆利用快速扩增cDNA末端(RACE)技术克隆了牛AGPAT6基因5′cDNA，用PCR技术克隆到牛AGPAT6基因的8个内含子序列，获得了包含外显子和内含子的AGPAT6基因的基因组DNA片段，并为GenBank数据库收录，收录号为EF432784。2．牛AGPAT6基因SNPs及其与奶牛泌乳性能性状的关系检测牛AGPAT6基因部分序列，发现了11个SNPs，并分析了这些SNPs与泌乳性能性状的关系，结果如下：(1)在第2内含子发现了1个PCR-SSCP SNP位点(281T-281C)，此PCR-SSCP(281T-281C)位点不同基因型的个体的305天产奶成年当量(305dME)、乳脂率和乳蛋白率存在显著差异(P＜0.05)：杂合型个体的305dME显著高于纯合基因型个体(P＜0.05)，BB基因型个体的乳脂率和乳蛋白率显著高于AA基因型个体(P＜0.05)。(2)在第3内含子发现了5个SNPs位点(410G-410T、489G-489T、600G-600T、649G-649A、729-729A)，其中的1个PCR-RFLP SNP(410G-410T)位点不同基因型的个体，乳脂率和乳中体细胞评分(SCS)存在显著差异(P＜0.05)；BB基因型个体的乳脂率显著高于AA基因型(P＜0.05)，BB基因型个体的SCS显著低于AA基因型(P＜0.05)。第3内含子的另一个PCR-RFLP SNP(489G-489T)位点不同基因型的个体，305dME和乳脂率存在显著差异(P＜0.05)；CC基因型的305dME显著高于DD基因型(P＜0.05)，DD基因型个体的乳脂率显著高于CC基因型(P＜0.05)。(3)在第4内含子发现了2个PCR-SSCP SNP位点(153A-153G、606A-606G)，位点组合不同基因型的个体SCS存在显著差异(P＜0.05)；在第10、11内含子发现了3个PCR-SSCP SNP(341A-341G、430C-430T、451C-451G)，位点组合不同基因型的个体305dME、乳脂率和SCS存在显著差异(P＜0.05)：在第6、7、8、9内含子均来发现PCR-RFLP多态。3．牛PRLR基因SNPs及其与奶牛泌乳性能性状的关系检测牛PRLR基因部分序列发现了11个SNPs，分析这些SNPs与泌乳性能性状的关系，结果如下：(1)在第3外显子发现了3个PCR-SSCP SNPs位点(1104A缺失、1267A-1267G、1268C-1268T)，3个SNPs位点不同基因型的个体305dME存在极显著差异(P＜0.01)；AD基因型的305dME最高。(2)在第5外显子发现了1个PCR-SSCP SNP位点(3365T-3365C)，此SNP位点不同基因型的个体泌乳性能性状差异不显著(P＞0.05)。(3)在第7外显子发现了4个PCR-SSCP SNPs位点(5533C-5533T、5650A-5650G、5651G-5651A、5652A-5652T)，4个SNPs位点不同基因型的个体305dME、乳脂率、乳蛋白率和SCS存在显著差异(P＜0.05)：AA基因型个体的305dME最低，但乳脂率和乳蛋白率最高，AC基因型个体的SCS显著高于其他基因型个体。(4)在第8内含子发现了1个PCR-RFLP SNP位点(7740A-7740G)，该位点不同基因型的个体305dME存在显著差异(P＜0.05)、乳蛋白率存在极显著的差异(P＜0.01)：AA基因型个体的305dME较高、BB基因型个体的蛋白率极显著高于其他基因型个体。(5)在第10外显子发现的SNPs存在群体间差异。在澳大利亚荷斯坦牛群体发现1个PCR-SSCP SNP位点(9742A-9742G)，在加拿大荷斯坦个群体发现2个PCR-SSCP SNPs位点(9681C-9681T、9902G-9902C)，在陇州奶牛群体中发现3个PCR-SSCP SNP位点(9681C-9681T、9757G一9757T、9939G插入)，在中国荷斯坦群体发现3个PCR-SSCP SNP位点(9681C-9681T、9742A-9742G、9902G-9902C)，5个SNPs位点不同基因型的个体305dME存在极显著差异(P＜0.01)，乳脂率、乳中干物质含量和SCS存在显著差异(P＜0.05)：DD基因型个体的305dME极显著的高于其他基因型，EE基因型个体的乳脂率显著高于CC、DD基因型个体，CC基因型个体的SCS显著高于BB基因型个体。4．基因型的组合效应对奶牛泌乳性能的影响组合AGPAT6基因第2内含子和第3内含子的SNPs位点不同基因型分析其与奶牛泌乳性能的关系，结果表明：AGPAT6基因第2内含子和第3内含子的SNPs位点间存在互作效应，其组合效应值均高于第2内含子和第3内含子SNPs的单标记效应值：组合1(A1A1A2A2C2C2基因型)个体的305dME和乳脂率高于其他组合。因此，可初步确定SNPs组合位点(281T、410G、489G)为影响牛乳品质的最佳SNPs组合基因型。组合PRLR基因第3外显子、第7外显子、第8内含子和第10外显子的SNPs位点不同基因型分析其与奶牛泌乳性能的关系，结果表明：PRLR基因第3、7、10外显子和第8内含子的SNPs位点存在互作效应，其305dME组合效应值均高于第3、7、10外显子和第8内含子SNPs的单标记效应值：组合4(A1A1B3B3A5B5E4E4)个体的305dME高于其他组合，可初步确定组合4(A1A1B3B3A5B5E4E4)为影响牛产奶量的最佳组合基因型。5．AGPAT6、PRLR基因多态与中国荷斯坦奶牛群体泌乳性能的关系在中国荷斯坦牛品种内，分析了AGPAT6和PRLR基因上17个SNPs位点不同基因型与奶牛泌乳性能不同性状的关系，结果显示：PRLR基因第3外显子3个SNPs位点不同基因型个体的初次配种日龄存在显著差异(P＜0.05)、初次产犊日龄存在极显著差异(P＜0.01)，BB基因型个体的初次配种日龄显著地高于其他基因型个体，初次产犊日龄存在极显著地高于其他基因型个体。第5外显子的SNP位点不同基因型个体的乳脂率和乳蛋白率存在显著差异(P＜0.05)，AA基因型个体的乳脂率和乳蛋白率显著地高于BB基因型个体。第7外显子4个SNPs化点不同基因型的个体305dME和初次产犊日龄存在显著差异(P＜0.05)；BB基因型个体的305dME最高，AA基因型个体的初次产犊日龄最高。第10外显子的5个SNPs位点不同基因型个体的乳脂率存在显著差异(P＜0.05)；DD基因型个体的乳脂率和乳干物质含量显著低于FF基因型个体(P＜0.05)。更多还原

【Abstract】 395 blood samples from 4 dairy cattle populations, Chinese Holstein, Canadian Holstein, Australia Holstein and Longzhou dairy cattle were collected to extract genomic DNA in this study. The identification and sequence analysis to the bovine acyl-sn-glycerol-3-phosphate acyltransferaseζ(AGPAT6) gene was performed by homology cloning and bioinformatics approach combined with 5’ RACE technique. Afterwards, detection of SNPs within some genomic fragments of the AGPAT6 gene and PRLR gene were employed with the use of sequencing and PCR-SSCP, PCR-RFLP method. Finally, the association among AGPAT6 and PRLR gene with milk performance traits of dairy cattle in 4 populations were investigated. The advances have been acquired in 5 points.1. Cloning of bovine AGPAT6 geneThe RACE technology was used to clone bovine AGPAT6 gene 5’-cDNA, and PCR technology was used to clone AGPAT6 gene 8 introns. Exons and introns contained in AGPAT6 genomic DNA were rebuild one sequence that was accepted by GenBank database, its accession number was EF432784.2. Relativity of PCR-SSCP or PCR-RFLP SNPs of the bovine AGPAT6 gene with milk performance traits of dairy cattleIdentification for mutations in the amplified fragments of AGPAT6 gene was performed and the 11 SNPs were detected by PCR-SSCP or PCR-RFLP. These SNPs were genotyped in the experimental populations. The analysis of general lineal model was used to employ to association between the SNPs and milk performance traits of dairy cattle. The results showed as follows:(1) 1 SNP (281T-281C) were detected by PCR-SSCP in intron 2 of AGPAT6 genes. The different genotypes of PCR-SSCP (281T-281C) loci have the significant difference on 305d matured equivalency (305dME), milk fat percentage, and milk protein percentage (P＜0.05).(2) 5 SNPs (410G-410T, 489G-489T, 600G-600T, 649G-649A, 729-729A) were detected by PCR-RFLP in intron 3 of AGPAT6 gene. The different genotypes of PCR-RFLP (410G-410T) loci have the significant difference on milk fat percentage, and somatic cell score (SCS) (P＜0.05). The different genotypes of PCR-RFLP (489G-489T) loci have the significant difference on 305dME, and milk fat percentage (P＜0.05).(3) 2 SNPs (153A-153G, 606A-606G) were detected by PCR-SSCP in intron 4 of AGPAT6 gene. The different genotypes of SNPs loci have the significant difference on SCS (P＜0.05). 3 SNPs (341A-341G, 430C-430T, 451C-451G) were detected by PCR-SSCP in intron 10 of AGPAT6 genes. The different genotypes of PCR-SSCP SNPs loci have the significant difference on SCS (P＜0.05). No SNPs was found in intron 6, 7, 8, 9 of AGPAT6 gene.3. Relativity of PCR-SSCP SNPs of the bovine PRLR gene with milk performance traits of dairy cattleIdentification for mutations in the amplified fragments of PRLR gene was performed and the 11 SNPs were detected by PCR-SSCP. These SNPs were genotyped in the experimental populations. The analysis of general lineal model was used to employ to association between the SNPs and milk performance traits of dairy cattle. The results showed as follows:(1) 3 SNPs (1104A delete, 1267A-1267G, 1268C-1268T) were detected by PCR-SSCP in exon 3 of PRLR gene. The different genotypes of PCR-SSCP SNPs loci have the significant difference on 305dME (P＜0.01).(2) 1 SNPs (3365T-3365C) were detected by PCR-SSCP in exon 5 of PRLR gene. The different genotypes of PCR-SSCP SNPs loci have no significant difference on milk performance traits of dairy cattle.(3) 4 SNPs (5533C-5533T, 5650A-5650G, 5651G-5651A, 5652A-5652T) were detected by PCR-SSCP in exon 7 of PRLR genes. The different genotypes of PCR-SSCP SNPs loci have the significant difference on 305dME, milk fat percentage, milk protein percentage, and SCS(P＜0.05).(4) 1 SNP (7740A-7740G) were detected by PCR-RFLP in intron 8 of PRLR gene. The different genotypes of PCR-RFLP SNP loci have the significant difference on 305dME, and milk protein percentage (P＜0.05).(5) 5 SNPs (9681C-9681T, 9742A-9742G, 9757G-9757T, 9902G-9902C, 9939G insert) were detected by PCR-SSCP in exon 10 of PRLR gene. The different genotypes of PCR-SSCP SNPs loci have the significant difference on 305dME, milk fat percentage, and SCS (P＜0.05).4. Analysis on combination of genotype of 11 SNPs of AGPAT6 gene and genotype of 11 SNPs of PRLR gene effects(1) Combination of genotype of AGPAT6 gene intron 2 and intron 3 with milk performance traits of dairy cattle were analysis. Interaction between genotype of AGPAT6 gene intron 2 and intron 3 were found. The effect of combination 1 (A1A1A2A2C2C2) had significant positive relation with 305dME and milk fat percentage.(2)Combination of genotype of PRLR gene exon 3, 7, 10 and intron 8 with milk performance traits of dairy cattle were analysis. Interaction between genotype of PRLR gene exon 3, 7, 10 and intron 8 were found. The effect of combination 4 (A1A1B3B3A5B5E4E4) had significant positive relation with 305dME.5. Relativity of SNPs of the bovine AGPAT6 and PRLR gene with milk performance traits of Chinese HolsteinIn Chinese Holstein population, 17 SNPs of AGPAT6 and PRLR gene were associated to the milk performance traits of dairy cattle. The result showed as follow:The different genotypes of PCR-SSCP SNPs loci of PRLR gene exon 3 have the significant difference on days of first mate and days of first calving (P＜0.05).The different genotypes of PCR-SSCP SNP loci of PRLR gene exon 5 have the significant difference on milk fat percentage, milk protein percentage, and dry matter of milk content (P＜0.05).The different genotypes of PCR-SSCP SNPs loci of PRLR gene exon 7 have the significant difference on 305dME and days of first calving (P＜0.05).The different genotypes of PCR-SSCP SNPs loci of PRLR gene exon 10 have the significant difference on milk fat percentage (P＜0.05).更多还原