【作者】 张玉皓；

【导师】 李凤娥；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 根据基因生理生化功能、猪繁殖性状QTL定位结果和猪-人比较图谱,选取H2A.Z基因和DAZL基因作为影响猪繁殖性状的候选基因。应用比较基因组学和生物信息学等方法进行候选基因的分离鉴定;采用RT-PCR进行部分基因的组织表达研究;利用直接测序法对所分离片段进行多态性检测;以大白猪（322头,438窝）、中国瘦肉猪新品系DIV母猪（210头,409窝）和大梅F₂资源家系青年母猪（105头）为试验材料,进行基因多态性与猪繁殖性状的关联分析。研究结果如下:1.H2A.Z基因:（1）获得猪H2A.Z基因932bp的cDNA序列,其中包括387bp的编码区序列,185bp的5’UTR序列和360bp的3’UTR序列;获得932bp的基因组序列,其中包括完整的intron2、3、4。（2）对所获得序列进行多态性检测,在第二内含子发现一个连续4bp的缺失:建立了第2内含子的PCR-Bsu15I-RFLP分型技术。（3）遗传标记与繁殖性状的关联分析结果表明:Bsu15I多态位点与初产DIV系猪的NBA（P＜0.05）显著相关,基因加性效应显著（P＜0.05）;与大白猪的所有胎次的NBA（P＜0.01）极显著相关,基因加性效应显著（P＜0.05）。2.DAZL基因:（1）获得猪DAZL基因2805bp的cDNA序列,其中包括887bp的编码区序列和1917bp的3’UTR序列;获得DAZL基因的部分基因组序列,其中包括内含子2,3,4,5,6,7,8,9的序列。（2）对所获得序列进行SNP检测,建立了DAZL基因第7内含子385bp处的PCR-MspⅠ-RFLP分型技术和第九内含子167bp处的PCR-TaqⅠ-RFLP分型技术。（3）遗传标记与繁殖性状的关联分析结果表明:TaqⅠ多态位点与大白猪所有胎次产活仔数显著相关（P＜0.05）;与DIV猪的头胎产活仔数显著相关（P=0.06）。（4）构建了DAZL—PGEX—kg重组载体,并在大肠杆菌表达系统中进行了诱导表达;诱导表达的蛋白经过PAGE凝胶检测,结果表明:DAZL—PGEX—kg重组载体所表达的蛋白分子量与预测结果相一致。上述研究结果显示:H2A.Z基因Bsu15I;DAZL基因TaqⅠ多态位点可作为猪产仔数的分子遗传标记。对上述基因的分离、鉴定、多态性及表达调控研究有助于我们更深地了解基因的生物学功能,为揭示猪产仔数分子机理和标记辅助选择的实施提供理论依据。更多还原

【Abstract】 Litter size is one of the important factors which impact the benefit of porcine industry.In this study,the candidate gene approach has been emploied to discover new molecular markers associated with reproductive traits.According to physiological or biochemical functions of genes,QTL mapping results and pig-human comparative map, H2A.Z gene and DAZL gene were selected.The objective of this study was to isolate and character these candidate genes,to analyze the expression of these genes in different tissues,to identify mutations in these genes sequence and establish suitable method to detect polymorphisims,to determine associations between the polymorphisms and the reproductive performance in femal LargeWhite,Chinese lean type new line DIV and LargeWhite×Meishan F₂ resource populations.The main results are as following:1.The analysis results of genetic effect of H2A.Z gene（1） A 932bp cDNA sequence of porcine H2A.Z gene spanning complete coding sequences（CDS） was isolated by RT-PCR.A 1231bp genomic sequence encompassing complete intron 2,3 and 4 was amplified from total porcine genomic DNA.（2） A PCR-Bsu15I-RFLP assay was established to detect the 4bp deletion in intron 2.（3） Association analysis for the 4bp deletion with reproductive traits was preformed in nine independent populations.Statistical analysis demonstrated that Bsu15I polymorphism was associated with NBA in the first of DIV sows and Large White sows,an additive effect was detected（p＜0.05 or p＜0.01）.2.The analysis results of genetic effect of DAZL gene（1） A 2805bp cDNA sequence of pig DAZL gene was isolated including 887bp complete coding sequences and 1917bp 3’UTR sequences.An genomic sequence of porcne DAZL which encompass the introns of 2,3,4,5,6,7,8,9 were amplified from porcine genomic DNA.（2） For DAZL gene:a PCR-MspI-RFLP assay was established to detect the A/G mutation in intron 7;and a PCR-TaqI-RFLP assay was estabilished to detect the A/C mutation in intron 9.（3） Association study for these SNPs with reproductive traits was preformed in two independent populations.Statistical analysis demonstrated that:TaqI polymorphism is associated with NBA in the first litters of both Large White sows（p＜0.05） and Line DIV（p=0.06）,an additive effect was detected （p＜0.05）in Large White group.（4）An recombinated vector named DAZL-PGEX-kg was constructed and expressed in Escherichia coli expression system.Then the expressed protein was detected in PAGE gel,the result of which prove that the molecular weight of the expressed protein was in line with our prediction..The result shows that the Bsu15I-PCR-RFLP and TaqI-PCR-RFLP could be considered as the molecular markers of porcine litter size.The isolation and identification of these genes would give us a more profound.understanding of the functions of these genes,uncover the mechanism of pig litter size and give theoretics support for marker assistant selection（MAS）.更多还原