【作者】 韩立霞；

【导师】 孙少华；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2009， 硕士

【摘要】 多脊椎现象是存在于河北小尾寒羊中的有益突变,多数突变都会引起绵羊脊柱的加长和体尺增加,从而显著提高产肉量。本论文采用PCR-RFLP、dCAPS、PCR-SSCP以及直接测序等技术首次从分子水平上对小尾寒羊的Btg2基因和NR6A1基因的多态性与多脊椎性状的关系进行研究,以期找到与小尾寒羊多脊椎突变相关的分子遗传标记,并为进一步标记辅助选择提高河北小尾寒羊的肉用性能,培育我国肉用绵羊品种奠定基础。根据牛和猪的Btg2基因的DNA序列,选择保守区域设计引物,扩增绵羊Btg2基因的外显子3和3′UTR的部分序列,并对这些序列选取代表性个体进行测序,得到长度为1226bp的片段。全部片段测序结果经过同源性比对,共发现4个SNPs位点,突变位点依据拼接序列来命名分别为:A150G、C243T、A607G和T704C,其中有1个SNPs位于外显子,未引起氨基酸序列的改变。应用PCR-RFLP及dCAPS技术,对Btg2基因的A150G、C243T和A607G突变位点分别设计引物进行PCR扩增和群体检测。独立性卡方检测结果表明150位点的不同表型个体基因型频率差异不显著(P>0.05);243位点的差异极显著(P<0.01),卡方检验再分割结果表明,基因型频率在T14L6与T14L7、T14L5表型之间差异极显著,T14L6与T13L7之间差异显著;607位点的差异呈显著水平(P<0.05),卡方检验再分割表明基因型频率在T14L6与T14L7中差异显著。结果表明243位点、607位点可能与多脊椎性状有关。经过独立性卡方检验,表明单体型在不同表型中差异显著(P<0.05),分析表明单体型GTA可能与多脊椎性状有关。根据牛和猪的NR6A1基因的DNA及mRNA序列,设计8对引物,扩增绵羊的该基因的全部编码区序列。运用PCR-SSCP以及直接测序技术对扩增产物进行突变筛查,结果在外显子5的第25位碱基处上检测到1个T25G的单碱基突变,该突变未引起氨基酸序列的改变。对该位点进行群体检测分型,经独立性卡方检验后,发现基因型频率在5种不同多脊椎突变表型间差异达到显著水平(P<0.05),卡方检验再分割后发现基因型频率在T14L5与T14L6以及T14L5与T14L7中差异显著,表明该位点可能与多脊椎性状有关。更多还原

【Abstract】 Multi-vertebra trait is a positive mutation existing in the Hebei Small Tail Han Sheep, most of which can result in lengthening of the spine, which increases the body size and mutton production significantly. PCR-RFLP, dCAPS, PCR-SSCP and sequencing were used to study the correlation between the polymorphism of Btg2 and NR6A1 gene, and the Multi-vertebra trait. The associated molecular markers were expected to be found, which will be used in Marker-Assisted Selection in order to improve meat performance of Hebei Small Tail Han Sheep, and will lay the foundation for the breeding of meat sheep.The 1226bp fragment containing partial exon3 and partial 3′UTR was amplified using a pair of PCR primers designed based on the available nucleotide sequence of Btg2 gene from cattle and pig, and was sequenced in some representative individuals with distinct phenotypes. According to the homologous alignment of the sequenced results, 4 SNPs were detected (A150G, C243T, A607G and T704C), one of which was silent mutation existing in the exon.The dCAPS primers were designed according to A150G, C243T and A607G, respectively, followed by population analysis via PCR-RFLP. The results of independence Chi-square test showed that genotype frequencies at Site 150 had no significant difference among different vertebral patterning sheep (P>0.05). At Site 243, the genotype frequencies had extremely significant difference among different vertebral patterning sheep (P<0.05), and the resegmentation of Chi-square test showed that the genotype frequencies were extremely significantly different (P<0.01) between T14L6 and T14L7, and between T14L6 and T14L5, and were significantly different (P<0.05) between T14L6 and T13L7. At site 607, the genotype frequencies were significantly different (P<0.05), and according to the resegmentation of Chi-square test, the genotype frequencies between T14L6 and T14L7 were significantly different. The results showed that Site 243 and Site 607 might be associated with multi-vertebra trait. According to independence Chi-square test, haplotypes showed significant difference among different phenotypic individuals, and the haplotype GTA might be related to multi-vertebra trait.8 pairs of primers have been designed based on the available DNA and mRNA sequences of NR6A1 gene from cattle and pig, followed by amplification of the complete coding sequence (CDS) in Hebei Small Tail Han Sheep. Mutation screening has been carried out via PCR-SSCP and direct sequencing, which showed there was one silent mutation (T25G) at the 25th base in exon5. With respect to this site, the population genotyping and independence Chi-square test showed that the genotype frequencies were significantly different (P<0.05) among the five kinds of multi- vertebra phenotypes, and according to the resegmentation of Chi-square test, the genotype frequencies were significantly different (P<0.05) between T14L5 and T14L6, and between T14L5 and T14L7, showing this site might be related to the multi-vertebrae trait.更多还原