导　　师： 邱祥聘;曾凡同;杨关福

学科专业： 090501

授予学位： 博士

作　　者： ;

机构地区： 四川农业大学

摘　　要： HSP70基因编码的HSP70蛋白在鸡耐热性形成过程中起着重要的作用。在鸡的抗逆性育种中，HSP70基因有望成为改善热应激敏感性的候选基因。但目前关于HSP70基因序列中存在的单核苷酸多态性/(SNPs/)的研究报道很少。本研究选取我国20个地方鸡种共591个个体作为实验材料，采用变性高效液相色谱/(DHPLC/)技术，在鸡HSP70基因的全序列内进行未知SNPs的筛选，对典型峰型分别选取2个个体测序，再将同一对引物的所有测序样本的测序结果进行序列的同源性比较，从而识别和确认鸡HSP70基因的未知SNPs。并通过特异峰型与测序结果的对应关系，判定了每个个体在各SNPs位点的基因型，由此作者计算了每个SNPs位点在各品种中的基因型和等位基因的分布频率并对各品种间在SNPs位点的基因型和等位基因分布频率的差异进行了显著性检验。计算各品种的平均杂合度、各位点的基因多样度及其相关参数、各品种间的标准遗传距离/(D/_s/)和遗传一致性参数，衡量群体内以及群体间HSP70基因的遗传变异。作者还依据每个个体在各SNPs位点的基因型采用基于最大似然法原理编写的PHASE程序进行单倍型的确认，并计算每种单倍型在各品种中的分布频率。 结果分别在A5、A6、A7、A4引物扩增的PCR产物中检出并确认了10个SNPs：A258G、C276G、C507T、C1040A、G1044A、C143lA、T1476C、G1500A、A1529G、C1722T。所有检出的SNPs均位于编码区，其中1040位C→A的突变导致HSP70基因氨基酸序列347位发生脯氨酸/(Pro，P/)到组氨酸/(His，H/)的替换，1529位A→G的突变导致氨基酸序列510位发生赖氨酸/(Lys，K/)到精氨酸/(Arg，R/)的替换。 根据测序结果结合对应的DHPLC色谱峰型判定出测序的各样本在各SNP位点的基因型。其它未测序的个体与此色谱峰型相同的，将其在同一位点的基因型视为与测序个� The heat shock protein 70, which coded by the 70-kilodalton heat shock protein /(HSP70/) gene, is very important for the genesis of the heat tolerance of chicken. In the research of resistance breeding, HSP70 gene can be considered as the candidate loci of improving the sensibility of heat shock. But in present, researches about single nucleotide polymorphisms /(SNPs/) exist in chicken HSP70 gene are very limited. In this research, 591 individuals from 20 different Chinese native chicken breeds were studied. Denaturing High Performance Liquid Chromatography /(DHPLC/) technology was used to screen unknown SNPs existed in chicken HSP70 gene sequence. From every typical DHPLC peak, 2 individuals were selected to sequence. Homologous analysis was carried out based on the sequence information of samples that amplified from same primer to identity new unknown SNPs. From the compare of the typical DHPLC peak and sequence information, the genotype of every individual in each SNPs site was deduced. The genotype and gene fr equency of every SNPs site in different chicken breeds were calculated and the difference exist among different breeds were tested. Average heterozygosity of different breeds, genetic diversity and its associate parameters, standard genetic distance /(Ds/) and genetic identity parameters were calculated to measure the genetic variation exist among and between different chicken breeds in HSP70 gene. Based on the genotype of every individual in each SNPs sites, the PHASE program, which compiled by the theory of most likelihood, was used to affirm the haplotype and the distribution frequency of every haplotype in different chicken breed was calculated. In the PCR products amplified by primer A5, A6, A7 and A4, 10 SNPs sites /(A258G, C276G, C507T, C1040A, G1044A, C1431A, T1476C, G1500A, A1529G and C1722T/) were detected. All of these SNPs exist in the code region of HSP70 gene. Especially, the mutation of C-A happened in 1040 site lead to a substitution of the amino acid histidine for prol

关 键 词： 鸡 热应激蛋白 基因 单核苷酸多态性 变性高效液相色谱 基因频率 单倍型

领　　域： [农业科学] [农业科学]