利用捕获测序技术筛选影响鸡血清IgY含量的候选基因

发布时间：2018-05-29 02:03

本文选题：鸡 + 血清IgY含量　；参考：《扬州大学》2015年硕士论文

【摘要】：抗病性状的功能基因筛选和鉴定一直是现代家禽育种研究的热点。前期研究中,已利用60K SNP Bead chip技术,通过全基因组关联分析(GWAS)筛选到了与鸡免疫性状相关显著关联位点及重要区域,在此基础上,对与血清免疫球蛋白Y (Immunoglobulin Y, IgY)含量显著相关的染色体区域进行了重测序分析。以56日龄北京油鸡(样本数728只)和白来航鸡(样本数525只)为素材,分别选择IgY含量极高和极低个体各40个,组成两品种、高低表型各4个处理组,每个处理随机等量(100ng/μL)混成分为4个重复,每个重复(池)包含10个个体。筛选分布于11,16和19三条染色体上共3.3M的候选目标区域,运用捕获芯片、Solexa高通量测序等技术进行测序分析。共获得单碱基变异35154个,经注释,其中基因型为纯合的碱基变异为28862个,杂合的单碱基变异6292个,得到CDS区的同义突变SNP1045个,引起氨基酸发生变化的错义突变有2190个,位于基因上的SNP有18332个。筛选到829个Indel位点,其中绝大部分位于mRNA区域。随机挑选四个基因TRIM7.2, BFIV21, AKIP以及RBL2,运用PCR-Sanger法进行混池测序得到24个SNP,包含二代测序结果中的21个SNP位点,证实捕获测序SNP结果可靠。对2个Indel进行验证,其中16号染色体上BF1基因上的9个碱基(CCACTGCCA)的缺失是在两品种高表型组共有的Indel突变,与捕获测序结果一致。对BF1基因Indel所在的内含子5的多态性进行检测,结果表明,在进行捕获测序的北京油鸡和白来航鸡样本中,该Indel区域存在三种基因型,即野生型(9个碱基全部存在,AA型)、突变型(9个碱基其中三个发生突变,BB型)、缺失型(9个碱基全部缺失,CC型),但基因型IgY均值间呈现AABB, AACC的变化,但差异并不显著(P0.05),两个品种具有相同的变化趋势。在高度选育的白来航鸡马立克氏病易感品系B19单倍型(37只)的基因型全部为AA型,抗病品系B21单倍型(34只)的基因型全部为BB型,基因型频率各为100%。MHC BF1基因影响Ⅰ类抗原分子与抗原肽结合的特异性,本研究发现的9bp碱基的突变可能引起的可变剪接或者基因表达差异,继而改变鸡血清IgY抗体的生成以及马立克氏病的抗病性。
[Abstract]:Screening and identification of functional genes for disease resistance traits has been a hot topic in modern poultry breeding. In previous studies, significant association loci and important regions associated with immune traits were screened by using 60 K SNP Bead chip technique and GWAS-based genomic association analysis. The chromosomal regions associated with serum immunoglobulin Y (IgY) content were sequenced. Using 56-day-old Beijing fowl (728 samples) and Bailai hens (525samples) as materials, 40 individuals with extremely high IgY content and 40 individuals with extremely low IgY content were selected to form two breeds, four treatments with high and low phenotypes, respectively. The random equal amount of 100 ng / 渭 L in each treatment was 4 repeats, and each replica (pool) consisted of 10 individuals. The candidate regions of 3.3M were screened on 11C16 and 19 chromosomes. High throughput sequencing techniques such as capture chip Solexa were used to carry out sequencing and analysis. A total of 35154 single-base mutations were obtained, among which 28862 were homozygous, 6292 were heterozygous, and 2190 missense mutations were found in the CDS region. There are 18,332 SNP on the gene. 829 Indel loci were screened, most of which were located in the mRNA region. Four genes TRIM7.2, BFIV21, AKIP and RBL2 were randomly selected, and 24 SNPs were obtained by PCR-Sanger method, including 21 SNP loci of the second generation sequencing results, which confirmed the reliability of the captured and sequenced SNP results. The results of two Indel tests showed that the deletion of 9 bases CCACTG CCA on chromosome 16 was a common Indel mutation in the high phenotypic groups of the two cultivars, which was consistent with the results of capture and sequencing. The polymorphism of intron 5 of BF1 gene Indel was detected. The results showed that there were three genotypes in the Indel region in the samples of Beijing oil chicken and white Laihang chicken which were captured and sequenced. That is, wild type (all 9 bases all have AA type), mutation type (3 of 9 bases have mutation of BB type), deletion type (9 bases are all deletion of CC type), but the mean value of genotype IgY shows the change of AABB, AACC. However, the difference was not significant (P 0.05), and the two varieties had the same trend of change. The genotypes of B19 haplotype and B21 haplotype were all AA genotype and BB genotype, respectively. The genotypes of 100%.MHC BF1 genes affect the specificity of the binding of class I antigen molecules to antigenic peptides. In this study, the mutation of 9bp bases may cause variable splicing or gene expression differences. It then changes the production of serum IgY antibodies and the resistance to Marek's disease.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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