荷斯坦奶牛BRCA2基因、STAT3基因SNPs检测及其与乳房炎和产奶性状的关联分析

发布时间：2018-08-09 14:38

【摘要】：乳腺癌易感基因2(BRCA2)能在抑制恶性肿瘤的发生上发挥作用,并且拥有这个基因突变的家族具有较高的乳腺癌发生率。BRCA2蛋白主要参与DNA双链损伤修复。BRCA2能通过多种机制对细胞周期、基因转录表达、DNA损伤修复等发挥不同的调节作用。牛的BRCA2基因定位于牛12号染色体上,全长约50kb,共有28个编码外显子。对BRCA基因的研究主要集中在人类以及小鼠上,而在奶牛上则未见相关报道,以BRCA基因作为影响奶牛产奶性状及乳房炎的候选基因,探索其与产奶性状及乳房炎的相关性,具有重要的研究价值。信号转导及转录活化因子3(STAT3)是一类脱氧核糖核酸(DNA)结合蛋白。STAT3的基因在牛中则定位于第19号染色体上。STAT3蛋白广泛表达于不同类型的细胞和组织中,既是一种胞质信号因子,又是胞核转录因子,参与细胞生长、恶性转化、凋亡等生理功能的调控。STAT3基因可被瘦素激活,在白介素-6信号通路中作为急性期反应因子调控免疫响应。本研究先利用DNAMAN软件、在线软件ExPASy及在线软件PSORT II Prediction对奶牛BRCA2基因及STAT3基因进行生物信息学分析。再以374份荷斯坦奶牛颈静脉血样为材料提取基因组DNA,利用基因组DNA扩增目的基因序列,使用DNA测序技术检测候选基因中的多态位点。采用PCR-RFLP和Forced PCR-RFLP技术对多态性位点进行分型,最后与荷斯坦奶牛乳腺炎和产奶性状进行关联分析,旨在筛查与奶牛多种产奶性状及乳房炎相关的多态位点,为中国荷斯坦奶牛的高效选育、种质资源保存与利用提供遗传学依据。主要研究结果及结论如下:1.BRCA2基因与STAT3基因生物信息学分析对BRCA2和STAT3进行奶牛、水牛、绵羊、野马和家鼠之间的核苷酸同源性分析,发现同源性大都在90%以上,说明基因在物种间有很高的保守性。对奶牛上述候选基因编码的蛋白质进行结构和功能预测,结果表明BRCA2和STAT3蛋白均为亲水性蛋白,且均主要存在于细胞质中。BRCA2蛋白包括了7个BRCA2重复域,而STAT3蛋白在第580-670氨基酸处有一个SH2结构域。2.BRCA2基因与STAT3基因遗传变异检测本研究对BRCA2基因与STAT3基因的共18个外显子和部分内含子进行分析检测,最终检测到4个SNPs位点。BRCA2基因的T48349A位点与C51808T位点,STAT3基因的A43087G位点与T75353C位点。其中C51808T与T75353C均位于基因的CDS区,且T75353C位点处于CDS区的3’非编码区,其余两个位点则位于内含子区。3.BRCA2基因与STAT3基因群体遗传学分析对BRCA2基因与STAT3基因进行群体遗传学分析发现,这四个SNP位点均是中度多态。BRCA2基因的T48349A位点与C51808T位点都不处于Hard-Weinberg平衡,而STAT3基因的A43087G位点与T75353C位点均处于Hard-Weinberg平衡。4.BRCA2基因与STAT3基因多态性与产奶性状、乳房炎的关联分析关联分析表明BRCA2基因的C51808T位点与奶牛的日均产奶量和体细胞评分显著相关,CT型个体有较高的产奶量,TT型具备较低的体细胞数;而STAT3基因的A43087G位点与乳蛋白率极显著相关,AG型个体的乳蛋白率相对较高。中国荷斯坦奶牛BRCA2基因和STAT3基因的生物信息学分析、多态位点检测及其与产奶性状和乳房炎的关联分析,为进一步研究BRCA2基因和STAT3基因的生物学功能提供了理论依据,为了解其在乳房炎抗性中的作用机理提供一定的理论基础。
[Abstract]:Breast cancer susceptibility gene 2 (BRCA2) can play a role in inhibiting the occurrence of malignant tumors, and the family with this gene mutation has a high incidence of breast cancer,.BRCA2 protein is mainly involved in DNA double chain damage repair,.BRCA2 can be regulated by a variety of mechanisms for cell cycle, gene transfer expression, and DNA damage repair. The BRCA2 gene of cattle was located on the bovine chromosome 12, with a total length of about 50kb, and there were 28 exons. The study on BRCA gene was mainly concentrated in human and mice, but no related reports were reported on the cows. The BRCA gene was used as a candidate gene for milk production and mastitis of dairy cows and was explored with milk production and mastitis. The signal transduction and transcription activator 3 (STAT3) is a class of DNA (DNA) binding protein.STAT3, which is located on chromosome nineteenth in cattle and is widely expressed in different types of cells and tissues, not only a cytoplasmic signaling factor but also a nuclear transcription factor. The regulation of.STAT3 gene with cell growth, malignant transformation, apoptosis and other physiological functions can be activated by leptin, and regulate the immune response as an acute phase reaction factor in the il--6 signaling pathway. This study first uses DNAMAN software, online software ExPASy and online software PSORT II Prediction to carry out biological letters to cow BRCA2 and STAT3 genes. The genomic DNA was extracted from the jugular vein blood samples of 374 Holstein cows. The sequence of the target gene was amplified by genomic DNA, and the polymorphic loci in the candidate genes were detected by DNA sequencing. The polymorphic loci were typed by PCR-RFLP and Forced PCR-RFLP technology. Finally, the mastitis and milk production of Holstein cows were made. Association analysis was conducted to screen polymorphic loci related to milk production traits and mastitis in dairy cows. It provides genetic basis for the efficient breeding of Chinese Holstein cows and the conservation and utilization of germplasm resources. The main results and conclusions are as follows: 1.BRCA2 and STAT3 gene bioinformatics analysis of dairy cows and water for BRCA2 and STAT3 The analysis of nucleotide homology between cattle, sheep, wild horses and domestic mice showed that the homology was more than 90%, indicating that the genes were highly conserved among the species. The structure and function of the protein encoded by the above candidate genes were predicted. The results showed that both BRCA2 and STAT3 proteins were hydrophilic proteins and were mainly found in the cytoplasm. The.BRCA2 protein contains 7 BRCA2 repeat domains, and the STAT3 protein has a SH2 domain.2.BRCA2 gene and the STAT3 gene genetic variation detection at the 580-670 amino acid. The 18 exons and the partial introns of the BRCA2 gene and STAT3 gene are analyzed and detected, and the T48349A loci of the.BRCA2 gene of the 4 SNPs loci are detected. The A43087G loci and the T75353C loci of the C51808T locus, the STAT3 gene, and the C51808T and T75353C are located in the CDS region of the gene, and the T75353C loci are in the 3 'non coding region of the CDS region, and the other two loci are in the genetic analysis of the.3.BRCA2 and STAT3 genes in the intron. It was found that both of these four SNP loci were not in Hard-Weinberg equilibrium between the T48349A loci and the C51808T loci of moderate polymorphic.BRCA2 gene, while the A43087G loci and T75353C loci of the STAT3 gene were both in Hard-Weinberg balanced.4.BRCA2 gene and STAT3 gene polymorphism and milk producing traits, and the association analysis of the association analysis of mastitis showed that the BRCA2 base was based on the association analysis of the mastitis The C51808T locus is significantly related to the daily milk production and somatic cell score of the dairy cows. The CT type has a higher milk production and the TT type has a lower body cell number, while the A43087G site of the STAT3 gene is closely related to the milk protein rate, and the milk protein rate of the AG type individuals is relatively high. The BRCA2 gene and STAT3 gene of the Chinese Holstein cows have a relatively high rate of milk protein. The analysis of Informatics, polymorphic loci and its association with milk production and mastitis provide a theoretical basis for further study of the biological functions of BRCA2 and STAT3 genes, and provide a theoretical basis for understanding the mechanism of its role in the resistance to mastitis.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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