猪Bic基因中Foxp3结合位点突变对miR-155表达影响探究

发布时间：2018-04-29 16:55

本文选题：抗病育种 + SNP　；参考：《华中农业大学》2015年硕士论文

【摘要】：近年来抗病育种研究成为国内外动物遗传育种专家高度关注的领域。中国地方猪种是抗病选育中亲本的重要来源,因此从遗传角度分析中国地方猪种与外来猪种的免疫差异对解析中国地方猪种是否具有较强抗逆性和猪的抗病新品种培育等是十分必要的。mi R-155在免疫系统发生发育及调控中扮演重要角色,其在不同猪种中差异表达可能引起整个免疫系统的差异。本研究旨在利用染色质免疫共沉淀技术探索猪Bic基因中与Foxp3蛋白相结合的区域,利用双荧光素酶报告系统分析已发现的大白猪和梅山猪中Bic基因的一个A/C突变导致的相应序列与转录因子Foxp3的结合差异并在个体水平利用定量PCR对mi R-155及其候选靶基因表达量进行检测,之后对mi R-155的候选靶基因进行双荧光实验验证。我们得到的具体结果如下:(1)在实验室之前扩增得到的5个SNP的基础上,在大白猪和梅山猪中扩增pre-mi R-155前后约3kb左右的片段,通过测序得到20个疑似SNP的位点。(2)初步确定猪中转录因子Foxp3与pre-mi R-155上游305bp左右的AAACA基序结合。(3)由于SNP的存在不同猪种中的基序与Foxp3结合程度有差异,AAACC型片段的结合能力强于AAACA型片段,并且这种结合可能需要一定的条件,例如片段长度或基序数量。(4)转录因子Foxp3与两种单倍型猪的Bic基因的结合能力的差异影响mi R-155的表达,使CC型个体中的mi R-155-5p和mi R-155-3p的表达水平均高于AA型个体。(5)初步验证mi R-155的候选靶基因在AA型个体中表达量高于CC型个体,初步确定mi R-155与Sept11、Phf17基因的靶向关系。本研究初步证明了大白猪和梅山猪由于Bic基因中Foxp3结合位点的差异导致mi R-155的表达及靶基因的改变,为揭示这两个猪种间的免疫能力差异性提供一些理论依据,同时为抗病新品种的选育工作提供新的分子鉴定依据。
[Abstract]:In recent years, the research of disease resistance breeding has become a highly concerned field of animal genetics and breeding experts at home and abroad. Local pig breeds in China are an important source of parents in breeding for disease resistance. Therefore, it is necessary to analyze the immunological difference between Chinese local pig breeds and foreign pig breeds from a genetic point of view. It is necessary to analyze whether Chinese local pig breeds have strong resistance to stress and to breed new breeds of pig disease resistance, etc., which are necessary for the occurrence of .mi R-155 in the immune system. Play an important role in education and regulation, The differential expression in different pig breeds may cause differences in the whole immune system. The aim of this study was to explore the binding region of porcine Bic gene to Foxp3 protein by chromatin immunoprecipitation. Double luciferase reporting system was used to analyze the binding difference between the corresponding sequence of Bic gene and transcription factor Foxp3 caused by an A / C mutation of Bic gene in white pig and Meishan pig, and to use quantitative PCR to detect mi R-155 and its candidate at individual level. Target gene expression was detected, The candidate gene of mi-155 was verified by double fluorescence assay. Our specific results are as follows: 1) on the basis of five SNP amplified before the laboratory, we amplified about 3kb fragments around pre-mi R-155 in large white pigs and Meishan pigs. Sequencing of 20 suspected SNP loci. 2) preliminary identification of AAACA motif binding between the transcription factor Foxp3 and 305bp upstream of pre-mi R-155. 3) due to the presence of SNP in different pig breeds, the degree of binding between the motif and Foxp3 is different. The combination ability is stronger than the AAACA fragment. And this binding may require certain conditions, such as fragment length or motif number. 4) the difference in binding ability of transcription factor Foxp3 to Bic gene of two haplotypes pigs affects the expression of mi R-155. The expression levels of mi R-155-5p and mi R-155-3p in CC type individuals were higher than those in AA type individuals.) the candidate gene expression of mi R-155 in AA type individuals was higher than that in CC type individuals, and the targeting relationship between mi R-155 and Sept11PPhf17 gene was preliminarily confirmed. This study preliminarily proved that the expression of miR-155 and the change of target gene were caused by the difference of Foxp3 binding sites in Bic gene between big white pig and Meishan pig, which provided some theoretical basis for revealing the difference of immune ability between the two pigs. At the same time, it provides a new molecular identification basis for the breeding of new varieties of disease resistance.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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