猪Bic基因两种单倍型与Foxp3结合能力差异对miR-155表达影响的研究

发布时间：2018-03-28 01:21

本文选题：Foxp3　切入点：猪　出处：《华中农业大学》2017年硕士论文

【摘要】：猪的抗病育种一直是养猪科研的重点研究内容,尤其是中外猪种的免疫力差异,从遗传的角度来研究中国猪种的抗病力对猪的抗病新品种培育非常重要。miR-155在免疫平衡和免疫系统中起着关键的作用,并广泛参与T细胞的活化、增殖和分化。本研究旨在利用凝胶电泳迁移实验探究猪miR155宿主基因Bic内含子的SNP与转录因子Foxp3的结合能力差异,在个体水平利用流式细胞仪分选了两种单倍型的Treg细胞并提取总RNA进行转录组测序,对两种单倍型个体的表达谱芯片进行了功能聚类,还检测了Poly I:C刺激情况下对miR155表达的影响及转录因子Foxp3的调控作用。具体结果如下:(1)根据宿主基因Bic内含子区域的Pre-miR155上游305bp和168bp这两处发生A/C突变的SNP位点,利用PCR-RFLP技术在培育品种-湖北白猪中酶切鉴定了两种单倍型AA和CC个体。(2)利用流式细胞仪通过标记CD3+CD4+CD25+三阳分选了AA和CC两种单倍型的调节性T细胞(Treg),并利用微量RNA提取试剂盒抽提了Treg细胞的总RNA,将RNA进行转录组测序,数据分析得到与免疫系统相关的基因有126个,在CC单倍型Treg细胞内上调表达的基因有122个,而下调表达的基因有4个;通过差异基因聚类分析发现与免疫应答相关的基因有23个;找到10个miR155的靶基因,其中3个靶基因有显著差异,另外7个没有显著差异,SOCS1以及SLA-6在CC单倍型Treg细胞内表达量较高,而MYLK在CC单倍型Treg细胞内不表达。测序所得的核糖体RNA比例较高,有效数据量不大。(3)在杜洛克x二花脸F2代群体酶切分型鉴定了两种单倍型的个体,利用Affymetrix表达谱芯片数据进行了AA单倍型和CC单倍型之间的差异基因筛选,通过对33d和35d的杜二F2群体表达谱芯片数据分析我们分别共检测到554个和232个两种单倍型之间差异表达的探针(p0.05),然后将差异表达基因进行功能聚类,筛选到与miR-155功能调控相关的基因包括ETS2、ATF3、TGFβ-1、TGFB1、IL15、SLA-DRB1、SOCS1和CASP8。(4)利用凝胶电泳迁移实验技术探讨猪的Foxp3蛋白与Bic基因中A/C型片段的结合能力差异,结果发现Foxp3与AA型片段的结合能力较CC型要强。(5)在巨噬细胞上验证Poly I:C刺激情况下miR155的表达变化及转录因子Foxp3对miR155的表达调控,结果表明miR155-5p的表达量在刺激12h内有所上调,但差异不显著;且Poly I:C刺激之后,Foxp3超表达发现miR155-5p的表达量显著上调。上述研究结果表明位于宿主基因Bic内含子区域的SNP突变是功能突变,能够影响转录因子Foxp3的结合能力,为揭示我国地方猪种与国外猪种的免疫力差异提供了理论依据,为抗病育种提供了分子标记。
[Abstract]:Pig breeding for disease resistance has always been the focus of scientific research in pig breeding, especially the difference in immunity between Chinese and foreign pig breeds. From the perspective of heredity, it is very important to study the resistance of Chinese pig breeds to the breeding of new varieties of pig disease resistance. MiR-155 plays a key role in the immune balance and immune system, and widely participates in the activation of T cells. The aim of this study was to investigate the difference of binding ability between SNP of porcine miR155 host gene Bic intron and transcription factor Foxp3 by gel electrophoresis migration assay. At individual level, two haplotypes of Treg cells were selected by flow cytometry and total RNA was extracted for transcriptional sequencing. Functional clustering was performed on the expression microarray of two haplotypes. The effects of Poly I / C stimulation on the expression of miR155 and the regulation of transcription factor Foxp3 were also examined. The results are as follows: 1) according to the SNP sites of Pre-miR155 upstream 305bp and 168bp in the Bic intron region of host gene, Two haplotypes, AA and CC, were digested in Hubei White Pig by PCR-RFLP technique. Two types of haplotypes, AA and CC, were identified by flow cytometry, and the regulatory T cells of AA and CC haplotypes were separated by flow cytometry. The total RNAs of Treg cells were extracted by RNA extraction kit, and RNA was sequenced. The results showed that there were 126 genes related to immune system, 122 genes upregulated in CC haplotype Treg cells, and 4 genes down-regulated. By cluster analysis of differentially expressed genes, 23 genes related to immune response were found, and 10 target genes of miR155 were found, 3 of which were significantly different. There was no significant difference in the expression of SOCS1 and SLA-6 in CC haplotype Treg cells, while MYLK was not expressed in CC haplotype Treg cells. Two haplotypes were identified by restriction endonuclease typing in the F2 generation of Duroc x Erhualian population. The difference genes between AA haplotypes and CC haplotypes were screened by Affymetrix expression microarray data. We detected 554 differentially expressed probes (p0.05) between the two haplotypes at 33d and 35d, and then clustered the differentially expressed genes by functional clustering. The genes related to the regulation of miR-155 function, including ETS2TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1TGFB1SLA-DRB1SSOCS1 and CASP8.4were screened to study the difference of binding ability between Foxp3 protein and A-% C fragment in Bic gene by gel electrophoresis. The results showed that the binding ability of Foxp3 to AA fragment was stronger than that of CC type. (5) the changes of miR155 expression in macrophages stimulated by Poly I / C and the regulation of miR155 expression by transcription factor Foxp3 were verified. The results showed that the expression of miR155-5p was up-regulated within 12 h after stimulation. But the difference was not significant, and the overexpression of miR155-5p was found to be significantly up-regulated after Poly I: C stimulation. The results indicated that the SNP mutation located in the intron region of host gene Bic was a functional mutation, which could affect the binding ability of transcription factor Foxp3. It provides a theoretical basis for revealing the difference of immunity between Chinese local pig breeds and foreign pig breeds, and provides molecular markers for disease resistance breeding.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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