發(fā)布時間：2018-03-28 01:21

  本文選題：Foxp3　切入點(diǎn)：豬　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：豬的抗病育種一直是養(yǎng)豬科研的重點(diǎn)研究內(nèi)容,尤其是中外豬種的免疫力差異,從遺傳的角度來研究中國豬種的抗病力對豬的抗病新品種培育非常重要。miR-155在免疫平衡和免疫系統(tǒng)中起著關(guān)鍵的作用,并廣泛參與T細(xì)胞的活化、增殖和分化。本研究旨在利用凝膠電泳遷移實(shí)驗(yàn)探究豬miR155宿主基因Bic內(nèi)含子的SNP與轉(zhuǎn)錄因子Foxp3的結(jié)合能力差異,在個體水平利用流式細(xì)胞儀分選了兩種單倍型的Treg細(xì)胞并提取總RNA進(jìn)行轉(zhuǎn)錄組測序,對兩種單倍型個體的表達(dá)譜芯片進(jìn)行了功能聚類,還檢測了Poly I:C刺激情況下對miR155表達(dá)的影響及轉(zhuǎn)錄因子Foxp3的調(diào)控作用。具體結(jié)果如下:(1)根據(jù)宿主基因Bic內(nèi)含子區(qū)域的Pre-miR155上游305bp和168bp這兩處發(fā)生A/C突變的SNP位點(diǎn),利用PCR-RFLP技術(shù)在培育品種-湖北白豬中酶切鑒定了兩種單倍型AA和CC個體。(2)利用流式細(xì)胞儀通過標(biāo)記CD3+CD4+CD25+三陽分選了AA和CC兩種單倍型的調(diào)節(jié)性T細(xì)胞(Treg),并利用微量RNA提取試劑盒抽提了Treg細(xì)胞的總RNA,將RNA進(jìn)行轉(zhuǎn)錄組測序,數(shù)據(jù)分析得到與免疫系統(tǒng)相關(guān)的基因有126個,在CC單倍型Treg細(xì)胞內(nèi)上調(diào)表達(dá)的基因有122個,而下調(diào)表達(dá)的基因有4個;通過差異基因聚類分析發(fā)現(xiàn)與免疫應(yīng)答相關(guān)的基因有23個;找到10個miR155的靶基因,其中3個靶基因有顯著差異,另外7個沒有顯著差異,SOCS1以及SLA-6在CC單倍型Treg細(xì)胞內(nèi)表達(dá)量較高,而MYLK在CC單倍型Treg細(xì)胞內(nèi)不表達(dá)。測序所得的核糖體RNA比例較高,有效數(shù)據(jù)量不大。(3)在杜洛克x二花臉F2代群體酶切分型鑒定了兩種單倍型的個體,利用Affymetrix表達(dá)譜芯片數(shù)據(jù)進(jìn)行了AA單倍型和CC單倍型之間的差異基因篩選,通過對33d和35d的杜二F2群體表達(dá)譜芯片數(shù)據(jù)分析我們分別共檢測到554個和232個兩種單倍型之間差異表達(dá)的探針(p0.05),然后將差異表達(dá)基因進(jìn)行功能聚類,篩選到與miR-155功能調(diào)控相關(guān)的基因包括ETS2、ATF3、TGFβ-1、TGFB1、IL15、SLA-DRB1、SOCS1和CASP8。(4)利用凝膠電泳遷移實(shí)驗(yàn)技術(shù)探討豬的Foxp3蛋白與Bic基因中A/C型片段的結(jié)合能力差異,結(jié)果發(fā)現(xiàn)Foxp3與AA型片段的結(jié)合能力較CC型要強(qiáng)。(5)在巨噬細(xì)胞上驗(yàn)證Poly I:C刺激情況下miR155的表達(dá)變化及轉(zhuǎn)錄因子Foxp3對miR155的表達(dá)調(diào)控,結(jié)果表明miR155-5p的表達(dá)量在刺激12h內(nèi)有所上調(diào),但差異不顯著;且Poly I:C刺激之后,Foxp3超表達(dá)發(fā)現(xiàn)miR155-5p的表達(dá)量顯著上調(diào)。上述研究結(jié)果表明位于宿主基因Bic內(nèi)含子區(qū)域的SNP突變是功能突變,能夠影響轉(zhuǎn)錄因子Foxp3的結(jié)合能力,為揭示我國地方豬種與國外豬種的免疫力差異提供了理論依據(jù),為抗病育種提供了分子標(biāo)記。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S828

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本文編號：1674130