本文選題：抗病育種 + SNP��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：近年來(lái)抗病育種研究成為國(guó)內(nèi)外動(dòng)物遺傳育種專家高度關(guān)注的領(lǐng)域。中國(guó)地方豬種是抗病選育中親本的重要來(lái)源,因此從遺傳角度分析中國(guó)地方豬種與外來(lái)豬種的免疫差異對(duì)解析中國(guó)地方豬種是否具有較強(qiáng)抗逆性和豬的抗病新品種培育等是十分必要的。mi R-155在免疫系統(tǒng)發(fā)生發(fā)育及調(diào)控中扮演重要角色,其在不同豬種中差異表達(dá)可能引起整個(gè)免疫系統(tǒng)的差異。本研究旨在利用染色質(zhì)免疫共沉淀技術(shù)探索豬Bic基因中與Foxp3蛋白相結(jié)合的區(qū)域,利用雙熒光素酶報(bào)告系統(tǒng)分析已發(fā)現(xiàn)的大白豬和梅山豬中Bic基因的一個(gè)A/C突變導(dǎo)致的相應(yīng)序列與轉(zhuǎn)錄因子Foxp3的結(jié)合差異并在個(gè)體水平利用定量PCR對(duì)mi R-155及其候選靶基因表達(dá)量進(jìn)行檢測(cè),之后對(duì)mi R-155的候選靶基因進(jìn)行雙熒光實(shí)驗(yàn)驗(yàn)證。我們得到的具體結(jié)果如下:(1)在實(shí)驗(yàn)室之前擴(kuò)增得到的5個(gè)SNP的基礎(chǔ)上,在大白豬和梅山豬中擴(kuò)增pre-mi R-155前后約3kb左右的片段,通過(guò)測(cè)序得到20個(gè)疑似SNP的位點(diǎn)。(2)初步確定豬中轉(zhuǎn)錄因子Foxp3與pre-mi R-155上游305bp左右的AAACA基序結(jié)合。(3)由于SNP的存在不同豬種中的基序與Foxp3結(jié)合程度有差異,AAACC型片段的結(jié)合能力強(qiáng)于AAACA型片段,并且這種結(jié)合可能需要一定的條件,例如片段長(zhǎng)度或基序數(shù)量。(4)轉(zhuǎn)錄因子Foxp3與兩種單倍型豬的Bic基因的結(jié)合能力的差異影響mi R-155的表達(dá),使CC型個(gè)體中的mi R-155-5p和mi R-155-3p的表達(dá)水平均高于AA型個(gè)體。(5)初步驗(yàn)證mi R-155的候選靶基因在AA型個(gè)體中表達(dá)量高于CC型個(gè)體,初步確定mi R-155與Sept11、Phf17基因的靶向關(guān)系。本研究初步證明了大白豬和梅山豬由于Bic基因中Foxp3結(jié)合位點(diǎn)的差異導(dǎo)致mi R-155的表達(dá)及靶基因的改變,為揭示這兩個(gè)豬種間的免疫能力差異性提供一些理論依據(jù),同時(shí)為抗病新品種的選育工作提供新的分子鑒定依據(jù)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S828

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 陳清森;劉小紅;楊紅杰;薛明;鄭友民;陳瑤生;;中國(guó)地方豬資源保護(hù)現(xiàn)狀與建議[J];中國(guó)豬業(yè);2012年01期

2 何芳明;溫朝暉;劉坤;;單核苷酸多態(tài)性在豬育種中的應(yīng)用[J];中國(guó)豬業(yè);2014年06期

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本文編號(hào)：1820782