不同品種豬感染圓環(huán)病毒Ⅱ型后肺組織差異miRNA的鑒定及功能分析
發(fā)布時(shí)間：2019-01-06 06:20
【摘要】：豬圓環(huán)病毒(porcine circovirus type,PCV)是無囊膜的單股環(huán)狀DNA病毒的一種,包括豬圓環(huán)病毒I型(PCV1)和豬圓環(huán)病毒II型(PCV2)兩個(gè)基因型,其中豬圓環(huán)病毒II型具有強(qiáng)的致病性,能夠引起斷奶后仔豬多系統(tǒng)衰竭綜合征(PMWS)等多種疾病,并可以引起豬體的免疫抑制,該病毒往往與其他病原混合或繼發(fā)感染,給我國養(yǎng)豬業(yè)造成了巨大損失,嚴(yán)重影響了我國養(yǎng)豬業(yè)的發(fā)展。豬肺組織是PCV2感染的主要病發(fā)部位。miRN A作為一種轉(zhuǎn)錄后水平上的基因調(diào)控因子,在動(dòng)物的進(jìn)化過程中高度保守且具有表達(dá)的時(shí)空特異性,廣泛地參與抗病反應(yīng)、細(xì)胞凋亡、細(xì)胞分化、激素分泌等多種生命過程。萊蕪豬作為中國的地方豬種在抗病抗逆性等方面優(yōu)于西方豬種�，F(xiàn)今,miRNA的表達(dá)分析模式已經(jīng)在很多重要的豬病上有所報(bào)道,但是PCV2感染后miRNA表達(dá)變化仍未有報(bào)導(dǎo)。本實(shí)驗(yàn)建立了四個(gè)肺組織miRNA的表達(dá)文庫,分別為萊蕪豬對照組(Laiwu),萊蕪豬PCV2感染組(Laiwu-i),大約克-長白豬對照組(YL)及大約克-長白豬感染組(YL-i)。隨后通過Solexa高通量測序及生物信息學(xué)分析的方法研究了感染后miRN A的表達(dá)豐度及差異表達(dá)情況。尋找到了282個(gè)已知和108個(gè)新發(fā)現(xiàn)的miRNA,并對這些miRNA進(jìn)行了進(jìn)一步的研究。(1)感染PCV2后萊蕪豬與大約克-長白雜交豬miRNA表達(dá)變化分析。在總共測出的282個(gè)已知miRNA中發(fā)現(xiàn)了23個(gè)顯著差異表達(dá)的miRNA(P-value0.05,Fold-change=log2(實(shí)驗(yàn)組/對照組)1 or-1)。后續(xù)對5個(gè)表達(dá)豐度較高且變化幅度較大的miRNA進(jìn)行了q RT-PCR驗(yàn)證分析,發(fā)現(xiàn)這5個(gè)miRNA(miR-122,miR-192,miR-451,miR-486,miR-504)表達(dá)模式均與測序結(jié)果一致(p0.05),表明測序結(jié)果是可信的。其中,miR-122,192,451,486均在攻毒后顯著性的上調(diào)表達(dá),而miR-504則呈顯著性的下調(diào)表達(dá)。(2)運(yùn)用DIANA-mirPath分析了miR-122,192,451,486四個(gè)顯著上調(diào)表達(dá)的miRN A靶基因及通路富集情況。在分析的過程中發(fā)現(xiàn),這四個(gè)miRNA的靶基因主要富集于Insulin signaling pathway,mTOR signaling pathway,Non-small cell lung cancer和Leukocyte transendothelial migration等與細(xì)胞增殖凋亡和疾病免疫等相關(guān)的信號(hào)通路。在這四個(gè)miRNA預(yù)測的靶基因中進(jìn)一步的研究發(fā)現(xiàn)NFAT5與IGF1基因均可以靶向四個(gè)上調(diào)miRNA中的兩個(gè)以上,且參與著多個(gè)相關(guān)通路。NFAT5和IGF1基因的qRT-PCR分析發(fā)現(xiàn),這兩個(gè)基因表達(dá)趨勢與上調(diào)的miRN A相反,呈顯著上調(diào)表達(dá)。綜上所述,本研究通過miRNA高通量測序的方法,對中國地方豬種萊蕪豬和大約克-長白雜交豬感染PCV2后肺組織miRNA表達(dá)譜變化進(jìn)行分析,確定了豬PCV2感染相關(guān)的數(shù)個(gè)miRNA,并通過DIANA-mirPath分析發(fā)現(xiàn)4個(gè)上調(diào)顯著的miRNA靶基因主要富集于細(xì)胞凋亡和免疫相關(guān)通路,并確定了兩個(gè)重要的靶基因。為今后PCV2相關(guān)疾病的研究提供了重要的參考,并證明了我國地方豬在抗PCV2方面與西方豬種間存在顯著差異,為我國地方豬遺傳多樣性的保護(hù)提供依據(jù),本研究也填補(bǔ)了PCV2感染miRNA表達(dá)譜方面的空白,為今后研究miRNA在PCV2感染過程中的作用奠定了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Porcine circovirus type (PCV) is one of the single-strand circular DNA viruses without a capsule membrane, including two genotypes of porcine circovirus type I (PCV1) and porcine circovirus type II (PCV2), wherein the porcine circovirus type II has strong pathogenicity, can cause various diseases such as the multi-system failure syndrome (PMWS) and the like of the post-weaning piglets, and can cause the immunosuppression of the pig body, and the virus is often mixed with other pathogens or secondary infection, thus causing great loss to the pig industry in China, and seriously affecting the development of the pig industry in China. Porcine lung tissue is the main disease-producing site of PCV2 infection. miRRN A, as a gene regulation factor at the post-transcriptional level, is highly conserved in the evolution of the animal and has the time-space specificity of expression, and is widely used in various life processes such as disease resistance, cell apoptosis, cell differentiation, hormone secretion and the like. Laiwu pig, as a local pig in China, is superior to the western pig species in the aspects of disease resistance and stress resistance. The expression analysis model of miRNA has been reported on many important pig diseases, but the expression of miRNA after PCV2 infection has not been reported. The expression library of four lung tissue miRNAs was established in this experiment, which were Laiwu pig control group (Laiwu), Laiwu pig PCV2 infection group (Laiwu-i), Great York-Wisconsin pig control group (YL), and the large-sized Yorkshire-pig infection group (YL-i). The expression abundance and differential expression of miRN A after infection were studied by Solexa high-throughput sequencing and bioinformatics analysis. 282 known and 108 newly discovered miRNAs were found and further studies were made to these miRNAs. (1) Analysis of the change of the expression of miRNAs of Laiwu and Yorkshire-long white hybrid pigs after PCV2 infection. A total of 23 miRNAs (P-value.05, fold-change = log2 (experimental group/ control group) 1 or-1) were found in a total of 282 known miRNAs. The expression patterns of 5 miRNAs (miR-122, miR-192, miR-451, miR-486, and miR-504) were found to be consistent with the sequencing results (p0.05). Of these, miR-122, 192, 451, 486 were significantly up-regulated after challenge, while miR-504 was significantly down-regulated. (2) The miRN A target gene and the pathway enrichment of miR-122, 192, 451 and 486 were analyzed by DIANA-mirPath. In the process of the analysis, the target genes of the four miRNAs are mainly enriched in the signaling pathways associated with cell proliferation and disease immunity, such as in the form of Inulin signaling pathway, mTOR signaling pathway, Non-small cell lung cancer, and Leukocyte transfixion migration. Further studies in the target genes predicted by the four miRNAs found that both the NFAT5 and the IGF1 gene can target more than two of the four up-regulated miRNAs and participate in a plurality of related pathways. The qRT-PCR analysis of the NFAT5 and IGF1 genes found that the expression of these two genes, in contrast to the up-regulated miRRN A, was significantly up-regulated. To sum up, this study analyzed the changes of the expression profiles of the lung tissue miRNAs after PCV2 infection with the local pig breeds of Laiwu and Big York-long white hybrid pigs by the method of high-throughput sequencing of the miRNA, and identified several miRNAs related to the infection of the porcine PCV2, Four up-regulated miRNA target genes were mainly enriched in the cell apoptosis and immune-related pathways through the DIANA-mirPath analysis, and two important target genes were identified. In order to provide an important reference for the study of PCV2-related diseases in the future, it is proved that there is a significant difference between the anti-PCV2 and the Western swine among the local pigs in China, which provides the basis for the protection of the genetic diversity of local pigs in China. The study also fills the blank of the expression profile of PCV2-infected miRNAs. and lays a solid foundation for researching the role of the miRNA in the PCV2 infection process in the future.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28
本文編號(hào)：2402455
[Abstract]:Porcine circovirus type (PCV) is one of the single-strand circular DNA viruses without a capsule membrane, including two genotypes of porcine circovirus type I (PCV1) and porcine circovirus type II (PCV2), wherein the porcine circovirus type II has strong pathogenicity, can cause various diseases such as the multi-system failure syndrome (PMWS) and the like of the post-weaning piglets, and can cause the immunosuppression of the pig body, and the virus is often mixed with other pathogens or secondary infection, thus causing great loss to the pig industry in China, and seriously affecting the development of the pig industry in China. Porcine lung tissue is the main disease-producing site of PCV2 infection. miRRN A, as a gene regulation factor at the post-transcriptional level, is highly conserved in the evolution of the animal and has the time-space specificity of expression, and is widely used in various life processes such as disease resistance, cell apoptosis, cell differentiation, hormone secretion and the like. Laiwu pig, as a local pig in China, is superior to the western pig species in the aspects of disease resistance and stress resistance. The expression analysis model of miRNA has been reported on many important pig diseases, but the expression of miRNA after PCV2 infection has not been reported. The expression library of four lung tissue miRNAs was established in this experiment, which were Laiwu pig control group (Laiwu), Laiwu pig PCV2 infection group (Laiwu-i), Great York-Wisconsin pig control group (YL), and the large-sized Yorkshire-pig infection group (YL-i). The expression abundance and differential expression of miRN A after infection were studied by Solexa high-throughput sequencing and bioinformatics analysis. 282 known and 108 newly discovered miRNAs were found and further studies were made to these miRNAs. (1) Analysis of the change of the expression of miRNAs of Laiwu and Yorkshire-long white hybrid pigs after PCV2 infection. A total of 23 miRNAs (P-value.05, fold-change = log2 (experimental group/ control group) 1 or-1) were found in a total of 282 known miRNAs. The expression patterns of 5 miRNAs (miR-122, miR-192, miR-451, miR-486, and miR-504) were found to be consistent with the sequencing results (p0.05). Of these, miR-122, 192, 451, 486 were significantly up-regulated after challenge, while miR-504 was significantly down-regulated. (2) The miRN A target gene and the pathway enrichment of miR-122, 192, 451 and 486 were analyzed by DIANA-mirPath. In the process of the analysis, the target genes of the four miRNAs are mainly enriched in the signaling pathways associated with cell proliferation and disease immunity, such as in the form of Inulin signaling pathway, mTOR signaling pathway, Non-small cell lung cancer, and Leukocyte transfixion migration. Further studies in the target genes predicted by the four miRNAs found that both the NFAT5 and the IGF1 gene can target more than two of the four up-regulated miRNAs and participate in a plurality of related pathways. The qRT-PCR analysis of the NFAT5 and IGF1 genes found that the expression of these two genes, in contrast to the up-regulated miRRN A, was significantly up-regulated. To sum up, this study analyzed the changes of the expression profiles of the lung tissue miRNAs after PCV2 infection with the local pig breeds of Laiwu and Big York-long white hybrid pigs by the method of high-throughput sequencing of the miRNA, and identified several miRNAs related to the infection of the porcine PCV2, Four up-regulated miRNA target genes were mainly enriched in the cell apoptosis and immune-related pathways through the DIANA-mirPath analysis, and two important target genes were identified. In order to provide an important reference for the study of PCV2-related diseases in the future, it is proved that there is a significant difference between the anti-PCV2 and the Western swine among the local pigs in China, which provides the basis for the protection of the genetic diversity of local pigs in China. The study also fills the blank of the expression profile of PCV2-infected miRNAs. and lays a solid foundation for researching the role of the miRNA in the PCV2 infection process in the future.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.28
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 夏偉;曹國軍;邵寧生;;MicroRNA靶基因的尋找及鑒定方法研究進(jìn)展[J];中國科學(xué)(C輯:生命科學(xué));2009年01期
，本文編號(hào)：2402455
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