不同品种猪感染圆环病毒Ⅱ型后肺组织差异miRNA的鉴定及功能分析
发布时间：2019-01-06 06:20
【摘要】：猪圆环病毒(porcine circovirus type,PCV)是无囊膜的单股环状DNA病毒的一种,包括猪圆环病毒I型(PCV1)和猪圆环病毒II型(PCV2)两个基因型,其中猪圆环病毒II型具有强的致病性,能够引起断奶后仔猪多系统衰竭综合征(PMWS)等多种疾病,并可以引起猪体的免疫抑制,该病毒往往与其他病原混合或继发感染,给我国养猪业造成了巨大损失,严重影响了我国养猪业的发展。猪肺组织是PCV2感染的主要病发部位。miRN A作为一种转录后水平上的基因调控因子,在动物的进化过程中高度保守且具有表达的时空特异性,广泛地参与抗病反应、细胞凋亡、细胞分化、激素分泌等多种生命过程。莱芜猪作为中国的地方猪种在抗病抗逆性等方面优于西方猪种。现今,miRNA的表达分析模式已经在很多重要的猪病上有所报道,但是PCV2感染后miRNA表达变化仍未有报导。本实验建立了四个肺组织miRNA的表达文库,分别为莱芜猪对照组(Laiwu),莱芜猪PCV2感染组(Laiwu-i),大约克-长白猪对照组(YL)及大约克-长白猪感染组(YL-i)。随后通过Solexa高通量测序及生物信息学分析的方法研究了感染后miRN A的表达丰度及差异表达情况。寻找到了282个已知和108个新发现的miRNA,并对这些miRNA进行了进一步的研究。(1)感染PCV2后莱芜猪与大约克-长白杂交猪miRNA表达变化分析。在总共测出的282个已知miRNA中发现了23个显著差异表达的miRNA(P-value0.05,Fold-change=log2(实验组/对照组)1 or-1)。后续对5个表达丰度较高且变化幅度较大的miRNA进行了q RT-PCR验证分析,发现这5个miRNA(miR-122,miR-192,miR-451,miR-486,miR-504)表达模式均与测序结果一致(p0.05),表明测序结果是可信的。其中,miR-122,192,451,486均在攻毒后显著性的上调表达,而miR-504则呈显著性的下调表达。(2)运用DIANA-mirPath分析了miR-122,192,451,486四个显著上调表达的miRN A靶基因及通路富集情况。在分析的过程中发现,这四个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等与细胞增殖凋亡和疾病免疫等相关的信号通路。在这四个miRNA预测的靶基因中进一步的研究发现NFAT5与IGF1基因均可以靶向四个上调miRNA中的两个以上,且参与着多个相关通路。NFAT5和IGF1基因的qRT-PCR分析发现,这两个基因表达趋势与上调的miRN A相反,呈显著上调表达。综上所述,本研究通过miRNA高通量测序的方法,对中国地方猪种莱芜猪和大约克-长白杂交猪感染PCV2后肺组织miRNA表达谱变化进行分析,确定了猪PCV2感染相关的数个miRNA,并通过DIANA-mirPath分析发现4个上调显著的miRNA靶基因主要富集于细胞凋亡和免疫相关通路,并确定了两个重要的靶基因。为今后PCV2相关疾病的研究提供了重要的参考,并证明了我国地方猪在抗PCV2方面与西方猪种间存在显著差异,为我国地方猪遗传多样性的保护提供依据,本研究也填补了PCV2感染miRNA表达谱方面的空白,为今后研究miRNA在PCV2感染过程中的作用奠定了坚实的基础。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
本文编号：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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
【参考文献】
相关期刊论文 前1条
1 夏伟;曹国军;邵宁生;;MicroRNA靶基因的寻找及鉴定方法研究进展[J];中国科学(C辑:生命科学);2009年01期
，本文编号：2402455
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