通城猪和长白猪对猪繁殖与呼吸综合征病毒耐受性差异研究

发布时间：2018-03-10 13:32

本文选题：猪繁殖与呼吸综合征病毒　切入点：RNA测序　出处：《中国农业大学》2015年博士论文　论文类型：学位论文

【摘要】：猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome, PRRS)是由猪繁殖与呼吸综合征病毒(PRRS Virus, PRRSV)引起的猪传染疾病。该病已成为危害养猪业最严重的疾病之一。近来研究表明,不同猪品种感染PRRSV后表现出的临床症状的严重程度有明显差异。然而,猪遗传变异对PRRSV耐受性的作用分子机理尚不清楚。基于合作单位华中农业大学对不同猪种感染PRRSV后临床症状比较分析,本论文选取了对PRRSV耐受性较强的通城猪和耐受性较差的长白猪作为研究材料,采用高通量测序的方法,对感染PRRSV后第3、5和7天猪和对照组猪的肺组织,进行mRNA-seq和miRNA测序,从编码基因和非编码RNA两个方面研究通城猪和长白猪对PRRSV耐受性差异的分子机理。为了研究通城猪和长白猪在感染PRRSV后基因表达调控的差异,本文将感染PRRSV后第3、第5和第7天的猪肺组织基因表达量分别和对照组进行比较,鉴定得到通城猪在各时间点差异基因数为:244、652、764,合并得到1288个差异显著表达基因,少于长白猪合并后差异显著基因个数2746个(2035、2037、843)的一半。这1288和2746个差异显著表达基因中只有913个基因是重叠的,这表明,PRRSV感染导致两种猪对基因的表达调控有明显差异。进一步研究发现,感染组与对照组相比,通城猪基因的表达变化幅度明显小于长白猪。根据以上结果,本文挑选出了在两个品种猪之间存在表达模式差异的6个候选基因:BID、BTG2、MX1、BCL-XL、IFIT1和SOD1。在Marc-145细胞中过表达这6个基因,均能显著抑制PRRSV在Marc-145细胞中的复制。与猪的参考基因组比较,本文在通城猪和长白猪中分别鉴定得到了 98416和34994个SNPs,发现了两个位于BTG2和BID基因的3'UTR的miRNA保守结合位点序列上的通城猪具有,而长白猪没有的SNPs。双荧光素酶活性检测表明,与长白猪基因型相比,这两个SNPs均能显著影响双荧光素酶活性。通城猪和长白猪在感染PRRSV后对BTG2和BID基因表达调控的差异,可能是与位于BTG2和BID基因的3'UTR的SNPs有关的。本文也利用此数据对两种猪与PRRSV感染相关的lncRNA (4024个)的表达进行了分析。感染后第3天与对照组相比,本文鉴定得到了 305个在两种猪中有相反表达模式的lncRNAs。同时,本论文发现了与人类中参与HIV感染过程的NEAT1-lncRNA序列相似的NEAT1-like在两个猪品种中感染PRRSV后第3天的表达量均大幅度上调,表明猪NEAT1-like可能参与了PRRSV感染过程。此外,本文还对两种猪PRRSV感染组和对照组的miRNA表达谱进行了比较分析,并得到了以下结果:1、两种猪中共检测到278个已知和294个新的miRNA表达;2、将通城猪感染PRRSV后的第3、5和7天猪肺组织的miRNA表达量与对照组进行比较,得到了 52、62、72个差异显著表达miRNA;将长白猪感染PRRSV后的第3、5和7天肺组织的miRNA表达量与对照组相比,得到了 72、75、109个差异显著表达miRNA; 3、与对照组相比,qRT-PCR验证了六个miRNA的差异显著表达,其结果与测序数据分析结果一致;4、本文通过预测发现,miR-2320-5p可能直接与PRRSV基因组3'UTR保守序列结合;5、与对照组相比,PRRSV感染会导致两种猪的miRNA编辑水平均显著上升。综上所述,本文鉴定得到了与PRRSV感染相关的编码基因(BID、BTG2、MX1、BCL-XL、IFI71和SOD1)、lncRNAs和miRNAs、可能与猪对PRRSV耐受性有关的SNPs。此项研究为筛选得到新的抗PRRSV基因提供了新的思路;为进一步探明遗传变异对不同猪种感染PRRSV表现出的不同耐受性提供了科学依据;同时也为阐明非编码序列(lncRNA和miRNA)参与PRRSV感染的机制和抗病育种提供了重要的科学理论基础。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) is composed of porcine reproductive and respiratory syndrome virus (PRRS Virus, PRRSV) of infectious diseases caused by swine. The disease has become one of the most serious diseases of swine. Recent research shows that there are significant differences in different pig breeds after PRRSV infection the clinical severity of symptoms. However, the molecular mechanism of porcine genetic variation in PRRSV tolerance is not clear. The cooperation unit of Huazhong Agricultural University in different pig breeds comparative analysis of clinical symptoms of PRRSV infection based on the select of PRRSV strong tolerance of Tongcheng pigs and less tolerance of Landrace as the study material. By using the method of high-throughput sequencing, the infection of PRRSV 3,5 after 7 days and pigs and pigs in the control group lung tissue, mRNA-seq and miRNA sequencing, from the two aspects of gene encoding and RNA encoding Study on Tongcheng and Landrace pigs PRRSV molecules on the difference of tolerance mechanism. In order to study the difference of gene expression regulation of Tongcheng and Landrace pigs at PRRSV after infection, the infection of PRRSV after third, respectively, compared with the control group lung tissue gene fifth and seventh days of expression, the number of genes identified at each time point between Tongcheng pig: 244652764, 1288 differentially expressed genes were significantly less than the merger, after the merger of significant differences between Landrace gene number 2746 (20352037843) of the half. The 1288 and 2746 significant differences in gene expression of only 913 genes overlap, suggesting that PRRSV infection leads to the regulation of the expression of the gene was two pigs differences. Further studies showed that the infection group compared with the control group, the expression changes of Tongcheng gene was significantly less than that of Landrace. According to the above results, this paper selected two varieties There are 6 candidate gene expression pattern differences between pigs: BID, BTG2, MX1, BCL-XL, IFIT1 and SOD1. in Marc-145 cells expressing the 6 genes could significantly inhibit the PRRSV replication in Marc-145 cells. Compared with the reference in the pig genome, Tongcheng pigs and Landrace pigs were identified 98416 and 34994 SNPs, found two in the BTG2 and BID genes of 3'UTR conserved miRNA binding site sequence of Tongcheng and Landrace has indicated that no SNPs. dual luciferase activity compared with Landrace genotypes, the two SNPs was significantly affected by dual luciferase activity. The expression of Tongcheng and Landrace pigs regulation of the differences of BTG2 and BID gene in PRRSV infection, and may be located in the BTG2 and BID gene of 3'UTR SNPs. This paper also use this data to lncRNA two pigs infected with PRRSV (4024) surface Was analyzed. Compared with the control group third days after infection, we identified 305 in two pigs have opposed the expression pattern of lncRNAs. at the same time, this paper found the expression similar to the NEAT1-lncRNA sequence of HIV infection in the process involved in human NEAT1-like in two pig breeds in third days after PRRSV staining are a big increase, indicate that porcine NEAT1-like may be involved in the process of PRRSV infection. In addition, the expression of the two pigs PRRSV infection group and control group of miRNA spectrum are analyzed, and the results are as follows: 1, two pigs were detected in 278 known and 294 new miRNA expression; 2, the Tongcheng pigs infected by PRRSV after 3,5 and 7 days of lung tissue miRNA expression were compared with control group, the 52,62,72 expression of miRNA was significantly different; the Landrace after infection with PRRSV at 3,5 and 7 days of lung tissue miRNA expression and Compared with the control group, the expression of miRNA 72,75109 significantly; 3, compared with the control group, qRT-PCR show the difference between the six miRNA significant expression, and the results of sequencing data analysis results are consistent; 4, this paper predicted that miR-2320-5p may be directly combined with the conserved sequences of PRRSV gene of group 3'UTR; 5, compared with the control group PRRSV infection leads to two swine miRNA editing level increased significantly. In summary, this paper identified encoding gene related with PRRSV infection (BID, BTG2, MX1, BCL-XL, IFI71 and SOD1), lncRNAs and miRNAs, and pigs to PRRSV tolerance related SNPs. this study screened for resistance a new PRRSV gene provides a new way of thinking; to provide a scientific basis for the different pigs infected with PRRSV showed different tolerance to further explore the genetic variation; also to clarify the non encoding sequence (lncRNA and miRNA) in PRR The mechanism of SV infection and the breeding of disease resistance provide an important scientific theoretical basis.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.28

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