通城猪抗蓝耳病的microRNAs筛选及其调控网络分析

发布时间：2018-01-19 02:09

  本文关键词： PRRSV PAM microRNAs 高通量测序 通城猪　出处：《华中农业大学》2015年硕士论文　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)俗称蓝耳病,以流行地域广、传播速度快、感染率高等特点成为全球规模化猪场的主要疫病之一,主要造成妊娠母猪的繁殖障碍及引起各种年龄猪特别是仔猪的呼吸道疾病。PRRS是由猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)引起的,PRRSV是一种单股正链RNA病毒,具有易变性且能引起宿主的免疫抑制,给兽医临床进行该病的控制带来很大困难,目前尚无有效的防控办法。通城猪是我国优良的地方品种,具有抗逆性强,肉质鲜嫩等特点,在HP-PRRS爆发期间,地处湖北省通城县境内的通城猪却无一例是因感染HP-PRRS而死亡的报道,故推测通城猪对HP-PRRS具有一定的抗性。Micro RNA(mi RNA)作为一类由内源基因编码的长度约为20-24个核苷酸的非编码单链RNA分子,在原核生物到哺乳动物中广泛分布并普遍参与机体内多种生命过程的调控,已有报道mi R-181、mi R-125b等能抑制PRRSV在宿主中的增殖,在PRRSV感染机体后是否有大量的mi RNAs参与抗病毒作用尚且不知。本研究以通城猪和大白猪为材料,利用人工感染PRRSV后和感染前的肺泡巨噬细胞(Porcine Alveolar Macrophages,PAMs)进行小RNA高通量测序,筛选在两品种感染前后和品种间PAMs中差异表达的mi RNAs并分析其调控网络,为进一步筛选通城猪中的抗PRRSV的mi RNAs奠定基础。本研究的主要内容及结果如下:(1)人工感染PRRSV后,通城猪的肺部病变和炎症比大白猪轻,肺组织中的病毒载量低于大白猪。利用5周龄且PRRSV、PCV、PRV三种病毒都为阴性的健康断奶仔猪通城猪22头和大白猪12头,按照同胞关系均匀随机分为感染组和对照组,经过预试验后进行人工感染,试验组猪肌肉注射PRRSV-Wu H3毒株,对照组猪肌肉注射DMEM;感染后第7天剖检,取肺脏等组织器官,每个肺脏的一侧采用PBS经支气管肺泡灌洗技术来收集PAMs,提取总RNA后用于小RNA的高通量测序,另一侧采集肺组织样品后用4%的多聚甲醛固定,用来制作病理切片并进行免疫组化分析。临床观察发现,6头感染组猪在2-5天内先后出现精神沉郁,采食下降、呕吐、喘气、高温、发热等症状,感染组的大白猪比通城猪体温升高更多,病症更明显;解剖时观察肺部大体病变,发现感染组的大白猪肺脏明显水肿,表面有大量出血点,肺叶出现深红色实变区,淋巴结广泛肿大,通城猪肺部肿胀,实变程度比大白猪轻,粉色有弹性,呈海绵状。用采集的肺组织样制作石蜡切片,经HE染色后于倒置显微镜下观察,发现通城猪和大白猪感染PRRSV后,肺脏均出现间质性肺炎的病变,细支气管和血管周围有单核巨噬细胞浸润,大部分肺泡壁显著增厚,肺泡腔狭窄,腔内主要可见坏死细胞,有均质红染的浆液及巨噬细胞,从组织学病变的严重程度比较,大白猪较通城猪更严重。对照组的通城猪和大白猪肺脏组织结构清楚,未见异常变化。用辣根过氧化物酶标记的PRRSV山羊抗鼠Ig G抗体通过免疫组化法来观察PRRSV抗原在肺脏中的分布,在显微镜完全相同的图像采集条件下,每张切片在40倍镜下随机选取至少5个视野,用软件IPP6.0进行定量分析,观察发现PRRSV抗体阳性反应产物在感染组猪肺脏中主要分布于肺泡巨噬细胞、肺泡II型上皮细胞、淋巴细胞及脱落细胞,在同一品种内,相对于对照组而言,感染组的猪肺脏中PRRSV阳性信号明显分布,而在感染组内,通城猪肺脏中PRRSV的分布量少于大白猪。(2)通城猪和大白猪感染PRRSV前后的12个PAM样本进行小RNA组的高通量测序,每个样本得到至少0.5G的数据,获得至少10327597条原始序列,比对得到391个已知猪的成熟mi RNA,其中67个在不同组合中差异表达。选择12头猪的PAM作为测序样本,提取总RNA后构建12个c DNA文库,于Illumina Hi Seq2000平台进行小RNA组的高通量测序,每个样本得到的原始数据量都在0.5G以上,至少获得10327597条原始序列,过滤后得到的纯净序列reads数均占原始序列总reads数的95%以上,20bp左右的碱基错误率都在0.02%以内。利用mi RDeep2将测序序列与Gene Bank中猪基因组序列及mi RBase中成熟mi RNA序列进行比对,发现在12个样本中均得到391个已报道的猪的成熟mi RNA。两两组合T检验筛选出了67个差异表达mi RNAs,筛选标准为T-test的P值≤0.05,fold change1为上调,fold change1为下调,其中通城猪感染组与对照组(TC-INJ-CON)相比显著下调表达的mi RNA有4个(mi R-101、mi R-146b、mi R-296-3p和mi R-664-5p),而上调表达的mi RNA有5个(mi R-22-3p、mi R-24-2-5p、mi R-27b-5p、mi R-423-5p和mi R-7137-5p);而大白猪感染组与对照组(LW-INJ-CON)相比显著下调表达的mi RNA有36个,上调表达的mi RNA有20个;通城猪感染组与大白猪感染组(TC-INF-LW)相比,有5个下调的mi RNA和3个上调的mi RNA;通城猪对照组和大白猪对照组(TC-CON-LW)比较,有4个下调mi RNA和3个上调mi RNA。其中,12个mi RNAs出现在多个组合中,例如mi R-101在TC-INF-CON中下调,而在LW-INF-CON和TC-CON-LW中上调,mi R-296-3p和mi R146b在TC-INF-CON和LW-INF-CON两个组合中均下调,而mi R-423-5p和mi R7137-5p在TC-INF-CON和LW-INF-CON两个组合都是上调。(3)预测到67个差异表达mi RNAs的宿主靶基因有22234个,在PRRSV基因组上的结合靶点有119个,宿主靶基因富集到多条GO Terms和KEGG通路中,并形成mi RNA-m RNA调控网络。利用软件Target Scan Human6.2预测差异表达mi RNAs在猪基因组中的靶基因,得到22243个m RNA-mi RNA互作位点,利用软件mi Randa预测到差异表达mi RNA在PRRSV基因组上有119个结合靶点。通城猪感染组与对照组差异表达mi RNAs的靶基有4474个,富集到801条GO Terms和50个KEGG通路中,大白猪感染组与对照组差异表达mi RNAs的靶基有12856个,富集到901条GO Terms和57个KEGG通路中。在二者共有的42个通路中,有12个与信号转导相关的通路和18个与疾病癌症相关的通路,还有巨噬细胞内受体FcγR介导的吞噬作用通路,推测此通路可能与宿主抗病毒的先天性免疫或PRRSV侵入机制有关。结合本课题组转录组测序数据的分析结果(另文见博士生梁婉的论文),将通城猪的差异表达mi RNA和m RNA联合分析,发现有8个差异表达mi RNAs能靶向143个差异表达基因,其中4个下调的mi RNA对应37个上调的基因,4个上调的mi RNA对应38个下调的基因,将143个差异基因用在线软件DAVID作GO和KEGG分析,富集到51个生物学过程(BP)、17种细胞组分(CC)和10种生物学功能(MF)的分类中,参与到p53信号通路、JAK-STAT信号通路、癌症通路、细胞因子及其受体的相互作用中,利用Cytoscape软件制作mi RNA——m RNA的蛋白调控关系网络,发现mi R-101和mi R-25b-5p等对多个靶基都具有调控作用。
[Abstract]:Porcine reproductive and respiratory syndrome (Porcine Reproductive and Respiratory Syndrome, PRRS) commonly known as blue ear disease, in broad areas, spread fast, high rate of infection has become one of the main diseases of global scale pig farms, mainly caused by reproductive failure of pregnant sows and pigs of all ages, especially in piglets caused by respiratory disease caused by porcine.PRRS reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) caused by PRRSV, is a kind of positive single strand RNA virus, which is changeable and can cause host immune suppression, control to the veterinary clinic for the disease has caused great difficulties, there is no effective prevention and control measures is Tongcheng. China's fine local variety, with strong resistance, fresh meat and other characteristics, during the HP-PRRS outbreak, the territory is located in Tongcheng County of Hubei Province Tongcheng but no example is due HP-PRRS infection and death have been reported, suggesting that Tongcheng resistant.Micro RNA on HP-PRRS (MI RNA) is a kind of endogenous gene encoding by the length of single stranded RNA molecules encoding non 20-24 nucleotides, in the regulation of prokaryotes to mammals are widely distributed and widely participate in a variety of life process in the body, have been reported mi R-181, MI R-125b can inhibit PRRSV proliferation in the host, whether in the PRRSV infection of a large number of MI RNAs is involved in the antiviral effect unknown. In this study, Tongcheng and large white pigs as materials, using human alveolar macrophages after PRRSV infection and infection before (Porcine Alveolar Macrophages, PAMs) are small RNA high-throughput sequencing, screening in two varieties of PAMs infection in MI and Varietal Difference before and after RNAs and analysis of the regulatory network for further screening of Tongcheng pigs against PRRSV mi R NAs foundation. The main contents and results of this study are as follows: (1) PRRSV after artificial infection, Tongcheng lung lesions and inflammation than large white light load in lung virus below Yorkshire. By 5 weeks of age and PRRSV, PCV, PRV three kinds of virus were negative healthy weaned piglets Tongcheng pigs 22 large white and 12 head, according to the uniform sibling were randomly divided into infection group and control group, after the pre test after artificial infection test group porcine intramuscular injection of PRRSV-Wu H3 strains, the control group pigs by intramuscular injection of DMEM infection; seventh days after the autopsy, the lungs and other organs, each side of the lung using PBS technology to collect by bronchoalveolar lavage PAMs, the total RNA was extracted for high-throughput sequencing of small RNA, the other side to collect lung tissue samples were fixed with 4% paraformaldehyde, used to make pathological and immunohistochemical analysis. Clinical observation, 6 The pig infection group in 2-5 days has appeared in depression, vomiting, decreased food intake, breathing, temperature, fever and other symptoms, the infection group increased more than the large white pigs of body temperature, symptoms are more obvious; anatomical observation of pulmonary pathological changes, found that the infection group significantly large white lung edema, surface bleeding deep red, lobar consolidation, extensive lymph node enlargement, Tongcheng pig lungs swelling and consolidation degree than the large white light pink, elastic, spongy. With the acquisition of the lung tissue samples in paraffin, stained by HE in inverted microscope observation, found in Tongcheng and large white pigs infected after PRRSV, the lungs showed interstitial pneumonia lesions around the bronchioles and vascular infiltration of mononuclear macrophage, most of alveolar wall thickening of alveolar cavity stenosis, mainly necrotic cells, and macrophages have serous homogeneous red staining, from Comparison of the severity of histological lesions, a large white Tongcheng more serious. The control group of Tongcheng and large white lung tissue structure is clear, no abnormal changes. Using horseradish peroxidase labeled Goat anti mouse G antibody PRRSV Ig to observe the distribution of PRRSV antigen in the lung by immunohistochemical method, image acquisition conditions is the same in the microscope, each slice in 40 magnification were randomly selected at least 5 horizons were quantitatively analyzed by IPP6.0 software, PRRSV antibody positive reaction in pig lung infection group distributed mainly in alveolar macrophages was observed, alveolar type II epithelial cells, lymphocytes and epithelial cells in the same variety in compared with the control group, the positive signal of PRRSV infection of pig lungs obviously in the distribution, and in the infection group, the distribution amount of Tongcheng pigs in the lung of PRRSV less than largewhite. (2) through the city and large white pigs The infection of 12 PAM samples before and after PRRSV for high-throughput sequencing of small RNA group, each sample obtained at least 0.5G data obtained at least 10327597 original sequence alignment, mature mi RNA 391 known pig, 67 of them in different combinations. Differentially expressed in 12 pigs as PAM sequencing samples, construct 12 C DNA library after total RNA extraction, for high-throughput sequencing of small RNA group on Illumina Hi Seq2000 platform, the original data of each sample obtained at the volume of more than 0.5G, at least 10327597 original series, more than the number of pure reads sequences obtained after filtration were accounted for 95% of the number of the original sequence of total reads, about 20bp the base error rate is less than 0.02%. The use of MI RDeep2 and Gene Bank in the sequencing sequence of the pig genome sequence and MI RBase mi RNA in the mature sequence alignment, found that 391 reported swine are in 12 samples 鐔焟i RNA.涓や袱缁勫悎T妫，

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