【作者】 张萍；

【导师】 姜运良；

【作者基本信息】 山东农业大学 ， 动物遗传育种与繁殖， 2017， 硕士

【摘要】 每年因为猪流行病的传播和发病给世界的养猪业造成了巨大的经济损失,在这些流行病中,PCV2是一种广泛存在的猪流行病病毒,具有很强的致病性,它可以与许多病原并发和继发感染,是引起仔猪断奶后多系统衰竭综合征(postweaning multisystemic wasting syndrome,PMWS)的主要病原。有研究表明,不同的猪品种由于遗传差异对PCV2的抗病反应也不同。我国的地方猪种莱芜猪较西方猪种有更好的抗PCV2的特性。miRNA作为一类单链非编码小RNA分子具有在转录后水平的调控作用,在动物的发育、增殖、分化和凋亡过程中有着重要的作用。本研究主要针对莱芜猪和大长二元杂交猪对PCV2的不同抗性开展进一步的研究。在RNA-seq的基础上,通过检测预测靶基因的m RNA和蛋白水平的分析,荧光素酶活性检测,确定了ssc-miR-122的靶基因。针对莱芜猪和大长杂交猪肺组织中差异表达的miR-122的表达变化和所调控的基因进行分析,确定了ssc-miR-122启动子活性及其关键调控区,并对启动子的关键调控区的SNP进行了分析。最后对ssc-miR-122与PCV2复制的关系进行了探讨。研究结果如下:1.miR-122表达量的检测及靶基因的验证。(1)利用qPCR对猪不同组织中ssc-miR-122的表达量进行了检测。结果表明与未攻毒莱芜猪相比,PCV2攻毒的莱芜猪在扁桃体、脾脏中上调表达,其中在心脏中上调较明显。在淋巴结、小肠、大肠、肝脏中有下调的表达趋势。与未攻毒大长猪相比,PCV2攻毒的大长猪在扁桃体、大肠、肝脏、和脾脏中有上调表达的趋势,其中在肝脏中上调表达十分显著。在淋巴结、小肠、心脏中有下调表达的趋势。(2)利用qPCR检测肺组织中预测靶基因NFAT5和NPEPPS的m RNA表达量变化。NFAT5和NPEPPS的表达量在攻毒与未攻毒的莱芜猪和大长猪肺组织中都是下调表达的。(3)预测了miRNA的靶基因。通过Targetscan、pic Tar、miRanda等在线预测数据库预测ssc-miR-122的靶基因为NPEPPS和NFAT5。其中NFAT5是激活的核因子5,是转录因子家族的一类,在哺乳动物的免疫应答中,NFAT5家族蛋白在诱导基因转录方面有着重要的作用。NPEPPS在MHC I类分子的抗原加工途径中起作用,涉及细胞毒性T细胞表位前体的N-末端修饰。(4)细胞水平上的验证。首先确定了ssc-miR-122的转染条件为30nmol并在转染后48h达到最佳转染条件。转染后,细胞内NFAT5和NPEPPS的m RNA水平都显著降低(P<0.05);Western blotting检测了NFAT5和NPEPPS的蛋白水平的变化,结果表明NFAT5和NPEPPS的蛋白水平都显著下调(P<0.05),与m RNA水平的表达变化一致。(5)Ssc-miR-122与NFAT5和NPEPPS的靶向关系。将构建的NFAT5和NPEPPS的野生型3’UTR和突变型3’UTR的双荧光素酶载体与ssc-miR-122 mimic共转染,结果表明野生型的荧光活性显著降低(P<0.01),表明NFAT5和NPEPPS是Ssc-miR-122的靶基因。2.莱芜猪与大长杂交猪的ssc-miR-122启动子的转录调控分析(1)利用测序发现莱芜猪和大长杂交猪的ssc-miR-122存在两种片段长度的启动子,其中一种比另一种长7bp,通过双荧光素酶报告基因分析两者活性存在显著差异(P<0.001)。通过启动子删除实验确定了ssc-miR-122的核心启动子区,发现在前体上游-4587至-3594bp处存在转录激活活性。并在该区域发现一个7bp(AACCCCC)的插入缺失突变。通过检测7bp的完全型和缺失型启动子发现,插入型的启动子活性明显高于缺失型的启动子活性(P<0.001)。通过在线预测(http://www.genomatix)分析在这7bp的插入缺失突变处存在转录因子CART家族的存在。(2)(AACCCCC)插入缺失多态性的群体水平检测。通过PCR扩增和测序检测了44头莱芜猪、49头杜洛克猪、56头长白猪、47头约克夏猪在该位点的多态性。发现杜洛克猪没有插入型,而长白猪和约克夏猪的插入型比例高于莱芜猪,但都不符合哈代温伯格平衡定律,说明存在选育的作用。3.miR-122对PCV2的复制效应不同类型启动子在PCV2接毒后的活性变化。双荧光素酶活性检测结果表明在PCV2接毒后插入型的启动子活性显著升高,而缺失型的启动子活性变化不明显。通过过表达miR-122,检测miR-122对PCV2复制的效应。结果表明miR-122能明显抑制cap蛋白的表达。说明miR-122能有效抑制PCV2的复制。综上所述,通过本研究我们确定了莱芜猪和大长二元杂交猪肺组织中差异表达的miR-122,确定了两个与免疫相关的靶基因。说明miR-122可能是通过作用于NFAT5和NPEPPS这两个靶基因的调控来参与机体免疫相关的反应。其次对miR-122的转录调控进行分析,筛选出一个启动子区的与抗PCV2相关的分子标记,为培育对PCV2抗病性的猪提供了依据。更多还原

【Abstract】 The frequent outbreak of epidemic in pigs has resulted in severe economic losses to the pig industry all over the world.Among these diseases,porcine circovirus virus 2 disease is one of the most important diseases which damage our country’s swine industry.Porcine circovirus type 2,which has strong pathogenicity,is the primary cause of Postweaning Multisystemic Wasting Syndrome(PMWS)in pigs.There are evidence that the clinical symptoms of PCV2 infection in different breeds of pigs were different because of the host genetic variations.There are proofs that the Chinese pig breed Laiwu(LW)pigs has more resistance to porcine circovirus virus 2 disease than Yorkshire/Landrace crossbred(YL)pigs.In this study,we aimed at to study the different resistance of PCV2 in LW pigs and YL pigs.On the basis of the RNA-seq,we analysised the different expression of ssc-miR-122 and its targets genes between LW pigs and LY pigs in lung tissue.We analysised the activity of of ssc-miR-122 promoter region and SNP in the crucial promoter region.We identified the predicting target genes by decting the mRNA and protein levels and detecting the activity of fluorescein.The results are as follows:1.Detection of miR-122 expression and verification of target genes.(1)The expression of ssc-miR-122 in different tissues of swine was detected by qPCR.The results showed that PCV2-infected Laiwu pigs were up-regulated in the tonsils and spleen compared with the PCV2-negetive control Laiwu pigs,significantly up-regulated in the heart.But in the lymph nodes,small intestine,large intestine,liver showed down-regulated expression trend.Compared with PCV2-negetive control pigs,PCV2-infected pigs showed a tendency to up-regulate in the tonsils,large intestine,liver,and spleen,significantly up-regulated expression in the liver.But in the lymph nodes,small intestine,heart were down-regulated expression.(2)The mRNA expression level of NFAT5 and NPEPPS in lung tissue were detected byqRT-PCR.The results showed that both NFAT5 and NPEPPS were significant decreased in PCV2-infected a PCV2-negative control LW and LY pigs.(3)Predicted the target genes of ssc-miR-122.We predicted NFAT5 and NPEPPS are two target genes of ssc-miR-122 through the the online prediction database of Targetscan,picTar and miRanda.The product of NFAT5 is a member of the nuclear factors of activated T cells family of transcription factors.Proteins belonging to this family play a central role in inducible gene transcription during the immune response.NPEPPS(Aminopeptidase Puromycin Sensitive)is a Protein Coding gene.Among its related pathways are immune system and antigen processing-Ubiquitination and Proteasome degradation.NPEPPS involves in proteolytic events essential for cell growth and viability and plays a role in the antigen-processing pathway for MHC class I molecules.It also involved in the N-terminal trimming of cytotoxic T-cell epitope precursors.(4)Validation in PK15 cells.We firstly explored the transfection condition of ssc-miR-122 was 30 nmol and the optimal transfection condition was reached 48 h after transfection.At this time,detecting the mRNA levels of NFAT5 and NPEPPS were significantly decreased(P <0.05).The protein level of NFAT5 and NPEPPS was analyzed by western blotting.The results showed that the NFAT5 and NPEPPS protein was significantly decreased after transfection with ssc-miR-122 mimic(p<0.05),was constant to the mRNA expression level.(5)NFAT5 and NPEPPS were directly targets of ssc-miR-122.Results from co-transfection of luciferase reporter vector containing either wild or mutant NFAT5 and NPEPPS 3′UTR with ssc-miR-122 mimic into PK15 cells showed that,the luciferase activity harboring the wild NFAT5 and NPEPPS 3′UTR rather than the mutant NFAT5 and NPEPPS3′UTR was significantly decreased(p<0.001),indicating NFAT5 and NPEPPS was the directly target of ssc-miR-1222.The transcriptional regulation of ssc-miR-122 promoter between LW and LY pigs.(1)By sequencing the reporter plasmids,we found there were two fragments of length in ssc-miR-122 promoter region between LW and LY pigs,one is 7 bp longer than the other,theactivity of the two different length promoters were significant different by double luciferase reporter gene analysis(P<0.01).We confirmed the core promoter region by constructing with5′ progressive deletion fragments of the ssc-miR-122 promoter,we found the existence of potential positive-regulatory elements from-4587 to-3594 bp of pre-miR-122.There was a7bp(AACCCCC)deletion mutation in this region,By transfecting with 7 bp indel or7 bp deletion fragments,the promoter activity of the shorter type was significantly higher than the longer type(P<0.001).The presence of the transcription factor CART family exists at the 7bp insertion deletion mutation by online prediction(http://www.genomatix).(2)The polymorphism of indel AACCCCC in different group pigs.We tested 44 Laiwu pigs,49 Duroc pigs,56 Changbai pigs and 47 Yorkshire pigs by RT-PCR,it was not detected in Duroc pigs,but in Laiwu pigs,the allelic frequency of AACCCC showed was smaller than the Landrace and Yorkshire pigs.All the three breeds pigs showed significant departure from Hardy-Weinberg equilibrium at this site.3.The luciferease activity of different types of ssc-miR-122 promoters after PCV2 infected.The results of double luciferase activity showed that the promoter activity of the inserted type was significantly increased after PCV2 administration,while the activity of the short promoter was not changed obviously.The effect of miR-122 on PCV2 replication was examined by overexpressing miR-122.The results showed that miR-122 could significantly inhibit the expression of cap protein.Indicating that miR-122 can effectively inhibit PCV2 replication.In conclusion,we identified miR-122,a differentially expressed miRNA,in lung tissues of LW and LY pigs,and analyzed the transcriptional regulation of miR-122,and identified two immunological related target genes of ssc-miR-122 in this study.Indicating that miR-122 may be involved in the immune response of the body by acting on the regulation of NFAT5 and NPEPPS.Screening of a promoter region with molecular markers associated with PCV2,which provided the basis for cultivating excellent PCV2 resistant pigs.更多还原