MicroRNAs在鸡感染肠炎沙门氏菌中的表达调控

发布时间：2018-07-20 21:49

【摘要】：MicroRNAs(miRNAs)大小为20~25个核苷酸,存在于真核生物体内,是一类内源性的具有调控功能的非编码RNA。miRNAs通过碱基互补配对的方式识别靶mRNA,并根据互补程度的不同指导沉默复合体降解靶mRNA或者阻遏靶mRNA的翻译。相关的研究显示,gga-mir-1662、gga-mir-215和gga-mir-1416与鸡肠炎沙门氏菌的感染相关,且gga-mir-1662与TLR1LA、gga-mir-1416与BCL10可能存在靶向关系。SNP(single nucleotide polymorphism)是基因组DNA碱基的变异,在基因组中的发生频率较高,有意义的变异会影响到基因组DNA组成元件的功能、转录产生的mRNA水平、蛋白的表达,进而影响基因的功能,导致生物性状改变甚至致病。因此SNP被广泛用于群体遗传学研究(如生物的起源、进化及迁移等方面),和疾病相关基因的研究,在药物基因组学、诊断学和生物医学研究中起重要作用。本研究从miRNA与靶基因的表达以及mi RNA的变异两个方面研究其在鸡肠炎沙门氏菌感染中的作用机制。用肠炎沙门氏菌感染2日龄济宁百日鸡,在感染后的第1天、第3天、第7天、第14天、第21天、第28天和第35天采集空肠组织样品,实验组和对照组各4只,提取组织总RNA后检测相关microRNA和靶基因的表达量;感染后第7天取200只济宁百日鸡的盲肠内容物和血液,分别用来检测肠炎沙门氏菌的含量和提取基因组DNA,设计相应引物,扩增miRNA的目的片段,进行测序、分型,对SNP位点与肠道内容物肠炎沙门氏菌含量进行关联分析。研究结果显示:1)不同的microRNA对照组和实验组差异表达的时间点不同,gga-mir-1662和gga-mir-215在感染后的第7天达到差异表达的高峰,试验组的表达量分别是对照组的1.6倍和1.66倍。gga-mir-1416则在21日龄表现出明显的上调表达,实验组是对照组的1.5倍。2)microRNA及其潜在靶基因不是在每个时间点能表现出明显的靶向关系,可能与机体的复杂调控有关系。感染后的第7天,gga-mir-1662明显上调,TLR1LA明显下调,靶向关系明显;gga-mir-1416与BCL10在感染后的第21天和第28天差异显著,但不能表现出明显的靶向关系。3)不同的microRNA在体内的变化趋势不同,对照组和实验组表现出相近的表达趋势。相对于各个不同时间点的表达,总体而言,感染后的第1天、第3天、第7天、第14天属于高表达时间点,而第21天、第28天、第35天属于低表达时间点。4)对个体分型之后与肠炎沙门氏菌含量相关联,发现了4个与肠炎沙门氏菌感染相关的SNP位点突变,分别为rs18823870、rs312707374、rs316396584、rs314185118。rs18823870位于gga-mir-215上游,rs312707374、rs316396584、rs314185118位于gga-mir-1416上游,进一步说明gga-mir-215和gga-mie-1416与肠炎沙门氏菌感染显著相关,可以作为抗肠炎沙门氏菌感染的分子标记。本研究鉴定出4个与肠炎沙门氏菌菌感染相关的位于miRNA上游的SNP位点,gga-mir-215和gga-mie-1416与肠炎沙门氏菌感染显著相关,可以作为抗肠炎沙门氏菌感染的分子标记。
[Abstract]:MicroRNAs (miRNAs) range from 20 to 25 nucleotides and exist in eukaryotes. It is a kind of endogenous non-coding RNA.miRNAs with regulatory function, which recognizes target mRNAs by base complementary pairing, and guides the translation of target mRNA or repressor target mRNA by silencing complex according to the degree of complementarity. Related studies have shown that gga-mir-1662 and TLR1LAGGa-mir-1416 may have a targeting relationship with BCL10, and that gga-mir-1662 and TLR1LAgga-mir-1416 may be a variation of genomic DNA base, and the frequency of occurrence in the genome is higher than that of TLR1LAGgga-mir-1416 and BCL10, and the relationship between gga-mir-1662 and TLR1LAgga-mir-1416 and BCL10 may be related to the infection of Salmonella enteritidis. Meaningful variation will affect the function of the components of genomic DNA, the level of mRNA produced by transcription, the expression of protein, and then affect the function of gene, leading to the change of biological traits and even the pathogenesis of the disease. Therefore, SNP is widely used in population genetics (such as the origin, evolution and migration of organisms) and disease related genes, which play an important role in pharmacogenomics, diagnostics and biomedical research. In this study, we studied the mechanism of miRNA and target gene expression and the variation of miRNA in salmonella enteritidis infection. Two days old Jining 100 day chickens infected with Salmonella enteritidis were collected on the 1st, 3rd, 7th, 14th, 21st, 28th and 35th day after infection. The cecum contents and blood of 200 Jining 100 day chickens were collected on the 7th day after infection to detect the content of Salmonella enteritidis and to extract the genomic DNA, and to design the corresponding primer, which were used to detect the content of Salmonella enteritidis and to extract the genomic DNA. The aim fragment of miRNA was amplified, sequenced and typed, and the correlation between SNP site and intestinal content of Salmonella enteritis was analyzed. The results showed that the different time points of different microRNA expression in control group and experimental group reached the peak of differential expression on the 7th day after infection. The expression levels of microRNA and its potential target genes in the experimental group were 1.6 times and 1.66 times as much as those in the control group, respectively, and were significantly up-regulated at 21 days of age, while those in the experimental group were 1.5 times higher than those in the control group. It may be related to the complex regulation of the body. On the 7th day after infection, TLR1LA was up-regulated and down-regulated, and the targeting relationship was significantly different between BCL10 and BCL10 on the 21st and 28th days after infection, but there was no obvious targeting relationship between BCL10 and BCL10 on the 21st and 28th days after infection. The control group and the experimental group showed similar expression trend. Compared with the expression at different time points, on the whole, the first day, the third day, the seventh day, the 14th day after infection were high expression time points, and the 21st day, the 28th day, On the 35th day, four SNPs related to salmonella enteritis were found, which were rs18823870rs312707374rs316396584rs316396584rs1838707374rs1838707374rs3127374rs312396584rs312707374rs312396584rs312707374rs316396584rs31485118 in the upper reaches of gga-mir-215. The results further indicated that gga-mir-215 and gga-mie-1416 were significantly correlated with Salmonella enteritis infection and could be used as molecular markers against Salmonella enteritidis infection. In this study, four SNPs located upstream of gga-mie-1416 and SNPs related to the infection of Salmonella enteritidis were identified, which could be used as molecular markers against Salmonella enteritidis infection.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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