AD相关基因Soat1、Ptgs2的e-QTL定位和遗传调控网络分析

发布时间：2018-03-04 10:09

  本文选题：甾醇氧-乙酰转移酶　切入点：前列腺素内过氧化物合酶2　出处：《南通大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的Soat1基因编码的乙酰辅酶A：胆固醇乙酰转移酶在维持细胞内胆固醇的动态平衡方面具有重要作用,直接或间接地影响了APP的分泌过程及Aβ的产生；Ptgs2基因编码的COX能将花生四烯酸代谢成各种前列腺素类,其中前列腺素E参与了胆固醇的合成和运输,Ptgs2过度表达可增强Aβ的作用强度,加强兴奋毒性引起的神经退行性病变。由此可推测,Ptgs2基因与Soat1基因可能在细胞内胆固醇的运输和转化上存在一定的联系,进而共同参与阿尔茨海默病(Alzheimer's disease, AD)的发病。为研究Soat1基因和Ptsg2基因在AD发病过程中协同作用的分子机制,本实验应用基因芯片技术、基因表达数量性状基因座定位(expression Qutative Trati Loci, eQTL)的方法和遗传相关度分析,借助BXD重组近交系(Recombinant Inbred, RI)小鼠,构建Soat1和Ptgs2基因的表达调控网络,从全基因组水平上研究这两个基因参与AD发病的分子机制。 方法本实验采用BXDRI小鼠,运用eQTL定位的方法对Soat1基因(Probe set1417697_at)及Ptgs2基因(Probe set1417263_at)的表达变异及表达调控进行研究,研究两基因在BXDRI小鼠及亲本(C57BL/6J和DBA/2)海马组织中的表达差异及差异的变化趋势。采用区间作图在全基因组水平分别定位控制两基因表达变化的基因座(e-QTL)。通过检索数据库获取B6和D2两品系间的单核苷酸多态性(Single Nucleotide Polymorphisms, SNP)资料,并分析SNP对eQTL定位的影响。利用ClusterMap在线分析工具检索BXDRI小鼠海马组织基因组中分别受Soat1和Ptgs2基因反式调节的基因,即寻找其下游候选基因。通过皮尔森相关系数法筛选出具有高度遗传相关性的基因,并构建基因表达遗传调控网络。 结果RT-PCR结果显示Soat1基因的表达水平在B6和D2两亲本的海马组织中存在显著差异,在B6中的表达量明显高于D2,在BXDRI小鼠海马组织中的平均表达量为7.959(7),表达量在各品系小鼠中呈线性变化趋势,该结果显示Soat1基因具备QTL定位的条件。通过全基因组区间作图法定位Soat1基因的上游调控位点,发现其自身所在的基因组位置上存在一个连锁值较高的eQTL, LRS值高达89.2,且相对应的探针靶序列上的SNP对eQTL检测无影响,初步判断该基因为顺式调控基因候选基因。运用cluster map在线分析工具检索BXD重组近交系小鼠海马组织基因组中受Soat1反式调节的基因,Soat1基因被定位到反式eQTL富集区,21个基因的反式eQTL被定位到此处,排除连锁不平衡对下游基因定位的影响,结果显示其中的7个基因与Soat1呈显著性偏相关,由此推测这7个基因为Soat1基因的下游候选调控基因。通过皮尔森相关系数法分析Soat1基因与BXD RI小鼠海马组织中其他基因的遗传相关度,获得遗传相关度较高的(0.43)前18个基因,Ptgs2包含在其中,遗传相关度高达0.61。 Ptgs2基因在BXD重组近交系小鼠海马组织中平均表达量高达8.32,在亲本B6小鼠海马组织中表达为8.54,D2中表达量较低为7.86,两亲本间表达差异达1.6倍,差异具有统计学意义,且在BXD重组近交系各个品系间的表达变化呈线性趋势,符合数量性状的特征。基因表达数量性状基因座定位分析发现控制该基因的eQTL为顺式eQTL,即调控该基因表达变化的为该基因自身。该区域同时还是反式eQTL的富集区,偏相关系数分析发现其中有8个基因为Ptgs2基因的下游调控候选基因。皮尔森相关系数分析发现BXD重组近交系小鼠基因组中有151个基因与Ptgs2高度相关,其中前13个基因与Ptgs2的遗传相关度到达0.59以上,Soat1基因包含在其中。 在构建的Ptgs2与Soat1基因遗传网络中,有10个基因同时与Soat1基因和Ptgs2基因呈现高度相关性。其中,Dnm3、 Glul两个基因均参与了AD的发病,Dnm3做为Dnm家族成员之一,还参与了胆固醇的运输与代谢。由此推测,Soat1基因可能通过Dnm及Glul基因与Ptgs2基因联系起来共同参与了AD的发病,进一步说明Soat1基因和Ptgs2协同参与了AD的发病。 结论本实验可初步判断Soat1、Ptgs2基因为顺式调控基因,Rab2,Taz等7个基因为Soat1基因的下游候选调控基因,Vcam1、Mlstd1等8个基因为Ptgs2基因的下游候选调控基因。无论在Soat1基因遗传相关基因网络中还是在Ptgs2基因遗传相关基因网络中,Ptgs2与Soat1基因都呈现高度相关性。在构建的Ptgs2与Soat1基因网络中,Dnm和Glu1基因同时与Soat1和Ptgs2呈现高度的相关性,并参与了APP的加工和Aβ的产生,由此推测,Soat1基因可能通过Dnm及Glu1基因与Ptgs2基因联系起来共同参与了AD的发病,由此进一步证明Ptgs2与Soat1基因协同参与了AD的发病过程。
[Abstract]:Objective: the Soat1 gene encoding acetyl coenzyme A: cholesterol acyltransferase plays an important role in maintaining the dynamic balance of intracellular cholesterol, directly or indirectly affect the secretion of APP and A beta; Ptgs2 gene encoding COX can be four arachidonic acid metabolism into various prostanoids, including prostaglandin E in synthesis and transport of cholesterol, overexpression of Ptgs2 can enhance the strength of A beta, neurodegeneration caused by excitotoxicity. Strengthen it can be concluded that Ptgs2 gene and Soat1 gene may be linked cholesterol transport and transformation in cells, and involved in Alzheimer's disease (Alzheimer's, disease, AD) incidence for study of the molecular mechanism of Soat1 gene and Ptsg2 gene synergistically in the pathogenesis of AD, the application of gene chip technology, gene expression quantity of gene. (expression Qutative Trati Loci base location, eQTL) correlation analysis method and genetic, using BXD recombinant inbred lines (Recombinant, Inbred, RI) mice, construct the expression regulation networks of Soat1 and Ptgs2 genes, the molecular mechanism of these two genes from the whole genome level in the pathogenesis of AD.
In this experiment we use BXDRI mice, using the method of eQTL localization of Soat1 gene (Probe set1417697_at) and Ptgs2 (Probe set1417263_at) gene expression variation and expression regulation of two genes in BXDRI mice and their parents (C57BL/6J and DBA/2) expression differences and changes in hippocampus. The interval mapping in the whole genome level were two loci positioning control gene expression changes (e-QTL). By searching the database to obtain the polymorphism of B6 and D2 between the two lines (the Single Nucleotide Polymorphisms, SNP), and analyze the effect of SNP on the eQTL location. Soat1 and Ptgs2 analysis tools to retrieve genes transactivated by gene respectively. BXDRI in the hippocampus in the mouse genome using ClusterMap online, searching for its downstream gene. Through the Pearson correlation coefficient was screened with high genetic correlation Sex gene and gene expression genetic regulation network.
Results RT-PCR results showed that the expression level of Soat1 gene had significant differences in B6 and D2 of two parents in the hippocampus, the expression in B6 was significantly higher than that of D2, the average expression in the hippocampus of BXDRI mice was 7.959 (7), the expression of linear change trend in different strains of mice, the results display with QTL localization of Soat1 gene. The condition of the upstream control sites of whole genome interval mapping method to locate the Soat1 gene, it is found that there is a higher eQTL of its own value chain the genomic position, LRS value is as high as 89.2, and the corresponding probe target sequence of SNP had no effect on the detection of eQTL, preliminary judgment this gene is a candidate gene CIS gene regulation. Using cluster map online analysis tools to retrieve BXD recombinant inbred mice hippocampus genome by Soat1 transregulated genes, the Soat1 gene was mapped to the trans eQTL enrichment zone, 21 trans eQTL gene was mapped to the exclusion of linkage disequilibrium effects on downstream gene localization, the results showed that there was a significant correlation of 7 genes and Soat1, which indicated that the 7 genes downstream of Soat1 gene as candidate gene. Through Pearson correlation coefficient method for analysis of genetic correlation of other Soat1 gene BXD and RI in hippocampus of mice in the inheritance of acquired high correlation (0.43) before the 18 genes, Ptgs2 included, genetic correlation as high as 0.61.
Inbred Ptgs2 gene in recombinant BXD strain mice in the hippocampus of the average level of up to 8.32, in the hippocampus of B6 mice in parents was 8.54. The expression of D2 gene expression is low 7.86, two differential expression between the parents was 1.6 times, the difference was statistically significant, and the BXD expression of recombinant inbred lines of various products between a linear trend, in line with the characteristics of quantitative traits. The gene expression of QTL mapping analysis showed that the control of the gene eQTL for CIS eQTL, which regulate the gene expression changes of the gene itself. The region is also the enrichment of trans eQTL, partial correlation analysis shows that there are 8 as a downstream gene candidate gene Ptgs2. Pearson correlation analysis showed that BXD recombinant inbred strain mouse genome with 151 genes highly correlated with Ptgs2, which is the first 13 Ptgs2 gene and genetic correlation to reach 0.59 On the other hand, the Soat1 gene is included in it.
In the Ptgs2 and Soat1 gene genetic network construction, there are 10 genes and Soat1 gene and Ptgs2 gene were highly correlated. Among them, Dnm3 and Glul two genes involved in the pathogenesis of AD, Dnm3 as a member of the Dnm family, is involved in the transport and metabolism of cholesterol. By this hypothesis, Soat1 gene may be linked by Dnm and Glul genes and Ptgs2 genes involved in the pathogenesis of AD, suggested that Soat1 gene and Ptgs2 co participate in the pathogenesis of AD.
Conclusion this experiment can determine the initial Soat1, Ptgs2 gene cis regulatory genes, Rab2, Taz and other 7 genes downstream of Soat1 gene candidate genes, Vcam1, Mlstd1 and other 8 genes as candidate gene Ptgs2 downstream regulated genes. Both genes involved in Soat1 gene genetic network in network or in the Ptgs2 gene genetic correlation gene, Ptgs2 gene and Soat1 gene are highly correlated. In Ptgs2 and Soat1 network construction in Dnm, and the Glu1 gene with Soat1 and Ptgs2 showed a high degree of correlation, and participated in the processing of the APP and A beta, it is speculated that Soat1 gene may be linked by Dnm and Glu1 gene and Ptgs2 gene participate in the pathogenesis of AD, which further proved that Ptgs2 and Soat1 genes involved in the pathogenesis of AD.

【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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