利用全基因組關(guān)聯(lián)分析鑒定雞腸炎沙門氏菌感染相關(guān)基因及其mRNA表達調(diào)控
發(fā)布時間：2018-11-22 18:54
【摘要】：腸炎沙門氏菌不僅是威脅家禽養(yǎng)殖業(yè)的一大危害性病原菌,而且是重要的食源性病原菌,容易造成食物中毒,引起人腸道疾病,危害著人類健康發(fā)展。畜禽產(chǎn)品、蛋類、肉制品是腸炎沙門氏菌重要的傳染源,而且腸炎沙門氏菌有著廣泛的傳播途徑,包括人類在內(nèi)的廣泛易感動物,是造成預防和控制腸炎沙門氏菌比較困難的重要原因。全基因組關(guān)聯(lián)分析(GWAS)是在基因組范圍內(nèi)篩選影響遺傳變異的高效能分析方法,具有統(tǒng)計效力高、精準度高等特點,并廣泛應用于動物重要經(jīng)濟性狀以及人類復雜疾病遺傳學的研究。本研究通過腸炎沙門氏菌感染200只濟寧百日雞,采用case-cntrol實驗設計,利用600K雞高密度芯片對盲腸內(nèi)沙門氏菌含量差異較大的濟寧百日雞進行全基因組關(guān)聯(lián)分析(GWAS),在全基因組范圍內(nèi)篩選與腸炎沙門氏菌感染相關(guān)的基因或分子標記,通過實時定量PCR,進一步分析每個相關(guān)基因不同基因型的表達量與腸炎沙門氏菌感染的相關(guān)性,探討腸炎沙門氏菌感染相關(guān)基因的表達調(diào)控。(1)通過GWAS分析,共檢測到3個SNPs與腸炎沙門氏菌感染顯著關(guān)聯(lián),分別位于3個不同的基因;其中rs313644723(C/T)位于1號染色上,基因Wnt7b(wingless-type MMTV integration site family)位于rs313644723(C/T)位點上游;rs80757564(G/T)位于5號染色體上,LRP5(low density lipoprotein receptor-related protein 5)基因第11段內(nèi)含子上;rs313281555(A/G)位于9號染色體上LPP(LIM domain containing preferred translocation partner in lipoma)第7段內(nèi)含子上。(2)差異SNPs位點不同基因型個體與盲腸沙門氏菌含菌量相關(guān)性分析結(jié)果:rs80757564(G/T)位點,GG基因型和TT、GT兩基因型相比差異顯著(P0.01),TT和GT兩基因型之間沒有顯著差異;rs313281555(A/G)位點基因型之間,GG基因型和AA、AG兩基因型之間有極顯著差異,AA和AG兩基因型之間沒有顯著差異;rs313644723(C/T)位點基因型之間,CC基因型和TT、CT基因型之間差異顯著(P0.05),TT和CT基因型之間沒有顯著差異,且該位點與Tilquin等人研究白來航雞感染沙門氏菌感染后泄殖腔內(nèi)沙門氏菌含量相關(guān)的QTL(CLOAC-ST)相一致。(3)熒光定量PCR結(jié)果表明:在盲腸和脾臟組織中,對照組和實驗組各候選基因不同基因型之間沒有顯著差異;而在不同基因型個體之間,在盲腸和脾臟組織中,LPP基因不同基因型之間沒有顯著差異;在脾臟組織中,基因Wnt7b,TT基因型顯著高于CC基因型(P0.05);LRP5基因中,位點G15802980T的TT基因型和TG基因型的表達量極顯著高于GG基因型(P0.01)。盲腸組織中Wnt7b各基因型之間具有顯著差異(P0.05),而LRP5各基因型之間沒有顯著差異,但是兩基因不同基因型之間具有相同的表達趨勢。綜上所述,通過GWAS篩選出的差異SNPs與腸炎沙門氏菌感染有重要作用,分別為:rs80757564(G/T);rs313281555(A/G)和rs313644723(C/T)。三個SNP所在基因LPP、Wnt7b與LRP5在雞腸炎沙門氏菌感染中發(fā)揮表達調(diào)控作用
[Abstract]:Salmonella enteritidis is not only a harmful pathogen threatening poultry industry, but also an important foodborne pathogen, which can easily cause food poisoning, cause human intestinal diseases and endanger the healthy development of human beings. Livestock and poultry products, eggs and meat products are an important source of infection of Salmonella enteritis, and Salmonella enteritis has a wide range of transmission routes, including human beings, a wide range of susceptible animals, It is an important reason that it is difficult to prevent and control Salmonella enteritis. Genome-wide association analysis (GWAS) is a highly efficient method for screening genetic variation in the genome, which has the characteristics of high statistical efficacy and high accuracy. And widely used in animal important economic traits and human complex disease genetics research. In this study, 200 chickens infected with Salmonella enteritidis were infected with Jining 100 day chickens. The (GWAS), was analyzed by using case-cntrol design and 600K chicken high density microarray for the whole genome association analysis of Chicken with large difference in the content of Salmonella in the Cecum. The genes or molecular markers associated with Salmonella enteritis infection were screened in the whole genome, and the correlation between the expression of different genotypes of each related gene and the infection of Salmonella enteritidis was further analyzed by real-time quantitative PCR,. To investigate the expression and regulation of Salmonella enteritidis infection related genes. (1) by GWAS analysis, a total of three SNPs were found to be significantly associated with Salmonella enteritidis infection, respectively located in three different genes; Among them, rs313644723 (C / T) is located on 1 staining, Wnt7b (wingless-type MMTV integration site family) is upstream of rs313644723 (C / T), rs80757564 (G / T) is on chromosome 5, and LRP5 (low density lipoprotein receptor-related protein 5 is on intron 11. Rs313281555 (A / G) is located on the intron of segment 7 of LPP (LIM domain containing preferred translocation partner in lipoma) on chromosome 9. (2) the relationship between different genotypes of SNPs loci and the quantity of Salmonella cecum: rs80757564 (G / T) loci. There was no significant difference between GG genotype and TT,GT genotype (P0.01), TT and GT genotype). There were significant differences between GG genotype and AA,AG genotype among rs313281555 (A / G) genotypes, but there was no significant difference between AA and AG genotypes. There were significant differences between rs313644723 (C / T) locus genotypes, CC genotypes and TT,CT genotypes (P0.05) between), TT and CT genotypes. This locus is consistent with QTL (CLOAC-ST), which is related to the content of Salmonella in cloacal cavity after infection with Salmonella by Tilquin et al. (3) fluorescence quantitative PCR results showed that: in cecum and spleen tissues, There was no significant difference between different genotypes of candidate genes in control group and experimental group. However, there was no significant difference between different genotypes of LPP gene in the caecum and spleen, while in spleen, the genotype of Wnt7b,TT was significantly higher than that of CC (P0.05). In LRP5 gene, the expression of TT genotype and TG genotype at G15802980T was significantly higher than that of GG genotype (P0.01). There was significant difference among the genotypes of Wnt7b in cecum (P0.05), but there was no significant difference among the genotypes of LRP5, but there was the same trend of expression between different genotypes of the two genes. In conclusion, the difference between SNPs and Salmonella enteritidis infection was found by GWAS, which were rs80757564 (G / T), rs313281555 (A / G) and rs313644723 (C / T), respectively. Expression and Regulation of three SNP genes LPP,Wnt7b and LRP5 in Chicken Salmonella enteritidis infection
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S858.31
本文編號：2350241
[Abstract]:Salmonella enteritidis is not only a harmful pathogen threatening poultry industry, but also an important foodborne pathogen, which can easily cause food poisoning, cause human intestinal diseases and endanger the healthy development of human beings. Livestock and poultry products, eggs and meat products are an important source of infection of Salmonella enteritis, and Salmonella enteritis has a wide range of transmission routes, including human beings, a wide range of susceptible animals, It is an important reason that it is difficult to prevent and control Salmonella enteritis. Genome-wide association analysis (GWAS) is a highly efficient method for screening genetic variation in the genome, which has the characteristics of high statistical efficacy and high accuracy. And widely used in animal important economic traits and human complex disease genetics research. In this study, 200 chickens infected with Salmonella enteritidis were infected with Jining 100 day chickens. The (GWAS), was analyzed by using case-cntrol design and 600K chicken high density microarray for the whole genome association analysis of Chicken with large difference in the content of Salmonella in the Cecum. The genes or molecular markers associated with Salmonella enteritis infection were screened in the whole genome, and the correlation between the expression of different genotypes of each related gene and the infection of Salmonella enteritidis was further analyzed by real-time quantitative PCR,. To investigate the expression and regulation of Salmonella enteritidis infection related genes. (1) by GWAS analysis, a total of three SNPs were found to be significantly associated with Salmonella enteritidis infection, respectively located in three different genes; Among them, rs313644723 (C / T) is located on 1 staining, Wnt7b (wingless-type MMTV integration site family) is upstream of rs313644723 (C / T), rs80757564 (G / T) is on chromosome 5, and LRP5 (low density lipoprotein receptor-related protein 5 is on intron 11. Rs313281555 (A / G) is located on the intron of segment 7 of LPP (LIM domain containing preferred translocation partner in lipoma) on chromosome 9. (2) the relationship between different genotypes of SNPs loci and the quantity of Salmonella cecum: rs80757564 (G / T) loci. There was no significant difference between GG genotype and TT,GT genotype (P0.01), TT and GT genotype). There were significant differences between GG genotype and AA,AG genotype among rs313281555 (A / G) genotypes, but there was no significant difference between AA and AG genotypes. There were significant differences between rs313644723 (C / T) locus genotypes, CC genotypes and TT,CT genotypes (P0.05) between), TT and CT genotypes. This locus is consistent with QTL (CLOAC-ST), which is related to the content of Salmonella in cloacal cavity after infection with Salmonella by Tilquin et al. (3) fluorescence quantitative PCR results showed that: in cecum and spleen tissues, There was no significant difference between different genotypes of candidate genes in control group and experimental group. However, there was no significant difference between different genotypes of LPP gene in the caecum and spleen, while in spleen, the genotype of Wnt7b,TT was significantly higher than that of CC (P0.05). In LRP5 gene, the expression of TT genotype and TG genotype at G15802980T was significantly higher than that of GG genotype (P0.01). There was significant difference among the genotypes of Wnt7b in cecum (P0.05), but there was no significant difference among the genotypes of LRP5, but there was the same trend of expression between different genotypes of the two genes. In conclusion, the difference between SNPs and Salmonella enteritidis infection was found by GWAS, which were rs80757564 (G / T), rs313281555 (A / G) and rs313644723 (C / T), respectively. Expression and Regulation of three SNP genes LPP,Wnt7b and LRP5 in Chicken Salmonella enteritidis infection
【學位授予單位】：山東農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S858.31
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