本文選題：柔嫩艾美爾球蟲 + IL-6基因　； 參考：《揚州大學》2017年碩士論文

【摘要】：本研究以京海黃雞為實驗材料,采用實時熒光定量法PCR法檢測IL-6、IL-8(CXCLi2)、CCLi2基因在雞柔嫩艾美爾球蟲攻毒和非攻毒群體間的組織表達譜差異,研究3個基因在球蟲感染中所起作用。進一步通過DNA測序技術,檢測炎性細胞因子IL-6(白介素6)基因上游-2200bp至下游500bp區(qū)域中的單核苷酸多態(tài)(single nucleotide polymorphism,SNP),并分析所測SNPs及其單倍型與京海黃雞柔嫩艾美爾球蟲抗性指標之間的關系,為京海黃雞抗病育種提供分子遺傳標記。主要研究結果如下:1、IL-6基因在各組織中均有表達,感染組IL-6基因在盲腸、胸腺、腎臟中表達量極顯著高于對(P0.01),在法氏囊中表達量顯著高于對照組(P0.05);IL-8基因在各組織中均有表達,在腺胃、盲腸和胸腺中表達量極顯著的高于對照組(P0.01),在脾和小腸中表達量顯著高于對照組(P0.05);CCLi2在各組織中均有表達,感染組在脾和胸腺中表達量極顯著高于對照組(P0.01),在腺胃、盲腸、腎臟中顯著高于對照組(P0.05)。2、以IL-6為候選基因測序共發(fā)現(xiàn)了 28個突變位點,與GenBank上進行比較,新發(fā)現(xiàn)了 4個突變位點。其中位于5'調控區(qū)的單核苷酸突變有19個,除去GenBank有記錄的SNPs,新發(fā)現(xiàn)3處突變;位于內含子的SNPs有2個;位于外顯子的SNPs有2個,均為同義突變;位于3'區(qū)的SNPs有5個。3、本試驗對京海黃雞IL-6基因28個SNPs位點與抗性指標進行關聯(lián)分析,發(fā)現(xiàn)其中有13個位點與抗性指標有關聯(lián)。其中與丙二醛(Malondialdehyde,MDA)有顯著關聯(lián)的SNPs有3個,分別為1、6、25號變異位點;與白介素2有顯著關聯(lián)的SNPs有6個,分別是7、12、16、17、20、21號變異位點;與谷胱甘肽過氧化物酶(Glutathione peroxidase,GSH-Px)有關聯(lián)的SNPs有3個,分別是12、15、17位點;14號位點SNP與一氧化氮有顯著關聯(lián);與白介素17有顯著關聯(lián)的SNPs位點是17和19;與過氧化氫酶(Catalase,CAT)有顯著關聯(lián)的SNPs位點為19和28;19號位點與白介素16有顯著相關。4、對5'調控區(qū)與抗性指標有關的9個SNPs位點進行單倍型分析:H2H3單倍型組合IL-17的濃度顯著高于其他單倍型組合的濃度(P0.05);H2H5單倍型組合GSH-Px濃度顯著高于H1H1、H1H2和H1H5單倍型組合(P0.05);H1H2、H1H4、H2H5單倍型組合MDA濃度顯著的高于單倍型組合H1H1、H1H5、H2H3(P0.05);單倍型組合H2H5IL-2濃度顯著的高于單倍型組合H1H1、H1H2和H1H5。對內含子和3'端與抗性指標有關的4個SNPs位點進行單倍型分析:H2'H3'單倍型組合的NO濃度顯著的低于H1'H3'單倍型組合(P0.05),CAT濃度顯著的低于H2'H2'單倍型組合(P0.05);H2'H2'單倍型組合超氧化物歧化酶(Superoxide Dismutase,SOD)濃度顯著高于H2'H3'、H3'H3'單倍型組合(P0.05),H1'H5'單倍型組合的SOD濃度顯著高于H3'H3'單倍型組合(P0.05);單倍型組合H1'H2'、H2'H3'IL-2的濃度顯著的高于H1'H1,單倍型組合(P0.05),IL-16的濃度顯著高于單倍型組合H1'H3'、H1'H5'(P0.05)。
[Abstract]:In this study, the tissue expression profiles of IL-6, IL-8, CXCLi2 and CCLi2 genes were detected by real-time fluorescence quantitative PCR method in Jinghai Yellow Chicken, and the effects of three genes on the infection of coccidia were studied. Further through DNA sequencing, The single nucleotide polymorphisms (SNPs) in the upstream to downstream 500bp region of the inflammatory cytokine IL-6 (IL-6) gene were detected. The relationship between the SNPs and its haplotypes and the resistance index of Eimeria tenella was analyzed. To provide molecular genetic markers for resistance breeding of Jinghai Yellow Chicken. The main results were as follows: the expression of IL-6 gene was significantly higher in the cecum, thymus and kidney than that in the control group, and the expression of IL-8 in the bursa of Fabricius was significantly higher than that in the control group. The expression of CCLi2 in the glandular stomach, caecum and thymus was significantly higher than that in the control group (P 0.01), and in the spleen and small intestine was significantly higher than that in the control group. The expression of CCLi2 in the spleen and thymus of the infected group was significantly higher than that in the control group (P 0.01), and in the glandular stomach, the expression of CCLi2 in the spleen and thymus was significantly higher than that in the control group. In the cecum, kidney was significantly higher than that in the control group (P0.05. 2). A total of 28 mutation sites were identified using IL-6 as a candidate gene. Compared with GenBank, 4 new mutation sites were found. There were 19 single nucleotide mutations in the 5 'regulatory region, 3 new mutations except for those recorded in GenBank, 2 in intron SNPs, 2 in exon SNPs, and 2 in exon, all of which were synonymous mutations. There are 5 SNPs in 3 'region. In this experiment, 28 SNPs loci of IL-6 gene and resistance index of Jinghai Yellow chicken were analyzed, and 13 of them were found to be related to resistance index. Among them, there were three SNPs with significant association with malondialdehyde malondialdehyde (malondialdehyde), six SNPs with significant association with interleukin 2, and three SNPs with glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase glutathione peroxidase GSH-Px. There was a significant correlation between SNP and nitric oxide at site 14. The number of SNPs sites significantly associated with interleukin 17 was 17 and 19; the number of SNPs sites with significant correlation with catalase catalase was 19 and 281.There was a significant correlation between IL-16 and IL-16 at locus 19, and 9 SNPs loci associated with resistance index for 5 'regulatory region. Haplotype analysis: the concentration of IL-17 in haplotype combinations was significantly higher than that in other haplotype combinations P0.05H2H5 haplotype GSH-Px was significantly higher than that of H1H1H1H2 and H1H5 haplotype combinations P0.05H2H2H2H5 haplotype combinations were significantly higher than that of haplotype combinations. The concentration of H2H5IL-2 in haplotype combination was significantly higher than that in haplotype combination H _ 1 H _ 1 and H _ 1 H _ 2 and H _ 1 H _ 5. The H2H5IL-2 concentration of haplotype combination was significantly higher than that of haplotype combination H _ 1 H _ 1 and H _ 1 H _ 5. Haplotype analysis of four SNPs loci related to resistance index in intron and 3 '-terminal haplotype: the concentration of no was significantly lower than that of H1 / H3' haplotype combination (P0.05) and the concentration of cat was significantly lower than that of H2H2'haplotype combination P0.05H2H2'haplotype combination P0.05H2H2'haplotype combination (P0.05H2H2'haplotype combination), which was significantly lower than that of H2H3'haplotype combination (P0.05H2' haplotype combination). The SOD concentration of H2H2H2H3H3H3'haplotype combination was significantly higher than that of H3H3H3'haplotype combination P0.05'H3H3'haplotype (P0.05N), and the concentration of H2H2H2H3H3H3H3H _ (3) H2H3H3H3H _ (3) (H2H2H3H3H _ (3)) was significantly higher than that of the haplotype combination (H0.05H _ (0. 05) IL-16), and the concentration of IL-16 was significantly higher than that of the haplotype combination (H0. 05).
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.31

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