绵羊MHC-DQB2单倍型的构建及其与布鲁氏菌病易感性相关分析

发布时间：2018-05-29 03:51

  本文选题：绵羊 + MHC-DQB2　； 参考：《石河子大学》2017年硕士论文

【摘要】：目的:通过对布鲁氏菌感染阳性组和阴性组哈萨克羊MHC-DQB2 exon2、exon3 SNP进行研究,并运用SHEsis软件对SNPs进行单倍型分析,以期得到两组个体间有显著差异的SNPs位点以及单倍型组合,以此作为与布鲁氏菌病易感性相关的遗传标记,为今后开展布鲁氏菌分子标记辅助选择研究提供一定的参考依据,为加快选育具有布鲁氏菌病抗性的绵羊新品种打下一定基础。方法:1.用虎红平板凝集实验对100只哈萨克羊进行血清布鲁氏菌检测,对布病感染阳性组和感染阴性组分别使用PCR-SSCP技术检测MHC-DQB2 exon2、exon3的SNPs,对具有不同基因型的个体进行克隆测序,然后运用序列比对软件寻找exon2、exon3 SNPs位点,通过统计分析,比较在布病感染阳性组和阴性组之间存在显著差异的位点,最终得出与布鲁氏菌病易感性相关的SNPs位点。2通过对检测到的所有的SNPs位点进行最小等位基因频率及Hardy-Weinberg平衡的吻合度检验,选出符合条件的SNPs位点,运用SHEsis在线软件对符合要求的SNPs进行单倍型分析,得到与布鲁氏菌病易感性相关的单倍型组合。结果:1.利用虎红平板凝集实验对100只哈萨克羊血清样本进行了布鲁氏菌感染检测,其中检出阳性血清样本16只,阴性血清样本84只,布病感染阳性率为16%。2.使用PCR-SSCP实验结果与测序结果结合分析,在哈萨克羊MHC-DQB2基因exon2 270bp中检测出44个SNPs位点;在exon3 282 bp中检测得到22个SNPs位点。3.对哈萨克羊MHC-DQB2基因进行SAP分析,得到exon2共编码90个氨基酸,其中有34个是SAPs位点;在34个SAPs位点中,有3个为同义突变位点,其余31个均为错义突变位点。得到exon3共编码93个氨基酸,其中有20个为SAPs位点。在20个SAPs位点中,有6个为错义突变位点,其余均为同义突变位点。4.通过对MHC-DQB2基因两个外显子SNP与布鲁氏菌病的关联分析,发现exon2只有9C/G位点基因频率在两组当中呈现出显著差异;通过对exon2每个SNP位点的基因型频率进行分析,发现9G/C、117G/T、131C/G、157C/A、180G/A5个位点在阳性组和阴性组之间存在显著差异(P0.05)。exon3 155T/C的等位基因在两组样本中的分布存在显著差异(P0.05);对exon3每个多态位点的基因型进行分析,发现各位点基因型在两组样本中的分布无显著差异。5.通过对哈萨克羊MHC-DQB2 exon2符合条件的18 SNPs个位点进行连锁不平衡分析,得到exon2存在13个连锁域,分别命名为Block1-Block13,其中有9个Block(Block2、Block3、Block4、Block6、Block7、Block8、Block10、Block12、Block13)个属于强连锁不平衡。对MHC-DQB2exon3进行连锁不平衡分析,得到exon3存在8个连锁域,分别命名为Block1-Block8,其中有4个Block(Block1、Block2、Block5、Block8)个属于强连锁不平衡。6.经SHEsis软件单倍型分析发现,由哈萨克羊MHC-DQB2 exon2 33个SNP位点构建得到了17种单倍型,其中Hap12、Hap13、Hap14、Hap15、Hap16、Hap17阳性组频率远高于阴性组频率,达到了显著性差异水平。由exon3的18个SNP位点构建得到21种单倍型,Hap4、Hap6的阳性组频率远低于阴性组频率达到了显著性差异水平。结论:1.哈萨克羊MHC-DQB2基因exon2、exon3均存在丰富的SNPs位点和SAPs位点,且exon2的SNPs位点和SAPs位点以及氨基酸错义突变位点都较之exon3更为丰富。2.初步推测哈萨克羊MHC-DQB2基因exon2 9C/G位点与布鲁氏菌易感性相关,9G/C、117G/T、131C/G、157C/A、180G/A 5个位点某种基因型与布病的易感性相关。初步分析认为MHC-DQB2 exon3的155T/C与绵羊布鲁氏菌病易感性相关。3.哈萨克羊MHC-DQB2基因exon2的9个强连锁不平衡Block以及exon3的4个强连锁不平衡Block内的基因位点可能是连锁遗传的。4.初步推测,对哈萨克羊MHC-DQB2 exon2而言,具有Hap12、Hap13、Hap14、Hap15、Hap16、Hap17这6种单倍型组合的个体对布病更易感;对哈萨克羊MHC-DQB2 exon3而言,具有Hap4、Hap6的个体对布病有更高的抗性,具有Hap9的个体对布病更易感。
[Abstract]:Objective: To study the MHC-DQB2 Exon2 and exon3 SNP of the positive group of Brucella infection and the negative group Kazakh sheep, and to analyze the haplotype of SNPs by SHEsis software in order to get the SNPs locus and haplotype combination of two groups of individuals with significant differences as a genetic marker related to the susceptibility of brucellosis. After carrying out the molecular marker assisted selection of Brucella, a certain basis was provided to speed up the selection of new breed of sheep with brucellosis resistance. Method: 1. Kazakh sheep were tested by Tiger red flat agglutination test on 100 Kazakh sheep. PCR-SSCP technique was used to detect MHC-DQB2 Exon2 and SNPs of exon3, and the individuals with different genotypes were cloned and sequenced. Then sequence alignment software was used to find Exon2 and exon3 SNPs loci. By statistical analysis, the significant differences between the positive and negative groups of the infected and negative groups were compared. Finally, the susceptibility to brucellosis was found. The SNPs site.2 was tested by the test of the minimum allele frequency and the Hardy-Weinberg balance of all the detected SNPs loci. The SNPs loci were selected, and the SHEsis online software was used to analyze the conforming SNPs, and the haplotype association with the susceptibility to brucellosis was obtained. Results: 1. The detection of Brucella infection in 100 Kazak sheep serum samples was carried out with 100 Kazak sheep serum samples, of which 16 positive serum samples and 84 negative serum samples were detected. The positive rate of brucellosis was 16%.2. using PCR-SSCP test results and sequencing results, and 44 S were detected in the MHC-DQB2 gene Exon2 270bp of harazak sheep. NPs loci; 22 SNPs loci.3. were detected in exon3 282 BP for SAP analysis of the Kazak sheep MHC-DQB2 gene, and Exon2 co coded 90 amino acids, of which 34 were SAPs loci; 3 of the 34 SAPs loci were synonymous mutation sites and the other 31 were missense mutation sites. Exon3 co coded 93 amino acids, of which 20 were 20. SAPs loci. In 20 SAPs loci, 6 are missense sites, and the rest are synonymous mutation sites.4. through the correlation analysis of two exon SNP of MHC-DQB2 gene and brucellosis. It is found that Exon2 only 9C/G loci gene frequency is significantly different in the two group; through the genotype frequency of each SNP loci of Exon2 The analysis showed that there was significant difference between the positive and negative groups of 9G/C, 117G/T, 131C/G, 157C/A and 180G/A5 (P0.05) the distribution of.Exon3 155T/C alleles in the two groups was significantly different (P0.05); the genotype of each polymorphic loci of exon3 was analyzed, and the distribution of the genotype in the two groups was found. There is no significant difference in the linkage disequilibrium analysis of the 18 SNPs loci of the Kazak sheep MHC-DQB2 Exon2. It is found that there are 13 chain domains of Exon2, named Block1-Block13, of which 9 Block (Block2, Block3, Block4, Block6, Block7, Block7, Block4, etc.) belong to the strong linkage disequilibrium. Xon3 has 8 chain domains, named Block1-Block8, and 4 Block (Block1, Block2, Block5, Block8) belong to the strong linkage disequilibrium.6. by SHEsis software haplotype analysis, and 17 haplotypes are obtained from 33 loci of the MHC-DQB2 Exon2 of Kazakh sheep. The frequency of AP14, Hap15, Hap16, Hap17 positive group was much higher than that of negative group, reaching a significant difference level. 21 haplotypes were obtained from 18 SNP loci of exon3. The frequency of Hap4, Hap6 positive group was much lower than that of negative group. Conclusion: 1. Hazak sheep MHC-DQB2 gene Exon2, exon3 all have rich SNPs positions. The point and SAPs loci, and the SNPs and SAPs loci of the Exon2 and the missense site of the amino acid are more abundant than exon3,.2. preliminarily conjectured that the 9C/G locus of the Kazak sheep MHC-DQB2 gene is associated with the susceptibility to Brucella, 9G/C, 117G/T, 131C/G, and the susceptibility of the 5 loci to the susceptibility of the disease. The 9 strong linkage unbalanced Block in.3. Kazak sheep MHC-DQB2 gene Exon2 for the susceptibility of MHC-DQB2 exon3 to the susceptibility to the sheep brucellosis, and the gene loci in the 4 strong linkage unbalanced Block of exon3, may be a preliminary conjecture of the linkage hereditary.4.. The 6 haplotype combinations of Hap17 are more susceptible to the disease; for the MHC-DQB2 exon3 of Kazakh sheep, there are Hap4, Hap6 individuals have higher resistance to the disease, and the individuals with Hap9 are more susceptible to the disease.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.26

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