本文選題：綿羊 + MHC-DQB2��； 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：目的:通過對布魯氏菌感染陽性組和陰性組哈薩克羊MHC-DQB2 exon2、exon3 SNP進(jìn)行研究,并運(yùn)用SHEsis軟件對SNPs進(jìn)行單倍型分析,以期得到兩組個體間有顯著差異的SNPs位點(diǎn)以及單倍型組合,以此作為與布魯氏菌病易感性相關(guān)的遺傳標(biāo)記,為今后開展布魯氏菌分子標(biāo)記輔助選擇研究提供一定的參考依據(jù),為加快選育具有布魯氏菌病抗性的綿羊新品種打下一定基礎(chǔ)。方法:1.用虎紅平板凝集實(shí)驗(yàn)對100只哈薩克羊進(jìn)行血清布魯氏菌檢測,對布病感染陽性組和感染陰性組分別使用PCR-SSCP技術(shù)檢測MHC-DQB2 exon2、exon3的SNPs,對具有不同基因型的個體進(jìn)行克隆測序,然后運(yùn)用序列比對軟件尋找exon2、exon3 SNPs位點(diǎn),通過統(tǒng)計(jì)分析,比較在布病感染陽性組和陰性組之間存在顯著差異的位點(diǎn),最終得出與布魯氏菌病易感性相關(guān)的SNPs位點(diǎn)。2通過對檢測到的所有的SNPs位點(diǎn)進(jìn)行最小等位基因頻率及Hardy-Weinberg平衡的吻合度檢驗(yàn),選出符合條件的SNPs位點(diǎn),運(yùn)用SHEsis在線軟件對符合要求的SNPs進(jìn)行單倍型分析,得到與布魯氏菌病易感性相關(guān)的單倍型組合。結(jié)果:1.利用虎紅平板凝集實(shí)驗(yàn)對100只哈薩克羊血清樣本進(jìn)行了布魯氏菌感染檢測,其中檢出陽性血清樣本16只,陰性血清樣本84只,布病感染陽性率為16%。2.使用PCR-SSCP實(shí)驗(yàn)結(jié)果與測序結(jié)果結(jié)合分析,在哈薩克羊MHC-DQB2基因exon2 270bp中檢測出44個SNPs位點(diǎn);在exon3 282 bp中檢測得到22個SNPs位點(diǎn)。3.對哈薩克羊MHC-DQB2基因進(jìn)行SAP分析,得到exon2共編碼90個氨基酸,其中有34個是SAPs位點(diǎn);在34個SAPs位點(diǎn)中,有3個為同義突變位點(diǎn),其余31個均為錯義突變位點(diǎn)。得到exon3共編碼93個氨基酸,其中有20個為SAPs位點(diǎn)。在20個SAPs位點(diǎn)中,有6個為錯義突變位點(diǎn),其余均為同義突變位點(diǎn)。4.通過對MHC-DQB2基因兩個外顯子SNP與布魯氏菌病的關(guān)聯(lián)分析,發(fā)現(xiàn)exon2只有9C/G位點(diǎn)基因頻率在兩組當(dāng)中呈現(xiàn)出顯著差異;通過對exon2每個SNP位點(diǎn)的基因型頻率進(jìn)行分析,發(fā)現(xiàn)9G/C、117G/T、131C/G、157C/A、180G/A5個位點(diǎn)在陽性組和陰性組之間存在顯著差異(P0.05)。exon3 155T/C的等位基因在兩組樣本中的分布存在顯著差異(P0.05);對exon3每個多態(tài)位點(diǎn)的基因型進(jìn)行分析,發(fā)現(xiàn)各位點(diǎn)基因型在兩組樣本中的分布無顯著差異。5.通過對哈薩克羊MHC-DQB2 exon2符合條件的18 SNPs個位點(diǎn)進(jìn)行連鎖不平衡分析,得到exon2存在13個連鎖域,分別命名為Block1-Block13,其中有9個Block(Block2、Block3、Block4、Block6、Block7、Block8、Block10、Block12、Block13)個屬于強(qiáng)連鎖不平衡。對MHC-DQB2exon3進(jìn)行連鎖不平衡分析,得到exon3存在8個連鎖域,分別命名為Block1-Block8,其中有4個Block(Block1、Block2、Block5、Block8)個屬于強(qiáng)連鎖不平衡。6.經(jīng)SHEsis軟件單倍型分析發(fā)現(xiàn),由哈薩克羊MHC-DQB2 exon2 33個SNP位點(diǎn)構(gòu)建得到了17種單倍型,其中Hap12、Hap13、Hap14、Hap15、Hap16、Hap17陽性組頻率遠(yuǎn)高于陰性組頻率,達(dá)到了顯著性差異水平。由exon3的18個SNP位點(diǎn)構(gòu)建得到21種單倍型,Hap4、Hap6的陽性組頻率遠(yuǎn)低于陰性組頻率達(dá)到了顯著性差異水平。結(jié)論:1.哈薩克羊MHC-DQB2基因exon2、exon3均存在豐富的SNPs位點(diǎn)和SAPs位點(diǎn),且exon2的SNPs位點(diǎn)和SAPs位點(diǎn)以及氨基酸錯義突變位點(diǎn)都較之exon3更為豐富。2.初步推測哈薩克羊MHC-DQB2基因exon2 9C/G位點(diǎn)與布魯氏菌易感性相關(guān),9G/C、117G/T、131C/G、157C/A、180G/A 5個位點(diǎn)某種基因型與布病的易感性相關(guān)。初步分析認(rèn)為MHC-DQB2 exon3的155T/C與綿羊布魯氏菌病易感性相關(guān)。3.哈薩克羊MHC-DQB2基因exon2的9個強(qiáng)連鎖不平衡Block以及exon3的4個強(qiáng)連鎖不平衡Block內(nèi)的基因位點(diǎn)可能是連鎖遺傳的。4.初步推測,對哈薩克羊MHC-DQB2 exon2而言,具有Hap12、Hap13、Hap14、Hap15、Hap16、Hap17這6種單倍型組合的個體對布病更易感;對哈薩克羊MHC-DQB2 exon3而言,具有Hap4、Hap6的個體對布病有更高的抗性,具有Hap9的個體對布病更易感。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.26

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