【作者】 申红；

【导师】 贾斌； 陈玉林；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 绵羊主要组织相容性复合体(Major histocompitibility complex,MHC)又称为绵羊淋巴细胞表面抗原(Ovine Lymphocyte Antigen,OLA),是由紧密连锁、高度多态的基因位点组成染色体上的一个遗传区域,其中MHC-Ⅱ类分子的DRB和DQB 2个基因所编码的MHC抗原在其免疫系统中发挥重要的作用,MHC与许多动物疾病的抗性、易感性、免疫应答以及生产性能都有密切关系。本试验对MHC-DRB1、DQB1基因在中国美利奴(新疆军垦型)羊(简称中国美利奴羊)和多浪羊群体中的分布进行检测,并且结合两绵羊品种的包虫病感染情况,探讨MHC-DRB1、DQB1基因PCR-RFLP多态性与包虫病遗传抗性间的相关性。为进一步研究MHC对包虫病的抗病免疫机制,以及抗病育种研究提供重要的依据,具有重要的学术理论价值和现实生产意义。1、本试验用ELISA试剂盒对塔城、伊犁和石河子部分地区的中国美利奴羊(868只)及喀什地区多浪羊(351只)包虫病感染情况进行了血清学诊断。结果,塔城、伊犁和石河子地区中国美利奴羊的平均感染率分别为22.33%、48.8%和6.9%,相互之间有显著差异(P﹤0.01)。相同饲养模式下,多浪羊和中国美利奴羊的包虫病感染率分别为26.84%和58.1%,差异极显著(P﹤0.01)。2、利用PCR-RFLP方法,对中国美利奴羊和多浪羊MHC-DRB1、DQB1基因第2外显子进行了遗传多态性检测,结果显示,中国美利奴羊和多浪羊的MHC-DRB1基因第2外显子在SacII、MvaI、SacI、Hin1I和HaeIII 5个酶切位点存在丰富的多态性。两绵羊品种均检测出了2、3、2、2和6种等位基因,但基因型分别为3、5、3、3、15和3、3、3、3、18种,MHC-DRB1第2外显子的229、225、208、210、178、173、159、87位碱基上存在多态性。而MHC-DQB1基因在MroxI、ScaI、SacII、NciI、TaqI、MvaI和HaeⅢ7个内切酶中,中国美利奴羊分别检测到2、2、4、2、3、6和3种等位基因和3、3、7、3、4、16和6种基因型,多浪羊在MroxⅠ、ScaⅠ、Sac II、TaqⅠ、MvaⅠ和HaeⅢ6个内切酶中检测到2、2、3、2、6、3种等位基因和3、3、5、2、14、5种基因型,MHC-DQB1第2外显子的266,246,231,217,213,198,192,174,161,140,96,34位碱基上存在多态性。中国美利奴羊和多浪羊MHC-DRB1、DQB1基因第2外显子在以上酶切位点均存在丰富多态性,其中,经克隆测序证实中国美利奴羊MHC-DQB1基因HaeⅢm、HaeⅢn、MvaⅠz和MvaⅠy是新等位基因。3、对中国美利奴羊和多浪羊包虫病阴性和阳性羊的MHC-DRB1和MHC-DQB1等位基因频率和基因型频率分别进行统计分析和差异性检验,结果显示,中国美利奴羊MHC-DRB1的MvaⅠb、HaeⅢe等位基因与包虫病抗性有相关(P﹤0.01),MvaⅠC(P﹤0.01)、HaeⅢd(P﹤0.05)与包虫病易感性相关。MvaⅠbb(P﹤0.01)、HaeIII ee和SacⅠab基因型(P﹤0.05)可能是包虫病的抗性基因型,而MvaⅠbc、HaeIII bd(P﹤0.01)、SacIIab和HaeIII df(P﹤0.05)可能是包虫病的易感基因型。MHC-DQB1基因的MvaⅠD(P﹤0.05)、SacIIb、TaqⅠA、HaeⅢN(P﹤0.01)为包虫病抗性相关等位基因,TaqⅠB、MvaⅠC、HaeⅢM(P﹤0.01)为易感性相关等位基因。MvaⅠDZ(P﹤0.05)、TaqⅠAA和HaeIII NN(P﹤0.01)为包虫病抗性相关基因型,而MvaⅠCZ(P﹤0.05)、TaqⅠAB和HaeIII MN(P﹤0.01)为包虫病易感性相关基因型。多浪羊MHC-DRB1的HaeⅢa(P﹤0.05)可能是包虫病的易感性等位基因,SacⅠab、HaeⅢcf、Hin1Ⅰaa(P﹤0.05)基因型对包虫病具有一定的抗性, HaeIII be和HaeIII ef(P﹤0.01)、SacⅠbb、HaeIII aa和Hin1Ⅰab(P﹤0.05)提示为包虫病的易感性基因型。MHC-DQB1的MvaⅠD(P﹤0.01)与包虫病抗性相关。MvaⅠDD和MvaⅠCD基因型(P﹤0.05)可能是包虫病的抗性基因型,而MvaⅠBZ(P﹤0.05)疑似为易感基因型。4、中国美利奴羊和多浪羊MHC基因座不同单倍型与包虫病抗性或易感性相关分析结果表明,中国美利奴羊MHC基因座单倍型DRB1-SacIAB、DRB1-MvaIbb、DQB1-TaqIAA、DQB1-HaeⅢNN与包虫病抗性密切关联(P﹤0.01),而单倍型DRB1-MvaIbc、DQB1-MvaIYY、DQB1-TaqIAB、DQB1-HaeⅢMN和单倍型DRB1-MvaIbb、DQB1-MvaICC、DQB1-TaqIAB、DQB1-HaeⅢMN与包虫病易感性相关(P﹤0.01)。多浪羊MHC基因座单倍型DRB1-SacIAB、DRB1-HaeⅢff、DQB1-MvaICD和单倍型DRB1-SacIAB、DRB1-HaeⅢff、DQB1-MvaIDD与包虫病抗性相关(P﹤0.05),然而,单倍型DRB1- Hin1Ⅰab、DRB1-HaeⅢbe和单倍型DRB1- Hin1Ⅰab、DRB1-HaeⅢef以及单倍型DRB1- Hin1Ⅰab、DRB1-HaeⅢaa分析认为与包虫病的易感性相关(P﹤0.01)。5、经对中国美利奴羊MHC基因单倍型DRB1-SacIAB、DRB1-MvaIbb、DQB1-TaqIAA、DQB1-HaeⅢNN个体人工感染攻虫试验,进一步验证了该单倍型为包虫病抗性相关基因单倍型。6、基因位点处于Hardy-Weinberg平衡对于群体遗传结构的稳定起着重要的作用。MHC-DRB1基因各酶切位点的Hardy-Weinberg平衡状态在两绵羊品种间不一致,中国美利奴羊在SacI和Hin1Ⅰ位点上达到Hardy-Weinberg平衡(P>0.05),多浪羊在MvaⅠ位点达到Hardy-Weinberg平衡(P>0.05),其余酶切位点均处于非平衡状态。而在MHC-DQB1基因各酶切位点,中国美利奴羊在TaqⅠ、MroxⅠ位点,多浪羊在TaqⅠ位点达到Hardy-Weinberg平衡(P>0.05),其余酶切位点都处于Hardy-Weinberg非平衡状态(P﹤0.01)。更多还原

【Abstract】 Major Histocompatibility Complex ( MHC ), also called Ovine Lymphocyte Antigen( OLA), is part of chromosomal region consisting of a group of closely linked and highly polymorphic loci. The antigens coded by DRB and DQB at MHC-Ⅱplay important roles in immune system, and have close relation with its functions. That the relationship between MHC and resistance/suseptibility to animal diseases/immune response/performance was closely linked. In this text, the polymorphisms of MHC-DRB1/DQB1 in Chinese merino and Duolang sheep with different intensity of infection hydatidosis were tested, and the relationship between MHC-DRB1/DQB1 and genetic resistance to hydatidosis was analyzed. Our research would help study the mechanisms of MHC-antidisease and provide data to anti-disease breeding, furthermore, which would have important theoretical and production value.1. The prevalence of E.granulosus was evaluated by ELISA diagnose Kit in Chinese merino sheep( 868 in total) in Tacheng, Yili, Shihezi and Duolang sheep( 351 in total) in Kashi. Results showed that the average infection rate of Chinese merino sheep were 22.33%, 48.8% and 6.9%, in Tacheng, Yili, Shihezi, respectively and that of total tested Dolang sheep and Chinese Merino sheep were 15.915% and 21.6% , both of which were significantly different (P﹤0.01).2. The polymorphisms of the second exon of MHC-DRB1 /DQB1 gene were investigated using the PCR-RFLP method in Duolang sheep and Chinese Merino sheep, results revealed that they were rich at SacII, Mval, SacI, Hin1I and HaeIII. In MHC-DRB1, 2, 3, 2, 2 and 6 RFLP patterns were found, and 3, 5, 3, 3, 15 and 3, 3, 3, 3, 18 genotypes were identified and base positions 229, 225, 208, 210, 178, 173, 159, 87 were polymorphic. In MHC-DQB1 gene, 2, 2, 4, 2, 3, 6 and 3 RFLP patterns were found with enzymes Mrox, ScaⅠ, SacII, NciI, TaqⅠ, MvaⅠand HaeⅢ, and 3, 3, 7, 3, 4, 16 and 6 genotypes were identified in Chinese merino sheep, and in Duolang sheep, 2,2,3,2,6,3 RFLP patterns with enzymes MroxⅠ, ScaⅠ, SacII, TaqⅠ, MvaⅠand HaeⅢ, and 3,3,5,2,14,5 genotypes, and base positions 266, 246, 231, 217, 213, 198, 192, 174, 161, 140, 96, 34 were polymorphic. According to above, we concluded the enzyme-digested sites of exon 2 of MHC-DRB1/DQB1 were in rich polymorphism, and in MHC-DQB1 in Chinese Merino sheep, allele HaeⅢm, HaeⅢn, MvaⅠz and MvaⅠy were new ones that found by cloning and sequencing.3. By comparing alleles and genotypic frequencies of MHC-DRB1 and MHC-DQB1 of healthy sheep with that of infectious ones in Chinese Merino sheep and Duolang sheep, we found that in Chinese Merino sheep, MvaⅠb, and HaeⅢe have some resistance to hydotydosis(P﹤0.01), genotype MvaⅠbb(p<0.01), HaeⅢee and SacⅠab may be resistant to hydotydosis, but MvaⅠbc, HaeIII bd, SacIIab and HaeIIIdf may be susceptible. MvaⅠD (P﹤0.05) , SacIIb, TaqⅠA and HaeⅢN were resistant to hydotidosis, TaqⅠB, MvaⅠC and HaeⅢM(P﹤0.01)were susceptible. MvaⅠDZ(P﹤0.05), TaqⅠAA and HaeIII NN were resistant, but MvaⅠCZ, TaqⅠAB(P﹤0.01)and HaeIII MN(P﹤0.01)were susceptible. In Duolang sheep, HaeⅢa(P﹤0.05)may be susceptible to hydotidosis, SacⅠab, HaeⅢcf and Hin1Ⅰaa(P﹤0.05) may be resistant. HaeIII be, HaeIII ef(P﹤0.01), SacⅠbb, HaeIII aa and Hin1Ⅰab(P﹤0.05)were susceptible in Duolang sheep at DRB1. MvaⅠD have resistance (P﹤0.01) to hydotidosis, MvaⅠDD and MvaⅠCD may be resistant, but MvaⅠBZ may be susceptible at DQB1.4. The relationship between halotype on MHC and hadytidosis was analyzed, results showed that in Chinese Merino sheep the halotype DRB-SacIAB, MvaIbb, DQB1-TaqIAA and HaeⅢNN have close relation with anti-hydytidosis ( P<0.01 ) , but DRB1-MvaIbc, DQB1-MvaIYY, DQB1-TaqIAB, DQB1-HaeⅢMN, DRB1-MvaIbb, DQB1-MvaICC, DQB1-TaqIAB and DQB1-HaeⅢMN were susceptible( P<0.01 ) . In Duolang sheep, DRB1-SacIAB, DRB1-HaeⅢff, DQB1-MvaICD, DRB1-SacIAB, DRB1-HaeⅢff and DQB1-MvaIDD were resistant to hydatidosis ( P<0.05 ) , but DRB1-HinIab, DRB1-HaeⅢbe, DRB1-HinIab, DRB1-HaeⅢef, DRB1-HinIab, DRB1-HaeⅢaa were susceptibl(eP<0.01).5. In this study, oncospheres were infected artifically with the healthy Chinese Merino sheep that halotype performed DRB1-SacIAB, DRB1-MvaIbb, DQB1-TaqIAA and DQB1-HaeⅢNN, all of which were confirmed to be resistant to hydytidosis.6. Hard-weinberg equilibrium plays an important role in maintaining the stability of genetic structure. It showed difference for Hardy-weinberg equilibrium of MHC-DRB1 in both sheep varieties. SacI and Hin1Ⅰin Chinese Merino sheep and MvaⅠin Dolang sheep have been attained to Hardy-weinberg equilibrium ( p>0.05 ) at MHC-DRB1, but others have not. TaqⅠand MroxⅠin Chinese Merino sheep and TaqⅠin Dolang sheep has been attained(P>0.05), but others have not(P<0.01)at MHC-DQB1更多还原