【摘要】 为研究鸡MHC B-LBⅡ基因的遗传多态性,首先在8个中国地方鸡种(藏鸡、仙居鸡、北京油鸡、固始鸡、斗鸡、丝羽乌骨鸡、白耳鸡和狼山鸡)B-LBⅡ基因第二外显子扩增了一长度为 175 bp 的 DNA 片段并进行 SSCP 基因型分析;在8 个地方鸡种共 467 个个体中检测到 37 个 PCR-SSCP 基因型;从被检样品中筛选出不同基因型的个体,并在其 B-LBⅡ基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,通过克隆和测序获得了该片段的核苷酸序列。经序列分析,在前述地方鸡种被筛选出的 30 个无血缘关系的个体中发现了 31 个 B-LBⅡ新等位基因,并参照哺乳动物 MHC II 类 B 等位基因命名规则进行了命名。对这 31 个 B-LBⅡ新等位基因长度为 374 bp 的 DNA 片段进行比对表明,在其第二外显子序列上共有 68 个多态性变异位点,其中简约性信息位点 51 个,单变异位点 17 个,具有丰富的遗传多态性。在这些多态性变异位点中,出现在遗传密码子第一和第二位上的碱基替换率分别为 36.76% 和 35.29%。等位基因序列间的相似性估测为 90.6%-99.5%;B-LBⅡ基因第二外显子的错义替换率和同义替换率分别为 14.64±2.67%和 2.92±0.94%。结果表明,B-LBⅡ基因的丰富遗传多态性主要是由基因重组和平衡选择效应所引起的。对 B-LBⅡ等位基因第二外显子所编码的 B-LBⅡ分子β1 结构域氨基酸序列比对发现,31 个 B-LBⅡ新等位基因属于 26 个等位基因主型;在β1结构域氨基酸序列的 33个变异位点上,存在 6 个同义替换和 27 个错义替换。分析认为,那些发生在多肽结合位点上的氨基酸错义替换与鸡 MHC B-LBⅡ分子的免疫特异性有关。该结果可为鸡的抗病育种研究提供分子生物学依据。更多还原

【Abstract】 Genetic polymorphism of the major histocompatibility complex (MHC) B-LBⅡ gene was studied by amplification of exon 2 using PCR, followed by cloning and DNA sequencing in eight indigenous Chinese chicken populations. To reveal the ge- netic variation of the B-LBⅡ gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LBⅡ sequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identifica- tion of 31 novel MHC B-LBⅡ alleles from 30 unrelated individuals of Chinese chickens that were sampled. These are the first designators for the alleles of chicken MHC B-LBⅡ gene based on the rule of assignment for novel mammalian alleles. Sequence alignment of the 31 B-LBⅡ alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informa- tive and 17 singleton variable sites were observed. Among the polymorphic sites, the nucleotide substitutions in the first and second positions of the codons accounted for 36.76% and 35.29%, respectively. The sequence similarities between the alleles were esti- mated to be 90.6%–99.5%. The relative frequencies of synonymous and nonsynonymous nucleotide substitutions within the region were 2.92%±0.94% and 14.64%±2.67%, respectively. These results indicated that the genetic variation within exon 2 appeared to have largely arisen by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of the β1 do- main coded by exon 2 revealed 6 synonymous mutations and 27 nonsynonymous substitutions at the 33 disparate sites. In particular, the nonsynonymous substitutions at the putative peptide-binding sites are considered to be associated with immunological specific- ity of MHC B-LBⅡ molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.更多还原