Characterization of Genetic Polymorphism of Novel MHC B-LBⅡ Alleles in Chinese Indigenous Chickens

中国地方鸡种MHC B-LBⅡ新等位基因的遗传多态性研究(英文)

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 genetic variation of the B-LB Ⅱ gene, 37 types of patterns detected by PCR-SSCP were investigated first, which would be used to screen novel B-LB Ⅱsequences within the breeds. The types of PCR-SSCP patterns and final sequencing allowed for the identification 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-LB Ⅱ alleles revealed a total of 68 variable sites in the fragment of exon 2, of which 51 parsimony informative 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 estimated 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 domain 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 specificity of MHC B-LB Ⅱ molecule in Chinese native chickens. These results can provide a molecular biological basis for the study of disease resistance in chicken breeding.

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。