藏鸡主要组织相容性复合体B-LBⅡ基因第二外显子序列遗传变异分析

根据鸡主要组织相容性复合体B-LBⅡ基因序列设计特异性引物,在藏鸡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,并通过克隆和PCR直接测序获得了该片段的核苷酸序列。发现了15个B-LBⅡ新等位基因。对18个B-LBⅡ等位基因核苷酸序列和其所编码的MHCB-LBⅡ分子β1结构域的氨基酸序列分析显示,第二外显子核苷酸序列遗传多态性异常丰富,存在着62个多态变异位点(共包括80个突变),其中41个为简约性多态位点;衡量该序列遗传多样性的π值为0.0718;反映其群体内遗传变异度的平均遗传距离为0.056±0.008,低于在5个外来品种所估算的平均遗传距离。该编码区核苷酸相对异义替换率(15.61±2.69%)显著高于其同义替换率(3.25±0.94%),进一步分析表明,基因重组和平衡选择机制可能是引起B-LBⅡ基因序列变异的主要因素。在β1结构域氨基酸序列中,存在11个同义替换和27个异义替换;在24个肽结合位点中有12个变异位点;与其他6个中国地方鸡品种和一个外来品种比较发现,有11个异义氨基酸替换仅出现在藏鸡群体中,并被认为与藏鸡的免疫特异性有关,可为鸡的抗病力研究提供分子依据。

Genetic Variation within Exon 2 of the MHC B-LBⅡ Gene in Tibetan Chicken

Genetic variation within exon 2 of chicken major histocompatibility complex B-LBⅡgenes was investigated by PCR amplification,cloning and sequencing of a 374 bp fragment of the indigenous Tibetan chicken genomic DNA.Fifteen novel B-LBⅡalleles were found.Alignment and comparison of 18 allelic sequences from the individuals sampled revealed a total of 62 variable sites (total of 80 mutations) in exon 2,of which 41 were parsimony informative sites.The nucleotide diversity (π) within the sequence of exon 2 was calculated to be 0.0718.Analysis of nucleotide variation confirmed a lower level of divergence (0.056±0.008) as estimated by average pairwise distance within the Tibetan chicken population than the five exotic breeds detected.The relative frequencies of synonymous and non-synonymous nucleotide substitutions within the region were 3.25±0.94% and 15.61±2.69%,respectively.These results indicated that the genetic variation within exon 2 seemed to have arisen largely by gene recombination and balancing selection.Alignment of the deduced amino acid sequences of β1 domain coded by exon 2 revealed 11 synonymous mutations and 27 non-synonymous substitutions at the 38 separate sites.Fifty percent (12/24) of the proposed peptide-binding sites were variable within β1 domain of chicken MHC B-LBⅡmolecules,of which 11 were unique non-synonymous amino acid substitutions.These particular non-synonymous substitutions are considered to be associated with immunological specificity of MHC B-LBⅡmolecule in Tibetan chicken,and they can provide a molecular biological basis for the study of disease resistance in chicken.