鸡MHC B-LBⅡ新等位基因检测及多态性研究

Identification of Novel Alleles and Polymorphism of MHC B-LBⅡGene in Chicken

在鸡的基因组中扩增了MHC B-LBⅡ基因第2外显子长度为374bp的片段,通过克隆、测序,发现了20个B-LBⅡ新等位基因.对第2外显子6个限制性酶切位点(AluⅠ、CaiⅠ、CfrⅠ、Hinl Ⅰ、HinfⅠ和RsaⅠ)的PCR-RFLP多态性进行分析,并运用在这6个位点所观测的纯合基因型组合对B-LBⅡ等位基因进行初步检测,结果显示,该方法的等位基因检出率可达65%,等位基因主型检出率为68.75%.对B-LBⅡ基因7个单倍型和20个等位基因第2外显子多态性分析表明,核苷酸的多态变异位点为63个,其中简约性信息位点48个,单变异位点15个;仅在中国地方鸡种所检测到的简约性信息位点13个,单变异位点11个.序列同源性达90.6%～99.5%;核苷酸异义替换率为14.64%±2.67%,高于同义替换率2.92%±0.94%.为鸡B-LBⅡ基因多态性进化机制研究提供了参考资料.

A 374hp fragment spanning over exon 2 of major histocompatihility complex B-LB Ⅱ gene was amplified, cloned and sequenced in eight Chinese indigenous chicken breeds and one introduced breed. Twenty new B-LB Ⅱ alleles were found in the chicken breeds sampled. The polymorphism of the exon 2 was investigated by PCR-RFLP technique at the six loci of Alu I, Cai Ⅰ , Cfr Ⅰ , Hinl Ⅰ , Hinf Ⅰ and Rsa Ⅰ. The observed homozygous genotypes at the six loci were utilized to tentatively identify the alleles of B- LB Ⅱ genes in the animals analyzed. It indicated that 65% of alleles were discernible by this method, and the alleles accounted for 68.75% of major type alleles sampled. Alignment of the exon 2 sequences of 7 haplotypes and the 20 alleles revealed 63 polymorphic sites, of which 48 sites were parsimony informative, and 15 sites were singleton variable. Among the variable sites, 13 of parsimony informative sites and 11 out of the singleton variable sites were unique to the Chinese indigenous breeds sampled. The sequences of the exon 2 shared 90.6%-99.5% homology to each other. The relative frequencies of nonsynonymous and synonymous substitutions of nucleotide at per site in the exon 2 were estimated, 14.64%±2.67%and 2.92%±0.94%, respectively; the former was highly exceeded the latter. The present study provided the basic data for investigation of the evolutionary mechanisms of MHC B-LB Ⅱ genes in chicken.