Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

XU Ri-fu; LI Kui; CHEN Guo-hong; QIANG-BA Yang-zong; XU Hui; MO De-lin; LI Chang-chun; FAN Bin; LIU Bang

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Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

机译：PCR-SSCP和RFLP对中国土鸡B-G基因遗传变异的研究

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摘要

To reveal genetic variation of MHC B-G gene at Chinese native chickens, two PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the B-G gene and used to amplify two DNA fragments in ten Chinese indigenous chicken breeds and one introduced breed. The fragments were cloned and sequenced to assure that the expected sequences of chicken B-G gene were isolated. Of which the 189 bp fragment encompassing the most variable region within exon 2 of B-G gene was employed for PCR-SSCP assay, this method provided evidence for the presence of at least 56 B-G genotypes in the Chinese chickens sampled. It revealed a high degree of diversity in B-G genes of Chinese local breeds; particularly, high variation of B-G gene was confirmed with the presence of 48 B-G genotypes within Tibetan chicken population. Not only can the B-G genotypes be used to preliminarily screen new B-G alleles, but also they would be utilized to investigate MHC haplotypes and matched unrelated donors for bone marrow transplantation in immune researches. Another fragment of 401 bp size spanning over partial intron 1 and exon 2 of B-G gene was employed for PCR-RFLP analysis with two restriction enzymes of Msp Ⅰ and Tas Ⅰ in the breeds sampled. In this part of the gene, three novel SNPs were detected at the two restriction sites. It was more generally found the transition of two nucleotides of A294G and T295C occurred at Tas Ⅰ restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that an alone mutation of A294G occurring at the site, which also caused the substitution of amino acid, asparagine 54-to-serine;and we haven't found only single mutation occurred at position 295 of the restriction site. At the Msp Ⅰ site, the transversion of G318C led to a non-synonymous substitution, glutamine 62-to-histidine. The variations at expression level caused by the genetic variability of B-G gene may bring about the changes in immune specificity of B-G antigen finally. Furthermore, two alleles, A and B, were identified at Msp Ⅰ and Tas Ⅰ loci of B-G gene, respectively. The allele frequencies were estimated, which gave a nonsymmetrical distribution either in the eight Chinese local breeds or in the introduced breed. By comparison, allele A at Msp Ⅰ locus was tended to be dominative, while, the allele B at Tas Ⅰlocus was tended to be prevalent in the breeds analyzed. It is concluded that the genetic variability of B-G gene revealed by the PCR-SSCP and RFLP assays in Chinese native chickens provide molecular data for further investigating the varied immune functions of B-G antigen; and the PCR-RFLPs at Msp Ⅰ and Tas Ⅰ loci of B-G gene might be used as genetic markers in selecting for the traits of disease resistance in chicken breeding.

机译：为了揭示中国本土鸡MHC B-G基因的遗传变异，设计了两个PCR引物对，与B-G基因内高变区周围的保守序列特异性杂交，并用于在10个中国本土鸡品种和一个引进品种中扩增两个DNA片段。将该片段克隆并测序以确保分离出鸡B-G基因的预期序列。其中B-G基因第2外显子中变化最大区域的189 bp片段用于PCR-SSCP分析，该方法提供了证据，表明在所采样的中国鸡中至少存在56个B-G基因型。这表明中国地方品种的B-G基因高度多样性。特别是，藏族鸡群中存在48种B-G基因型，证实了B-G基因的高度变异。 B-G基因型不仅可以用于初步筛选新的B-G等位基因，而且还可以用于研究MHC单倍型并与无关的供体进行免疫研究中的骨髓移植。用Bsp基因的部分内含子1和外显子2的另一个401 bp大小的片段，用MspⅠ和TasⅠ的两种限制性内切酶进行PCR-RFLP分析。在基因的这一部分，在两个限制性位点检测到三个新的SNP。更普遍地发现，A294G和T295C的两个核苷酸的转变发生在TasⅠ限制性酶切位点，因此导致推导的免疫球蛋白可变区样氨基酸序列中54位的天冬酰胺非同义取代为丝氨酸。 BG基因外显子2编码的结构域。在极少的频率下观察到该位点发生了单独的A294G突变，这也导致氨基酸天冬酰胺54取代为丝氨酸;而且我们还没有发现仅在限制位点295位发生单个突变。在MspⅠ位点，G318C的转座导致了一个非同义的取代，谷氨酰胺62-组氨酸。 B-G基因遗传变异引起的表达水平变化最终可能导致B-G抗原的免疫特异性发生变化。此外，在B-G基因的MspⅠ和TasⅠ位点分别鉴定了两个等位基因A和B。估计了等位基因频率，这在中国的八个地方品种或引进的品种中均给出了非对称分布。通过比较，在MspⅠ位点的等位基因A倾向于占优势，而在TasⅠ位点的等位基因B倾向于在所分析的品种中流行。结论：PCR-SSCP和RFLP检测显示中国本土鸡B-G基因的遗传变异性，为进一步研究B-G抗原的多种免疫功能提供了分子数据。 B-G基因MspⅠ和TasⅠ位点的PCR-RFLPs可作为遗传标记，用于选择鸡的抗病性状。

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来源
《农业科学学报（英文版）》 |2005年第8期|621-628|共8页
作者
XU Ri-fu; LI Kui; CHEN Guo-hong; QIANG-BA Yang-zong; XU Hui; MO De-lin; LI Chang-chun; FAN Bin; LIU Bang;
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作者单位

Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, P.R. China;

Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, P.R. China;

Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094, P.R. China;

Department of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. China;

Department of Animal Husbandry and Veterinary, College of Agriculture and Animal Husbandry, Tibetan University, Linzhi 860000,P.R. China;

Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, P.R. China;

Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, P.R. China;

Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, P.R. China;

Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, P.R. China;

Laboratory of Molecular Biology and Animal Breeding, College of Animal Science and Technology, Huazhong Agricultural University,Wuhan 430070, P.R. China;

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正文语种 chi
中图分类农业基础科学;
关键词
B-G genes; PCR-SSCP assay; Genetic variation; Chinese indigenous chicken; Genotype; Allele;

机译：B-G基因;PCR-SSCP法;遗传变异;中国土鸡;基因型;等位基因;

Investigation of Genetic Variation of B-G Gene with PCR-SSCP and RFLP in Chinese Indigenous Chickens

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