Characterization of BoLA class II <i>DQA</i> and <i>DQB</i> by PCR-RFLP, cloning, and sequencing reveals sequence diversity in crossbred cattle
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The BoLA class II <i>DQA</i> and <i>DQB</i> genes in crossbred cattle were studied using PCR-RFLP, cloning, and sequencing techniques. Seventy-two crossbred cattle (<i>Vrindavani</i>) were used in the current study. <i>Hae</i>III and <i>Xba</i>I restriction enzymes digested <i>DQA</i> exon 2-3, revealing seven (<i>Hae</i>III-A-G) and three (<i>Xba</i>I A-C) motifs, respectively. The BoLA-<i>DQB</i> gene was analyzed using PCR-RFLP with <i>Pst</i>I and <i>Taq</i>I restriction enzymes, yielding five restriction motifs for each restriction enzyme (<i>Pst</i>I-A-E and <i>Taq</i>I-A-E). In crossbred cattle, addition, deletion, and substitutions were observed in distinct sequences, resulting in variations in overall gene length. Changes in nucleotides at positions 64–80, 110–200, and 207–264 were largely responsible for polymorphism in <i>DQA</i> exon 2. The phylogenetic analysis predicted a high degree of nucleotide and amino acid changes in DQA exon 2-3 and DQB exon 2. <i>DQA</i> genes had a nucleotide dissimilarity of 0.3–25.4 percent, while <i>DQB</i> genes had a nucleotide dissimilarity of 1.5–14.3 percent. We cloned and sequenced 20 genotypes based on PCR-RFLP of the <i>DQA</i> and <i>DQB</i> genes. The current study observed variation in the <i>DQA</i> and <i>DQB</i> genes and will serve as a foundation for future research on the BoLA <i>DQA</i> and <i>DQB</i> genes.
本研究采用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）、克隆与测序技术，针对杂交牛的牛白细胞抗原（BoLA）II类DQA与DQB基因展开探究。本研究共纳入72头文达瓦尼（Vrindavani）杂交牛。采用HaeIII与XbaI限制性内切酶酶切DQA基因外显子2-3区域，分别获得7个（HaeIII-A至G）与3个（XbaI A至C）酶切基序。针对BoLA-DQB基因，本研究采用PstI、TaqI两种限制性内切酶开展PCR-RFLP分析，每种酶均得到5个酶切基序（PstI-A至E及TaqI-A至E）。在杂交牛的不同序列中，可观察到碱基的插入、缺失与替换现象，进而导致基因总长度发生变异。DQA基因外显子2区域的多态性主要由64~80、110~200及207~264位的核苷酸变异所介导。系统发育分析结果显示，DQA基因外显子2-3与DQB基因外显子2区域存在大量核苷酸与氨基酸变异。DQA基因的核苷酸差异率为0.3%~25.4%，而DQB基因的核苷酸差异率为1.5%~14.3%。本研究基于DQA与DQB基因的PCR-RFLP分型结果，共克隆并测序了20种基因型。本研究揭示了杂交牛BoLA-DQA与BoLA-DQB基因的遗传变异，可为后续牛白细胞抗原DQA、DQB基因的相关研究奠定基础。
