琅琊鸡Mx基因3′端部分序列PCR-RFLP与序列分析（摘要）

目的]从分子角度对琅琊鸡Mx基因的遗传变异进行研究,为琅琊鸡分子标记辅助选择、抗病育种提供依据。方法]采用常规的酚/氯仿抽提法提取基因组DNA。Mx基因3′端部分序列引物参考杨宁（上游引物P1：5′-GCCTACCAATACCTGG-TAGACTTT-3′,下游引物P2：5′-GCTCCCCCTCCTITCAAATA-3′）。琅琊鸡Mx基因3′端部分序列的PCR产物采用1%琼脂糖凝胶电脉检测,在紫外凝胶成像系统下观察并保存图像。利用3%琼脂糖凝胶电泳检测HaeⅢPCR-RFLP。根据酶切结果,选取不同基因型个体的PCR扩增产物,用DNA快速纯化回收试剂盒纯化。根据《分子克隆试验指南》,经连接、转化、克隆后,按照TaKaRa公司质粒DNA小量纯化试剂盒说明,对经PCR扩增鉴定为阳性的菌液提取质粒。鉴定后将重组质粒送TaKaRa公司测序。结果]琅琊鸡群体中存在Mx基因HaeⅢ酶切位点的多态性,且均有A、B两个等位基因,基因频率分别为0.562和0.438。经独立X~2性检验,Mx基因HaeⅢ酶切位点的基因型分布在琅琊鸡群体中极显著偏离Hardy-Weinberg平衡状态（P〈0.01）。对多态片段进行克隆测序分析,结果表明,该扩增片段长度大小为357 bp,通过DNAMAN分析软件对报道序列（NC_006088,杨宁）和Mx基因序列作比对分析,发现琅琊鸡Mx基因3′端部分序列扩增区域在20 506～20 862位点,变异序列在20771～20 802位点存在长度为31 bp的碱基缺失。HaeⅢ在本序列195 bp位点存在识别序列,对AB基因型（2条带）个体酶切产物是长度为195 bp和162 bp的2段,在3%琼脂糖凝胶中显示2条带;对AA基因型（1条带）个体发生酶切反应时,由于存在62～93位点长度为31 bp碱基缺失,所以酶切产物是长度为164 bp和162 bp的2段,与试验预期结果相一致。结论]在山东省地方鸡种琅琊鸡Mx基因3′端序列中发现存在HaeⅢ-RFLP。

PCR-RFLP and Sequence Analysis of Mx Gene 3'Sequence in Langya Chicken Breeds

Objective The aim was to provide basis for molecular marker assisted selection and resistance breeding of Langya chicken. Method The genetic polymorphism of Hae III site of Mx gene 3＇ sequence in Langya chicken was analyzed by PCR-RFLP. Result The results showed that Hae III site controlled by allele A and B were polymorphic in Langya chicken breeds and the allele frequencies of A and B were 0.562 and 0.438 respectively. The genotype distribution of Hae III site was significantly not in Hardy-Weinberg equilibrium （ P 〈0.01 ）. The polymorphic fragments were cloned and sequenced, and the results revealed that the fragment size was 357 bp and a deletion of 31 bp occurred in variation sequences. Conclusion It was found that Hae III-RFLP exists in Mx gene 3＇ sequence in Langya chicken breeds of Shandong Province.