CN116837104A - 与绵羊免疫性状相关的分子标记及其应用 - Google Patents
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Abstract
本发明公开了一种与绵羊免疫性状相关的分子标记及其应用,所述分子标记位于绵羊20号染色体Bola‑1a基因第8号外显子的核苷酸序列上。利用该分子标记可预测不同基因型羊之间免疫性状的差别,从而提高抗病育种筛选中标记辅助选择的效率。
Description
技术领域
本发明涉及化工领域,特别是,涉及一种与绵羊免疫性状相关的分子标记及其应用。
背景技术
随着肉羊养殖现代化的推进,抗病育种工作越发重要。筛选与免疫性状相关的基因是抗病育种常见手段。主要组织相容性复合体I(Major histicompatibility complexI,MHC-I)是存在于有核细胞表面的一类蛋白多肽,参与内源性抗原的递呈,诱导对病原微生物感染细胞的杀伤溶解,MHC-I结构变化影响细胞被杀伤(肖进,2017)。当机体针对病原微生物入侵发生炎症反应后,为了保护健康细胞组织不被过度侵害,中性粒细胞会分泌抗菌肽维持平衡(Koczulla等,2003;Colgan等,2013)。因此抗菌肽分泌的水平与细胞被杀伤率之间可能存在相关性。诸多研究也表明MHC-I基因与羊抗病能力相关(孙洪新等,2021)。Bola-1a是编码绵羊MHC-Iα链的基因,存在于细胞膜表面,是抗体标志组成部分,具有高度多态性。DNA分子标记技术的迅猛发展,为人们在分子水平上研究绵羊抗病能力的遗传机理奠定了基础,筛选获得相应的分子标记以便于更高效的用于绵羊抗病育种的筛选辅助,是本领域一个急需解决的技术问题。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种与绵羊免疫性状相关的分子标记及其应用,利用该分子标记可预测不同基因型羊之间免疫性状的差别,从而提高抗病育种筛选中标记辅助选择的效率。
为解决上述问题,本发明所采用的技术方案如下:
一种与绵羊免疫性状相关的分子标记,所述分子标记位于绵羊20号染色体Bola-1a基因第8号外显子的核苷酸序列上。
优选的,所述分子标记包括SNP位点,所述SNP位点位于ensembl基因组参考转录本收录号: ENSOART00000009855.1的cDNA序列第5035位核苷酸,所述SNP位点核苷酸为C或G。
优选的,所述分子标记的核苷酸序列如SEQ ID NO.1所示,所述SNP位点位于所述分子标记的第164位,所述SNP位点核苷酸为C或G。
本发明还包括用于检测上述所述分子标记的特异性引物组,其含具有如SEQ IDNO.2所示序列的上游引物以及具有如SEQ ID NO.3所示序列的下游引物。
本发明还包括上述任一项所述分子标记在绵羊抗病育种筛选中的应用。
相比现有技术,本发明的有益效果在于:采用本发明的分子标记可预测不同基因型羊之间免疫性状的差别,从而提高抗病育种筛选中标记辅助选择的效率。
具体实施方式
下面结合具体实施方式对本发明作进一步详细说明。
下述实施例中所有试验的实验方法如无特殊说明,均为常规方法;下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。本发明所述的SNP是单核苷酸多态性的简称,指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。
实施例1 绵羊免疫性状相关分子标记与绵羊免疫力的关系
一种与绵羊免疫性状相关的分子标记,所述分子标记位于绵羊20号染色体Bola-1a基因第8号外显子的核苷酸序列上。所述分子标记包括SNP位点,所述SNP位点位于ensembl基因组参考转录本收录号: ENSOART00000009855.1的cDNA序列第5035位核苷酸,所述SNP位点核苷酸为C或G。所述分子标记的核苷酸序列如SEQ ID NO.1所示,所述SNP位点位于所述分子标记的第164位,所述SNP位点核苷酸为C或G,该替换导致所述序列产生多态性。
1、引物的设计
根据SEQ ID NO.1所示的序列利用引物设计软件primer premier 5.0设计引物,获得上下游引物序列如下:
F:5’-CGCGGATCCTACTGCTGCTACCAACAAGG-3’(SEQ ID NO.2);
R:5’-CGGACTCTAAGGTCGGGAGCTCGCC-3’ (SEQ ID NO.3)。
2、鉴别具有不同免疫力性状的绵羊
2.1样品制备
采集100只相同月龄特藏寒杂交F2代绵羊的耳朵组织,并分别采用TIANampGenomic DNA Kit血液/细胞/组织基因组DNA提取离心柱型试剂盒(由天根生化科技(北京)有限公司生产),提取羊免疫性状测定相关群体的耳朵组织DNA样本。具体步骤为:
将耳朵组织用眼科剪剪碎至糊状,加入200μLGA(上述试剂盒自带),并震荡混匀;然后放入56℃水浴锅中消化过夜;
将消化后的组织样加入200μL缓冲液GB(上述试剂盒自带),充分颠倒混匀,于70℃放置10分钟,待溶液变清亮后,简短离心以去除管壁上的水珠;
加入200μL无水乙醇,充分震荡混匀15秒,此时可能会出现絮状沉淀,简短离心以去除管壁上的水珠;
将上一步所得溶液及絮状沉淀一并加入吸附柱CB3中,并将吸附柱放入收集管12000rpm离心30s,倒掉废液,并将吸附柱放回收集管中;
向吸附柱CB3中加入500μL缓冲液GD(上述试剂盒自带),12000rpm离心30s,倒掉废液,并将吸附柱放入收集管中;
向吸附柱CB3中加入600μL漂洗液PW(上述试剂盒自带),12000rpm离心30s,倒掉废液,并将吸附柱放入收集管中,并重复该步骤;
将吸附柱CB3置于室温下放置数分钟,以彻底晾干吸附柱材料中剩余的漂洗液;
将吸附柱CB3转入一个干净的离心管中,并向吸附柱膜中间部位悬空滴加50-200μL的洗脱液TE(上述试剂盒自带),室温下放置2-5分钟,12000rpm离心2分钟,将溶液收集到离心管中,获得耳朵组织DNA样本。
2.2 PCR扩增
利用步骤1的引物对对步骤2.1的100份耳朵样品DNA样本进行PCR扩增。
每份DNA样品的PCR扩增均采用下述25μL体系:10×PCR buffer 2.5μL;含有dATP、dTTP、dCTP和dGTP各2.5mM的4种dNTP混合物2μL;步骤1的浓度均为10μmol/L的SEQ ID No.2与SEQ ID No.3所示的引物各0.5μL;Taq DNA聚合酶( 2.5U/μL )0.5μL,DNA样品(100ng/μL)1μL,ddH2O 18μL。
PCR扩增的反应条件均为:94℃预变性5min;94℃30s,64℃30s,72℃30s,共35个循环;72℃延伸10min,4℃保存。
2.3 PCR产物测序
将PCR扩增得到的所有样品的PCR产物进行10%非变性聚丙烯酰胺凝胶电泳,获得三种带型,将含有不同带型的PCR产品的基因型分别命名为CC、CG和GG,挑选不同带型的PCR产物,经回收纯化后送北京六合华大基因科技股份有限公司测序,测得基因型CC的序列为序列表中SEQ ID No.1(SNP位点核苷酸均为C),测得基因型CG的序列为序列表中SEQ IDNo.1(SNP位点核苷酸为C和G),测得基因型CC的序列为序列表中SEQ ID No.1(SNP位点核苷酸均为G)。按照基因型对绵羊进行分类,将基因型CC的绵羊命名为CC基因型绵羊,将基因型CG的绵羊命名为CG基因型绵羊,将基因型GG的绵羊命名为GG基因型绵羊,结果显示,100只特藏寒杂交F2代绵羊中,22只CC基因型绵羊,36只CG基因型绵羊,42只GG基因型绵羊。
2.4 绵羊免疫力的鉴别
对上述100只特藏寒杂交F2代绵羊进行相同剂量的布鲁氏菌刺激,24H后采集上述100只特藏寒杂交F2代绵羊外周血,使用ELISA试剂盒(睿信生物 羊抗菌肽LL-37 ELISA试剂盒)检测抗菌肽水平,并对照基因分类发现,相对于GG基因型绵羊的抗菌肽表达水平,CG基因型绵羊抗菌肽表达水平提升9.8%,CC基因型绵羊抗菌肽表达水平提升26.9%。SEQ IDNO.1第164位核苷酸为C或G的突变与绵羊免疫力存在明显的相关性。且当突变位点为C时,抗菌肽表达水平最高,即具有最优的免疫力;当突变位点为G时,抗菌肽表达水平最低,即具有最差的免疫力。
通过对分子标记辅助选择,对群体内基因型为CG和GG的绵羊进行淘汰,显著提高了群体的免疫力,促进了绵羊的育种进程,增加绵羊群体的免疫性能,带动肉羊产业经济效益更大化,最终实现提高绵羊的经济效益,从而增加牧民或企业的收益。
对本领域的技术人员来说,可根据以上描述的技术方案以及构思,做出其它各种相应的改变以及形变,而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
Claims (5)
1.一种与绵羊免疫性状相关的分子标记,其特征在于,所述分子标记位于绵羊20号染色体Bola-1a基因第8号外显子的核苷酸序列上。
2.如权利要求1所述的分子标记,其特征在于,所述分子标记包括SNP位点,所述SNP位点位于ensembl基因组参考转录本收录号: ENSOART00000009855.1的cDNA序列第5035位核苷酸,所述SNP位点核苷酸为C或G。
3.如权利要求1所述的分子标记,其特征在于,所述分子标记的核苷酸序列如SEQ IDNO.1所示,所述SNP位点位于所述分子标记的第164位,所述SNP位点核苷酸为C或G。
4.用于检测权利要求3所述分子标记的特异性引物组,其特征在于,其含具有如SEQ IDNO.2所示序列的上游引物以及具有如SEQ ID NO.3所示序列的下游引物。
5.权利要求1-3任一项所述分子标记在绵羊抗病育种筛选中的应用。
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CN117051133A (zh) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | 用于检测绵羊布鲁氏菌病抗性的snp分子标记及其检测引物和应用 |
CN117051135A (zh) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | 一种绵羊布鲁氏菌病抗性snp分子标记及其应用 |
CN117070643A (zh) * | 2023-10-11 | 2023-11-17 | 中国农业科学院北京畜牧兽医研究所 | 与绵羊布鲁氏菌病抗性相关的snp分子标记及其应用 |
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2023
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN117051133A (zh) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | 用于检测绵羊布鲁氏菌病抗性的snp分子标记及其检测引物和应用 |
CN117051135A (zh) * | 2023-10-11 | 2023-11-14 | 中国农业科学院北京畜牧兽医研究所 | 一种绵羊布鲁氏菌病抗性snp分子标记及其应用 |
CN117070643A (zh) * | 2023-10-11 | 2023-11-17 | 中国农业科学院北京畜牧兽医研究所 | 与绵羊布鲁氏菌病抗性相关的snp分子标记及其应用 |
CN117070643B (zh) * | 2023-10-11 | 2024-01-30 | 中国农业科学院北京畜牧兽医研究所 | 与绵羊布鲁氏菌病抗性相关的snp分子标记及其应用 |
CN117051133B (zh) * | 2023-10-11 | 2024-01-30 | 中国农业科学院北京畜牧兽医研究所 | 用于检测绵羊布鲁氏菌病抗性的snp分子标记及其检测引物和应用 |
CN117051135B (zh) * | 2023-10-11 | 2024-01-30 | 中国农业科学院北京畜牧兽医研究所 | 一种绵羊布鲁氏菌病抗性snp分子标记及其应用 |
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