CN110079611A - 羊肌内脂肪和肌苷酸含量分子标记及其在品种培育中应用 - Google Patents
羊肌内脂肪和肌苷酸含量分子标记及其在品种培育中应用 Download PDFInfo
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- CN110079611A CN110079611A CN201910383978.3A CN201910383978A CN110079611A CN 110079611 A CN110079611 A CN 110079611A CN 201910383978 A CN201910383978 A CN 201910383978A CN 110079611 A CN110079611 A CN 110079611A
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Abstract
本申请提供了检测羊ANGPTL4上G303A和G396T多态性基因型位点来预测羊肌内脂肪和肌苷酸含量的方法，并提供了这种方法在品种培育中的应用。
Description
技术领域
本申请属于分子生物学和畜牧业肉羊品种培育领域，具体地，本申请提供了检测羊ANGPTL4上G303A和G396T多态性基因型位点来预测羊肌内脂肪含量的方法，并提供了这种方法在肉羊品种培育中的应用。
背景技术
肌内脂肪（IMF）是羊肉肌肉风味的主要指标之一，在正常范围内，肌内脂肪含量与羊肉的鲜嫩度、口感风味和多汁性呈现直接正相关的关系：肌纤维之间沉积一定量脂肪时，大理石纹增加，肉质鲜嫩性和多汁性得到改善（可见Gerbens F等，Assoeiations of heartand adipoeyte fatty acid-binding protein gene expression with intramuscularfat content in pigs，AnimSei，2001，79：347-354）。之前的研究表明，遗传上肌内脂肪含量与瘦肉率呈现负相关的关联，因此，培育高瘦肉率同时高机内脂肪含量（即肉质口感良好）的羊品种理论上完全可行。肌苷酸（IMP），又名次黄嘌呤核苷酸，其有多种来源，可在动物体内从头合成或者宰杀后由ATP形成，是鸡鸭鱼牛羊等多种肉类中重要的风味物质，主要为肉提供鲜味（1985年第一次国际鲜味问题讨论会即将肌苷酸提供的味道列为鲜味，也可见Yukiko M 等，Multiple taste functions of the umami substances in muscleextracts of yellowtail and bastard halibut，Fish Science，2010，76:521-528。但牛羊特别是具体品种绵羊的肌内脂肪和肌苷酸含量分子标记的筛选工作目前开展尚不多。
美利奴羊是是细毛绵羊品种的统称，在世界各地广泛饲养培育多年后衍生出了诸多毛用、毛肉兼用、肉毛兼用的品种。德国美利奴羊是我国自50年代起就开始引进养殖的优秀肉毛兼用品种，其在生长周期、繁殖性能、适应性、产肉性能上均有良好的表现，养殖实践中也已经与多种细毛杂种羊和本地羊尝试杂交以进一步改善其品种。针对德国美利奴羊肉质上的不足，使用分子标记高效筛选优良肉质个体用于繁殖和育种，是进一步改善德国美利奴羊品种的高效途径。
发明内容
针对我国常见的德国美利奴羊品种，申请人从现有技术出发，在ANGPTL4基因上寻找与羊肌内脂肪含量相关的多态性位点，在寻找到/现有技术中检索到的9个多态性位点中，发现了ANGPTL4两个多态性位点C724A和A601G与羊肉肌内脂肪明显相关。这两个分子标记可以作为培育优质肉用/肉毛德国美利奴羊品种时的参考依据。
一方面，本申请提供了检测ANGPTL4基因多态性的试剂在羊品种培育中的应用。
进一步地，所述羊为德国美利奴羊。
进一步地，所述ANGPTL4基因多态性为ANGPTL4上的G303A和/或G396T。
进一步地，所述试剂为PCR检测方法所用试剂。
进一步地，所述试剂为序列为SEQ ID NO.3-4的引物和测序用试剂。
进一步地，所述应用为选择ANGPTL4 303位置上基因型为GA或AA和396位置上基因型为GT或TT的羊用于品种培育。
另一方面，本申请提供了一种用于分子标记辅助羊品种培育的试剂盒，包括检测ANGPTL4基因多态性的试剂。
进一步地，所述ANGPTL4基因多态性为ANGPTL4上的G303A和/或G396T。
进一步地，所述试剂为PCR检测方法所用试剂。
进一步地，所述试剂为序列为SEQ ID NO. 3-4的引物和测序用试剂。
本申请的方法和试剂盒可用于各种肉羊或肉毛兼用、毛肉兼用羊，包括但不限于各种美利奴羊、小尾寒羊、杜泊羊、萨福克羊、湖羊、夏洛莱羊、波尔山羊、黄羊、大耳羊、努比亚山羊等。
本申请的方法和试剂盒可与其他分子生物学品种培育方法或传统品种培育方法联合使用。
本申请的方法和试剂盒筛选出的种羊可用于自然交配和人工授精的品种培育方法。
本申请的方法和试剂盒中检测多态性的试剂可以是基于PCR的检测方法，基于荧光能量共振的检测方法、基于质谱的检测方法、DNA芯片方法等中所用的试剂。
具体实施方式
实施例1 德国美利奴羊中ANGPTL4（类血管生成素羊蛋白4）多态性位点的发现
ANGPTL4中文名为类血管生成素样蛋白4，现有研究表明其广泛存在于哺乳动物中，与脂代谢、糖代谢、乃至肿瘤均有关联，申请人以此为基础，研究了德国美利奴羊中的ANGPTL4多态性及其与肌内脂肪和肌苷酸的关联。
实验对象选择申请人自有养殖场内的健康、成年纯种德国美利奴羊355只，屠宰前颈静脉采血5mL加入含抗凝剂而的离心管中保存于-20摄氏度下，屠宰后取背最长肌肉样-80摄氏度下保存/直接用于鲜肉肌苷酸含量测定。
肌内脂肪含量测定使用索氏抽提法，检测羊背最长肌肉样的肌内脂肪含量：（1）将3-5g样品在液氮中研至细粉，装入称重过的烘干滤纸包中，称重含湿样的滤纸包，干燥2h后，称重含干样的滤纸包；（2）使用索氏抽提器室温下乙醚抽提6h；（3）乙醚挥发后，将滤纸包干燥2h后称重。按照：（含干样滤纸包重-抽提后干燥滤纸包重）/（含湿样滤纸包重-滤纸包干重）计算肌内脂肪含量百分数。
羊背最长肌肉样的肌苷酸含量参考现有技术使用HPLC测定：（1）取2g肉样品，剪碎后加入10ml5%高氯酸，匀浆；（2）4000r/min离心5min，上清液过滤，向沉淀中加入5ml5%高氯酸，震荡后4000r/min离心5min，取上清液与之前的上清液合并；（3）调上清液pH至6.5，纯净水定容至25mL；（4）混匀过滤后用于HPCL检测；（5）Agilent 1200HPLC仪，C18 柱；流动相：5%甲醇、95%磷酸-三乙胺溶液，0.7 mL/min，25℃，检测波长：254 nm；（5）使用肌苷酸含量标准品建立标准曲线，计算肌苷酸含量（mg/g鲜肉）。
分子生物学实验部分委托北京微阅基因技术有限公司进行：
使用酚-氯仿法提取血液样品DNA，琼脂糖凝胶电泳检测DNA浓度后保存于-20摄氏度下。根据Genbank上绵羊ANGPTL4的序列信息（KF873613，CDS为237到1469，本申请SEQ IDNO.1-2）使用Primer Premier 5.1设计引物扩增，并进行PCR-SSCP分析，发现ANGPTL4上G303A（按KF873613全长mRNA序列计，Ala-Thr，由非极性氨基酸变为极性氨基酸）和G396T（按KF873613全长mRNA序列计，Gly-Trp，由非极性氨基酸变为临界极性氨基酸）两个与肌内脂肪和肌苷酸含量相关的多态性位点。
实施例2 多态性位点的基因型频率和等位基因频率
ANGPTL4上的G303A和G396T在实验用德国美利奴羊群（355只）中的基因型频率和等位基因频率见下表：
表1：ANGPTL4上的G303A和G396T的基因型频率和等位基因频率
经过卡方检验，实验用德国美利奴羊群在两个多态性位点处于Hardy-Weinberg 平衡状态（P>0.05）。两者均属于低度多态性位点。
实施例3不同基因型与肌内脂肪含量的关联
使用实施例1中所述的方法检测各肉样肌内脂肪和肌苷酸含量，各基因型与肌内脂肪和肌苷酸含量的关联如下表所示：
表2：不同基因型与肌内脂肪（背最长肌）含量的关联
表3：不同基因型与肌苷酸（背最长肌）含量的关联
从上表数据可见，两个位点背最长肌肌内脂肪含量最小二乘均值的关系为GG<GA<AA，GG<GT<TT，303位置处GG与GA，GA与AA之间差异均显著（P<0.05），396位置处GG与GT，GT与TT之间差异均显著（P<0.05），而AG与AA之间差异不显著（P=0.166）。两个多态性位点均有较好的作为品种培育分子标记的潜力。
从上表数据可见，两个位点背最长肌肌苷酸含量最小二乘均值的关系为GG<GA<AA，GG<GT<TT，303位置处GG与GA，GA与AA之间差异均显著（P<0.05），396位置处GT与TT之间差异显著（P<0.05），而GG与GT之间差异不显著（P=0.135）。
两个多态性位点均有较好的作为品种培育分子标记的潜力。
实施例4 多态性分子标记用于德国美利奴羊品种培育实践
确定多态性位点后，将种羊颈静脉采血样本送至北京微阅基因技术有限公司，以SEQID NO.5-8的引物扩增后测序检测ANGPTL4上的G303A和G396T处基因型。以基因型为标准选取303位置上基因型为AA和396位置上基因型为TT的种公羊1只，母羊5只，303位置上基因型为AA和396位置上基因型为GT的种公羊1只,母羊14只，303位置上基因型为GA和396位置上基因型为TT的种公羊2只,母羊6只，将上述公羊和母羊用于自然交配繁殖。截止到本申请申请日前，统计6月龄时8样本中背最长肌和臀大肌中肌内脂肪含量，平均值为9.31%±0.28%，5.82%±0.18%，肌苷酸含量为1.45 mg/g鲜肉从经验上看，超过申请人之前所养德国美利奴羊同期水平约1.3、0.8个百分点以及0.12 mg/g鲜肉，口感得到了一定提升，同时体重、羊毛等性状无显著变化。虽然尚未得到完全成年后的数据，但本申请分子标记辅助选择的实践效果也已经较为明确。
序列表
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Claims (10)
1.检测ANGPTL4基因多态性的试剂在羊品种培育中的应用。
2.根据权利要求1所述的应用，其中所述羊为德国美利奴羊。
3.根据权利要求1或2所述的应用，其中ANGPTL4基因多态性为ANGPTL4上的G303A和/或G396T。
4.根据权利要求3所述的应用，其中所述试剂为PCR检测方法所用试剂。
5.根据权利要求4所述的应用，其中所述试剂为序列为SEQ ID NO.3-4的引物和测序用试剂。
6.根据权利要求3-5任一项所述的应用，其中所述应用为选择ANGPTL4 303位置上基因型为GA或AA和396位置上基因型为GT或TT的羊用于品种培育。
7.一种用于分子标记辅助羊品种培育的试剂盒，包括检测ANGPTL4基因多态性的试剂。
8.根据权利要求7的试剂盒，其中所述ANGPTL4基因多态性为ANGPTL4上的G303A和/或G396T。
9.根据权利要求8的试剂盒，其中所述试剂为PCR检测方法所用试剂。
10.根据权利要求9的试剂盒，其中所述试剂为序列为SEQ ID NO. 3-4的引物和测序用试剂。
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