CN105950638B - TrkA基因作为绵羊产羔数性状的分子标记及其应用 - Google Patents
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Abstract
本发明属于绵羊分子标记筛选与应用技术领域，具体涉及TrkA基因作为绵羊产羔数性状的分子标记及其应用。本发明的分子标记克隆自TrkA基因(Gene Bank ID:101109763)。该分子标记的核苷酸序列如SEQ ID NO：1所述，在SEQ ID NO.1所示序列的的432位碱基处存在一个C>T的碱基替换突变，该突变导致SacⅡ‑RFLP酶切多态性。在湖羊和小尾寒羊进行相关的关联分析应用。本发明为绵羊产羔数性状的标记辅助选择提供了一个新的遗传标记资源。
Description
技术领域
本发明属于绵羊分子标记筛选技术领域，具体涉及TrkA基因作为绵羊产羔性状的分子标记及其应用。本发明的分子标记克隆自绵羊TrkA基因。
背景技术
传统育种方法中，对家畜性状选择的方法多依赖于家畜的表型，这种方法在大家畜选育中需要丰富的经验和长达几十年时间。对于一些由微效多基因控制的低遗传力的数量性状，例如绵羊产羔性状，不仅由母羊自身遗传因素决定，且受到不同环境和饲养条件的影响，常规育种方法遗传改良进展缓慢。形态学标记、细胞学标记、生物化学标记以及免疫学标记虽然已经在优良种畜选育中进行应用，但由于这些标记的多态性低和信息量小，且是对基因的间接反映，受到基因与环境互作的影响比较大，在生产实践中有很大的局限性。随着分子生物学技术的发展，尤其是分子遗传标记技术的发展和完善，利用与目标性状紧密连锁的分子遗传标记，对目标性状进行跟踪性选择，减少育种过程中选择的盲目性，并可以进行早期选育，极大提高育种效率。
TrkA(Tyrosine Kinase A)是由原癌基因trk编码的酪氨酸蛋白激酶受体，神经生长因子(NGF)以二聚体的形式与其相结合，诱导TrkA酪氨酸残基处磷酸化，通过激活磷脂酶Cγ(PLCγ)→磷脂酶肌醇激酶(PI3K)→衔接蛋白(Shc)信号通路将胞外信号传递到细胞核(马永和，2010)。TrkA不仅介导NGF对神经系统中胆碱能神经元的分化和发育的调控(Counts S等，2005)，而且在生殖系统也发挥功能，已经发现在人、鼠、金仓鼠、绵羊和山羊等动物卵泡均有TrkA基因表达(马永和等，2010)。不同发育阶段卵泡中TrkA表达丰度不同，猪的原始卵泡和初级卵泡的卵泡细胞和卵母细胞不表达TrkA，但在二级卵泡和三级卵泡的卵泡细胞及卵母细胞中有大量表达(Levanti MB等，2005)。NGF与TrkA参与早期卵泡的装配、颗粒细胞的增殖、卵母细胞的成熟及排卵等过程(Dissen GA等，2001)。在体外，NGF还可以促进牛腔前卵泡的发育(李海等，2005)和猪卵母细胞成熟(陆晨等，2013)。NGF-TrkA复合物对输卵管和子宫发育亦发挥重要作用，TrkA在子宫内膜细胞、腺细胞、基质细胞、输卵管黏膜上皮细胞均有表达，且卵泡期，输卵管中表达量显著高于卵巢(马永和等，2010)。
现有文献表明，TrkA基因参与了动物繁殖调控过程，尤其是卵子发生和卵泡发育。因此，寻找该基因中变异位点，通过与产羔性状间的关联分析，是进行分子遗传标记辅助选择的基础。此发明开展了绵羊TrkA基因部分基因组序列克隆和SNP筛查、检测及与产羔数性状关联分析，以提高繁殖力母羊选择的准确性，可望绵羊加快育种进程。
发明内容
本发明的目的在于克服现有技术的缺陷，特别是现有绵羊育种周期长、选择准确性低、无法进行早期选育等技术缺陷，提供一种提高绵羊产羔数性状关联的分子标记作为辅助选择的手段。本发明的核心在于通过PCR-RFLP技术对候选个体的TrkA基因部分序列进行基因型分析，为绵羊产羔数性状检测提供一种辅助选择的遗传标记。
本发明的技术方案如下所述：
申请人筛选得到一种绵羊产羔性状的分子标记，其核苷酸序列如下所述：
CTCCCGACTCTCACCGCCACCCCCTCCGGCCCACAGTCCCGGCCAGCGTGCATCTGCAGCCCGCCGTGGAGCAGCACCACTGGTGCATCCCCTTCTCGGTGGACGGGCAGCCGGCGCCCTCTCTGCGCTGGCTCTTCAATGGCTCCGCGCTCAACGAGACCAGCTTCATCTTCACCGAGTTCCTGGAGCTGGCGGCCAACGAGACGGTGCGCCACGGCTGCCTGCGCCTCAACCAGCCCACCCACGTCAACAACGGCAACTACACGCTGCTGGCGACCAACCCGCTGGGCCGGGCCGCCGCTTGGGTCATGGCTGCCTTCATGGACAACCCTTTCGAATTCAACCCTGAGGACCCCATTCCTGGTGTGAGGGCCACCCTGAACCCTGCTCCCGCTCCCTGGGCCCCAGCCGGGTGCAGATCCAGGTATCCGY(C/T)GGAGGCCTGGCCCACTTCCCACTGAGATCCTGACCCTGGAACCTGGAGGGCCTGGTCCCAGATGGAAAGTAGCCTGGAGTTCTGGTGTCCAGCTCTGTGCTGGCTTCCTCG，
在该序列的432碱基位处的Y是C或T，该突变导致RFLP-SacⅡ多态性。
申请人得到一种扩增TrkA基因片段或检测所述分子标记SNP的引物对，该引物对的DNA序列如下所示：
正向引物F：CTCCCGACTCTCACCGCC，
反向引物R：CGAGGAAGCCAGCACAGA。
申请人提供了一种筛选绵羊产羔性状分子标记的方法，所述的方法包括以下步骤：
从绵羊外周血液全血中提取基因组DNA，在位于Gene Bank登陆号ID:101109763所述的绵羊TrkA基因序列的突变位点附近设计一对引物，该引物对的DNA序列如SEQ ID NO：2和SEQ ID NO：3所示，对绵羊基因组DNA进行PCR扩增和序列测定，得到如SEQ ID NO：1所示的核苷酸序列，通过序列比对，筛查SNP，再利用SEQ ID NO：2和SEQ ID NO：3所示的引物对进行PCR扩增，将扩增获得片段利用限制性内切酶SacⅡ进行酶切分型及检测。
本发明的具体步骤如下：
采集湖羊和小尾寒羊外周血液，提取基因组DNA；设计了一种扩增上述TrkA基因(Gene Bank ID:101109763)片段和用于检测该基因片段的分子标记的引物对，该引物对DNA序列如下所示：
正向引物F：CTCCCGACTCTCACCGCC，
反向引物R：CGAGGAAGCCAGCACAGA。
申请人应用上述所得的引物对克隆得到绵羊(品种为湖羊和小尾寒羊)TrkA基因片段，其核苷酸序列如SEQ ID NO：1所述，在该序列的432位碱基处发生一个等位基因突变，即碱基C与碱基T替换，该突变导致PCR-SacⅡ-RFLP多态性。
更为详细的发明方案见《具体实施方式》所述。
本发明筛选的分子标记可在绵羊产羔性状辅助选择中应用。
附图说明
序列表SEQ ID NO：1是绵羊产羔性状相关的分子标记的核苷酸序列(也是扩增TrkA基因寻找SNP突变位点的部分核苷酸序列)，序列长度为543bp，在该序列的第432位碱基处存在一个等位基因突变(C>T)。
序列表SEQ ID NO：2和SEQ ID NO：3是扩增SEQ ID NO：1所示序列引物对的DNA序列，也是实现绵羊C>T突变的PCR-ScaⅡ-RFLP基因型分型方法的引物对的序列。
图1：是绵羊TrkA基因(如SEQ ID NO：1所述的序列)部分测序结果和SNP位点。
图2：是绵羊TrkA基因(如SEQ ID NO：1所述的序列)PCR-ScaⅡ-RFLP检测结果。琼脂糖浓度为1％。附图标记说明：泳道M为DL2000DNA Marker(包含100bp、250bp、500bp、750bp、1000bp和2000bp共6种带型)，其中：TT基因型片段大小为543bp，CC基因型片段大小为432bp，CT基因型片段大小分别为543bp和432bp。
图3：是本发明筛选的与绵羊产羔性状相关的分子标记的核苷酸序列。该序列的432碱基位处的Y是C或T，该突变导致RFLP-SacⅡ多态性。
具体实施方式
以下实施例用于对本发明作进一步说明，但不理解为对本发明的限制。在不背离本发明精神和实质的前提下，对本发明所做的修饰或替换，均属于本发明保护的范围。
1、血样采集
采集具有头胎产羔记录的绵羊(品种为湖羊和小尾寒羊)外周血液样品，血样采自甘肃省金昌中天羊业有限公司。
2、基因组DNA提取
采用传统DNA抽提方法(即酚/氯仿法)提取所采集绵羊血样中基因组DNA。具体步骤如下：
(1)取1mL采集的绵羊全血加入等体积的PBS缓冲液，温和震荡10min；
(2)4℃下，12000g离心10min后，弃上清；
(3)重复步骤(1)和步骤(2)至上清液透明，沉淀变为白色；
(4)加入700μL DNA抽提液(1M Tris-Cl(pH＝8.0)25mL、0.5M EDTA(pH＝8.0)100mL、10％SDS 50mL、2M NaCl 25mL，定容至500mL，常规高压灭菌)，悬浮沉淀物，并于37℃水浴1h；
(5)加入3μL蛋白酶K(20mg/mL)溶液，混匀，55℃水浴消化过夜，至细胞沉淀被全部消化；
(6)冷却步骤(5)溶液至室温，加入等体积的Tris饱和酚溶液，冰水浴并温和摇动15min；
(7)4℃下，12000g离心10min，将上清液转移到另一干净离心管中；
(8)加入0.5倍体积的饱和酚溶液和0.5倍体积的氯仿，冰水浴并温和摇动15min；
(9)4℃下，12000g离心10min，将上清液转移到另一干净离心管中；
(10)加入等体积的氯仿，冰水浴并温和震荡15min；
(11)4℃下，12000g离心10min，将上清液转移到另一干净离心管中；
(12)加入2倍体积的预冷无水乙醇(-20℃)，颠倒混匀多次至DNA沉淀析出，然后-20℃下静置30min；
(13)4℃下，12000g离心10min，弃上清；
(14)加入1mL 70％的乙醇，温和震荡10min；
(15)4℃下，12000g离心10min，弃上清；
(16)重复步骤(14)和(15)一次；
(17)待乙醇挥发干净后加入200μL超纯水溶解DNA，并检测质量。
3、引物设计
根据绵羊TrkA基因(Gene Bank登陆号ID:101109763)突变位点附近序列(SEQ IDNO：1)，利用Oligo 7软件设计一对特异性引物，所述的引物的DNA序列如下所示：
扩增SEQ ID NO：1的正向引物F：CTCCCGACTCTCACCGCC，
扩增SEQ ID NO:1的反向引物R：CGAGGAAGCCAGCACAGA。
4、PCR程序
采用PCR方法，以湖羊和小尾寒羊的基因组DNA为模板扩增目的片段。
PCR扩增反应总体系为25μL：其中DNA模板(50ng/μL)1μL，2×Easy Taq PCR SuperMix 12.5μL(TranGen)，上述制备的正向引物0.4μL(10μM)，反向引物0.4μL(10μM)，ddH2O10.7μL。
PCR扩增反应条件：94℃预变性5min；94℃变性30s，61℃退火30s，72℃延伸30s，35个循环后；72℃延伸5min，4℃保存。
对扩增得到PCR产物利用1％琼脂糖凝胶进行电泳检测。将不同绵羊个体得到的长度为543bp PCR产物混合测序，得到如SEQ ID NO：1所述的核苷酸序列，其测序峰图部分结果见图1。位于该序列的432位碱基处的C>T突变引起了SacⅡ酶切位点(CCG[C/T]↓GG)多态性。
5、限制性酶切片段多态性(RFLP)检测
采用SacⅡ-RFLP方法对绵羊TrkA基因SNP位点进行基因分型，酶切反应体系为10μL：其中PCR产物4μL，限制性内切酶(SacⅡ)0.5μL，10×buffer 1μL，用灭菌水补足10μL，样品混匀后离心，37℃孵育40min。
取5μL酶切产物用1％琼脂糖凝胶电泳，进行基因分型和鉴定，结果如图2所示。此扩增片段大小为543bp，该酶切多态位点位于该片段的432位碱基处，当该变异位点为C碱基时，会产生限制性内切酶SacⅡ的酶切位点(CCGC↓GG)，SacⅡ酶切PCR产物后会产生2个大小为432bp和111bp的条带，鉴定基因型为CC型；当该位点为T碱基时，则不能产生SacⅡ的限制酶酶切位点，SacⅡ酶切PCR产物后任然为一条543bp的条带，鉴定基因型为TT型；当该位点处为C/T杂合型时，由于同时存在C碱基和T碱基，则SacⅡ酶切PCR产物后产生3条大小分别为543bp、432bp和111bp的条带，鉴定基因型为CT型。由于1％琼脂糖凝胶对111bp大小条带分离效果不明显，故图2中为显示111bp的条带。
6、绵羊TrkA基因片段在产羔数性状关联分析中的应用
本试验共检测了963只绵羊，包括526只湖羊和437只小尾寒羊的多态性，确定其基因型后，进行了基因型与产羔数性状之间的关联性分析。利用SPSS 19.0软件中的广义线性模型(General Linear Model，GLM)程序，并采用方差分析的最小二乘分析法进行分析的。
具体的线性模型如下：
Yijk＝μ+Genotypei+Combinationj+εijk
其中，Yijk是性状观察值，μ为总体均数，Genotypei为基因型效应，Combinationj为组合的效应，εijk为随机误差，假定εij相互独立，服从N(0，σ2)分布。
基因型检测结果见表1.
表1TrkA基因不同基因型与产羔数的相关性
表1的说明：具有相同字母肩标的平均值间差异不显著(P>0.05)，具有不同字母肩标的平均值间差异显著(P<0.05)。
表1数据说明在963只绵羊个体中CC基因型有156只(其中湖羊98只，小尾寒羊58只)，CT型有541只(其中湖羊287只，小尾寒羊254只)，TT型有266只(其中湖羊141只，小尾寒羊125只)。基因型与产羔数性状分析结果见表1，绵羊TrkA基因多态性与产羔数性状呈显著相关(p<0.05)，在湖羊中，TT基因型(2.57±0.08)和CT基因型(2.38±0.06)产羔数显著高于CC基因型(1.67±0.10)。在小尾寒羊中，CT基因型(2.11±0.05)和TT基因型(2.21±0.07)显著高于CC基因型(1.83±0.11)。
主要参考文献：
李海,周虚,安铁洙.神经生长因子对牛腔前卵泡体外培养的影响.中国兽医科技,2005,35:139-142.
陆晨,李万宏,陈树雄,吕岩,周虚.神经生长因子对猪卵丘细胞扩散及卵母细胞体外成熟的影响.吉林农业大学学报,2013,35:351-354.
马永和.神经营养因子及其受体在母牛神经系统中的表达与功能研究.博士论文,2010,吉林大学,长春.
马永和,孙永峰,李纯锦,周虚.神经生长因子受体TrkA在母牛性腺及性腺外生殖器官中的表达研究.吉林农业大学学报,2010,32:330-334.
Counts S,Mufson EJ.The role of nerve growth factor receptors incholinergic basal forebrain degeneration in prodromal Alzheimerdisease.Neuropathol Exp Neurol,2005,64:263-272.
Dissen GA,Romero C,Hirshfield AN,Ojeda SR.Nerve growth factor isrequired for early follicular development in the mammalianovary.Endocrinology,2001,142:2078-2086.
Levanti MB,Germana A,Montalbano G,Vega JA,Germana G.TrkA and p75NTRin the ovary of adult cow and pig.Journal of Anatomy,2005,207:93-96。
Claims (2)
1.一种绵羊产羔数性状的分子标记，其核苷酸序列如下所述：
CTCCCGACTCTCACCGCCACCCCCTCCGGCCCACAGTCCCGGCCAGCGTGCATCTGCAGCCCGCCGTGGAGCAGCACCACTGGTGCATCCCCTTCTCGGTGGACGGGCAGCCGGCGCCCTCTCTGCGCTGGCTCTTCAATGGCTCCGCGCTCAACGAGACCAGCTTCATCTTCACCGAGTTCCTGGAGCTGGCGGCCAACGAGACGGTGCGCCACGGCTGCCTGCGCCTCAACCAGCCCACCCACGTCAACAACGGCAACTACACGCTGCTGGCGACCAACCCGCTGGGCCGGGCCGCCGCTTGGGTCATGGCTGCCTTCATGGACAACCCTTTCGAATTCAACCCTGAGGACCCCATTCCTGGTGTGAGGGCCACCCTGAACCCTGCTCCCGCTCCCTGGGCCCCAGCCGGGTGCAGATCCAGGTATCCGY(C/T)GGAGGCCTGGCCCACTTCCCACTGAGATCCTGACCCTGGAACCTGGAGGGCCTGGTCCCAGATGGAAAGTAGCCTGGAGTTCTGGTGTCCAGCTCTGTGCTGGCTTCCTCG，
该序列的432碱基位处的Y是C或T，该突变导致RFLP-SacⅡ多态性。
2.权利要求1所述的分子标记在绵羊产羔数性状辅助选择中的应用。
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