CN109652557B - 一种与猪腹股沟阴囊疝发生相关的分子标记及应用 - Google Patents
一种与猪腹股沟阴囊疝发生相关的分子标记及应用 Download PDFInfo
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Abstract
本发明属于猪的遗传标记制备技术领域，具体公开了一种与猪腹股沟/阴囊疝性状相关的分子标记及应用，该标记从猪PRKCE基因中筛选得到，在SEQ ID NO.1所示序列的230位碱基处发生一个C/T碱基突变,导致Taq α I‑RFLP多态性，利用该SNP位点设计引物，分析该SNP标记与腹股沟/阴囊疝的相关性，发现了猪PRKCE基因第十内含子区内的SNP突变位点与猪腹股沟/阴囊疝性状显著相关。本发明为猪标记辅助选择提供了新的标记资源。
Description
技术领域
本发明属于猪的标记辅助选择技术领域，具体涉及一种与猪腹股沟阴囊疝发生相关的分子标记及应用，该分子标记分离自猪PRKCE基因的第十内含子区域。
背景技术
腹股沟阴囊疝是在哺乳动物中普遍存在的一种复杂性疾病，主要表现为腹腔脏器(肠管或网膜)或腹水渗入腹股沟或阴囊内，造成局部膨大凸起。有研究表明，腹股沟/阴囊疝的发生是遗传和环境因素共同作用的结果，遗传力由于统计种群的不同而表现出较大变异，一般在0.2-0.6(Gatphayak,et al.Present Situation of PorcineHemiainguinalis/scrotalis in Thailand.The Global Food&Product Chain—Dynamics,Innovations,Conficts,Strategies.DeutscherTropentag.October 2005.),在纯种闭锁繁殖群中较多发，是一种典型的遗传缺陷病。该疾病会引起猪只长期食欲不振，消化不良，生长缓慢，影响猪只的使用性能，严重时可发生内毒素性休克而造成猪只死亡,给养殖户造成较大的经济损失。猪腹股沟/阴囊疝是由多基因控制的复杂遗传缺陷疾病，猪群的品种、饲养条件和来源差异以及人工表型选择干预均会使疝气的发病率表现出较大差异。单纯地依靠常规的表型选择只能小规模地降低患病率，而且需要很长的世代间隔。而以RCR-RFLP、微卫星标记和基因芯片等作为主要研究方法的标记辅助选择(MAS)技术可以很好地结合了分子抗病与育种，使常规育种获得了快速进展。
酶切扩增多态性序列(Cleaved Amplified Polymorphism Sequences，CAPS)标记又称为PCR-RFLP(PCR-restriction fragment length polymorphism)技术，是PCR技术与RFLP技术结合的一种方法。当SNP正好处于限制性内切酶位点上时，可将它转化为CAPS标记，并通过酶切片段大小的差异来检查PCR产物的多态性。由于内切酶的高度专一性，对待测的特定片段的针对性很强，只要酶切位点存在突变，在酶切条件合适的情况下就能检测出来，确认其是否发生了突变，受其他外界干扰因数影响较少，结果稳定，重复性好。作为一种分析速度快，成本低廉，信息量大的分子标记，PCR-RFLP技术已经广泛地应用于复杂疾病的鉴定，诊断及防治中。
腹股沟/阴囊疝作为一种常见的外科病，已经被研究了相当长的时间，研究者也尝试从解剖学、鞘状突未闭合及腹内压增高等不同的角度来解释其发病的原因，但目前为止，其发生的确切原因还未能找到。近年，随着生命科学技术的迅速发展，不少学者从生物化学的角度提出，腹股沟阴囊疝的发生主要由于睾丸下降异常和胶原代谢紊乱两个方面。其中在睾丸下降的过程中，引带平滑肌细胞未凋亡致使鞘状突未闭合萎缩，由此推测细胞凋亡在疝气的形成过程中发挥重要作用。蛋白激酶C-ε(protein kinase C-ε,PKCε)是一类由单一多肽链组成的磷脂和钙离子依赖性激酶，属于丝氨酸/苏氨酸激酶家族，是细胞信号传导的重要组成成分。大量研究发现，PKCε涉及细胞凋亡的调控。Knauf J等将部分缺失的PKCε导入PCCL3细胞中，发现细胞凋亡水平下降，提高了细胞对凋亡的抗性(1999)。Jones等发现用胆盐处理肝细胞后，PKCα、PKCδ和PKCε发生胞液到胞膜的易位，促进了肝细胞的凋亡(1997)。此外，Kajstura发现在平滑肌细胞中，血管紧张素能引起PKCδ、PKCε的膜易位，促使胞液内Ca2+水平增高，增加肌细胞凋亡(1997)。这些研究结果表明，PRKCE基因可以通过影响平滑肌细胞凋亡而促进阴囊疝的发生。因此，在猪育种进程中可以利用这些研究结果进行分子标记辅助选择，这将对猪腹股沟/阴囊疝病的个体筛选奠定良好的分子基础，为进一步解析该病发生的分子机理提供依据。本申请克隆了大白猪等PRKCE基因第十内含子区域的序列，并鉴定出一个SNP位点。通过关联分析发现，该位点与猪腹股沟/阴囊疝性状显著相关(P<0.05)。推测该SNP位点有可能与猪腹股沟阴囊疝病具有一定的关联性。
发明内容
本发明的目的在于提供了一种与猪腹股沟阴囊疝发生相关的分子标记，该分子标记位于猪PRKCE基因的第十内含子区，具体为SEQ ID NO.1所示序列的第230个碱基突变，SNPID为rs328681942。
本发明的另一个目的在于提供了一种与猪腹股沟阴囊疝发生相关的分子标记的应用，利用检测SEQ ID NO.1所示序列的第230个碱基，可用于猪育种筛选。
为了达到上述目的，本发明采取以下技术措施：
申请人通过克隆猪PRKCE基因的第十内含子序列，经关联分析，得到一个与猪腹股沟阴囊疝性状显著相关的遗传标记，从而为猪的腹股沟阴囊疝性状建立新的标记辅助选择位点。
以上所述的遗传标记，位于PRKCE基因的第十内含子序列内，SNP ID为rs328681942，具体为下述序列的第230位碱基，以下序列中的R为C或T，该SNP突变导致TaqαI-RFLP多态性；
ACCCTCAACGCTCTGTCCAGATCATTCCCTGTAACATTCTTGTTCTGTTACATCAAACTTCCAGGCTTAGCCATGCTTTATAATGATAAACAAAGCTCTACCGACTCCAAACATTGGATTGACAAAAATAGACAGTTTGTTTTGATTTGCTTTTGAAATGCTGATTTCCTTTTTGAATTTTCCTCCTCTTCATTGCAGGAACTGCCAATGTCTTCAAGTCTGGGGACCTR(C/T)GATCTTACTCATCAACATTTGCAAGGCCAGCATGGCTCCTGGCTTTTAGGTCAGATTCAGAAAGTGTCTTTCAAGTGCCATTTTTTTCTTCCAGTTCCTCAGGCATCCTATCTGCTGTTCACAGGCATTTTAGTGTGGTAGTAAGGTCACCGCCTTGGTTTCAATCCCATCTCCATTTTAGCTGGCTGTGTGATTTTGGTTAAGCGGCCTA。
一种与猪腹股沟阴囊疝发生相关的分子标记的应用，包括用于检测SNP ID为rs328681942的碱基的试剂在猪育种或猪腹股沟阴囊疝筛选中的应用；包含有SNP ID为rs328681942的碱基序列的试剂在猪育种或猪腹股沟阴囊疝筛选中的应用；利用包含有SNPID为rs328681942的碱基突变的基因序列在制备猪育种筛选药物或是猪腹股沟阴囊疝筛选药物中的应用；或是针对SNP ID为rs328681942的碱基突变设计的引物在制备猪育种筛选药物或猪腹股沟阴囊疝性状筛选药物中的应用。
以上所述的应用中，优选的，是利用下述引物
正向引物F：5'-ACCCTCAACGCTCTGTCC-3'
反向引物R：5'-TAGGCCGCTTAACCAAAA-3'
对猪基因组DNA中进行PCR扩增，将所得的PCR扩增片段用限制性内切酶TaqαI酶切分型，得到TaqαI-RFLP多态性核苷酸序列；CT基因型，221bp，229bp，441bp；CC基因型，212bp，229bp；TT基因型，441bp。
与现有技术相比，本发明具有以下优点：
申请人首次在大白猪，长白猪PRKCE基因序列中发现一个SNP位点(SNP ID:rs328681942)与猪腹股沟阴囊疝性状显著相关，为大白猪，长白猪的抗病育种相关的筛选工作提供了新方法。
附图说明
图1为针对PRKCE基因中的SNP ID为rs328681942的TaqαI-RFLP的检测结果示意图；
琼脂糖凝胶浓度为2﹪，图中泳道：M为DL2000Maker；泳道1为CT基因型，221bp，229bp，441bp；泳道2为CC基因型，212bp，229bp；泳道3和泳道4为TT基因型，441bp。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规技术；所述试剂或材料，如未特别说明，均来源于商业渠道。
实施例1：
猪PRKCE基因片段的获得及SNP检测方法的建立
试验选择外国血缘猪种大白猪，长白猪和二者的杂交猪种，根据猪PRKCE基因组序列(登陆号Gene ID：100524932)设计如下引物对，具体序列如下：
正向引物F：5'-ACCCTCAACGCTCTGTCC-3'
反向引物R：5'-TAGGCCGCTTAACCAAAA-3'
利用上述引物对在大白猪，长白猪和杂交猪基因组DNA中进行PCR扩增。
PCR反应体系见表1所示。
表1 PCR反应体系
PCR反应条件见表2所示。
表2 PCR反应条件
将所得的PCR产物进行纯化和克隆后进行序列测定，测序工作由北京奥科鼎盛生物生物科技有限公司完成。经blast比对分析，发现在该序列中第230位碱基处存在一个C/T碱基突变(SNP ID:rs328681942)，该突变引起限制性内切酶TaqαI酶切位点多态性。
取10ul PCR产物加入0.5ul限制性内切酶TaqαI(20U/ul)、2ul 10×buffer和7.5ul ddH2O，37℃酶切2小时，酶切产物经2％的琼脂糖凝胶电泳检测，在凝胶成像系统下观察并记录酶切结果。当此突变处碱基都为C时存在酶切位点，酶切结果检测到两条带记为CC基因型(212bp+229bp)；当此突变处碱基都为T时不识别该位点，酶切结果检测到一条带记为TT基因型(441bp)；当C和T都存在时，酶切结果为三条带记为CT基因型(212bp+229bp+441bp)。结果如图1所示。
实施例2：
本发明的遗传标记在不同猪群中的多态性分布检测验证
为了确定猪PRKCE基因第十内含子区域内的SNP与猪表型差异是否相关，选择法系大白猪(109头)、法系长白猪(53头)、美系大白猪(39头)和杂交猪(66头)为试验材料，样本采集及其数据收集于浙江开盛生态农业发展有限公司。采用常规TaqαI-RFLP方法进行多态性检测，并分析该SNP位点不同基因型与猪腹股沟阴囊疝性状的相关性。根据基因型频率和基因频率进行独立性检验(X2)。
表3 PRKCE基因TaqαI-RFLP基因型频率和等位基因频率在腹股沟/阴囊疝患病个体与正常个体中的分布比较
从表3中可以看出PRKCE基因的rs328681942位点多态性与猪腹股沟阴囊疝存在显著的关联性。其中，在总群体、法系大白猪和杂交猪群体中，PRKCE基因该位点的基因型和等位基因频率在患病、正常两组中的分布是具有显著差异的(P<0.05)。在患病群体中，等位基因T的频率显著高于正常个体，且在两个群体中的差异达到极显著水平(P<0.01)。但是在法系群体以及法系分群体中进行验证，发现其分布的显著性有所降低。说明与腹股沟阴囊疝病因相关的基因在不同群体中具有不同的表现，在大白猪中(包括美系大白猪和法系大白猪)，CC基因型对于腹股沟/阴囊疝的发生具有明显优势，为易患病基因型。
实施例3：
rs328681942位点在大白猪育种中的应用：
对某猪场中的腹股沟阴囊疝病阳性美系大白猪和腹股沟阴囊疝病阴性美系大白猪各50头，进行分子标记的检测，利用5'-ACCCTCAACGCTCTGTCC-3'和R：5'-TAGGCCGCTTAACCAAAA-3'对其DNA扩增后进行限制性内切酶TaqαI酶切，结果显示，共检测到50头基因型为CC的样本，均为患病猪，进一步证实了CC位点可作为大白猪腹股沟阴囊疝性状的潜在遗传标记。
序列表
<110> 华中农业大学
<120> 一种与猪腹股沟阴囊疝发生相关的分子标记及应用
<130> 一种与猪腹股沟阴囊疝发生相关的分子标记及应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 441
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
accctcaacg ctctgtccag atcattccct gtaacattct tgttctgtta catcaaactt 60
ccaggcttag ccatgcttta taatgataaa caaagctcta ccgactccaa acattggatt 120
gacaaaaata gacagtttgt tttgatttgc ttttgaaatg ctgatttcct ttttgaattt 180
tcctcctctt cattgcagga actgccaatg tcttcaagtc tggggacctr gatcttactc 240
atcaacattt gcaaggccag catggctcct ggcttttagg tcagattcag aaagtgtctt 300
tcaagtgcca tttttttctt ccagttcctc aggcatccta tctgctgttc acaggcattt 360
tagtgtggta gtaaggtcac cgccttggtt tcaatcccat ctccatttta gctggctgtg 420
tgattttggt taagcggcct a 441
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
accctcaacg ctctgtcc 18
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
taggccgctt aaccaaaa 18
Claims (2)
1.检测猪SNP ID为rs328681942的基因型的试剂在制备猪腹股沟阴囊疝性状筛选试剂中的应用，所述的猪为法系大白猪或美系大白猪，具有CC基因型的猪为易患病个体。
2.根据权利要求1所述的应用，所述的试剂包括引物，所述的引物为：正向引物F：5'-ACCCTCAACGCTCTGTCC-3'和反向引物R：5'-TAGGCCGCTTAACCAAAA-3'。
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| WO1996039538A1 (en) * | 1995-06-06 | 1996-12-12 | Dekalb Swine Breeders | Evaluating scrotal hernia in swine |
| EP1630236A2 (de) * | 2004-08-30 | 2006-03-01 | FBF- Förderverein Biologieforschung der Deutschen Schweineproduktion e.V. | Hernia inguinalis/scrotalis assozieerte Genomregionen |
| CN105671048A (zh) * | 2016-03-14 | 2016-06-15 | 江苏大学 | 干扰PRKCE基因表达的siRNA序列及其用途 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1996039538A1 (en) * | 1995-06-06 | 1996-12-12 | Dekalb Swine Breeders | Evaluating scrotal hernia in swine |
| EP1630236A2 (de) * | 2004-08-30 | 2006-03-01 | FBF- Förderverein Biologieforschung der Deutschen Schweineproduktion e.V. | Hernia inguinalis/scrotalis assozieerte Genomregionen |
| CN105671048A (zh) * | 2016-03-14 | 2016-06-15 | 江苏大学 | 干扰PRKCE基因表达的siRNA序列及其用途 |
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| Title |
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| "dbSNP build 138 rs328681942";WU_ABGC;《UCSC》;20140128;dbSNP至Submitter Handles * |
| "Genome-wide association study for conformation traits in three Danish pig breeds";Thu H. Le et al.;《Genetics Selection Evolution》;20170124;第49卷;摘要，第1页左栏第2-3段、第4页右栏倒数第1段至第7页左栏第1段 * |
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