CN114107517B - 一种与猪流感病毒抗性相关的snp标记及其应用 - Google Patents
一种与猪流感病毒抗性相关的snp标记及其应用 Download PDFInfo
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Abstract
本发明公开了一种与猪流感病毒抗性相关的SNP标记及其应用，本发明属于分子生物学技术领域。本发明所述SNP标记为猪MAN2A1基因中第481位碱基发生A/T碱基突变，所述猪MAN2A1基因的核苷酸序列的GeneBank登录号为：Gene ID:100514631，所述的猪流感病毒包括猪流感病毒H1N1和猪流感病毒H3N2。本发明提出了鉴定出猪流感病毒抗性相关的SNP分子标记，为猪流感抗性选育提供可利用分子标记资源。
Description
技术领域
本发明属于分子生物学技术领域，具体涉及一种与猪流感病毒抗性相关的SNP标记及其应用。
背景技术
猪流感病毒(Swine influenza virus,SIV)属于正黏液病毒，可以感染各个年龄、性别和品种的猪，引起发烧、呼吸困难、咳嗽、厌食和消瘦等症状，进而影响患病猪群生产和繁殖性能，给养猪业造成严重的经济损失。并且，该病毒具有传染人的潜能，对公共卫生及人类健康亦造成重大威胁。因此，筛选猪流感病毒抗性基因及分子遗传标记进而实施分子选育，从遗传本质上提高生猪自身免疫力与疾病抗性，对于从根本上防控猪流感的发生、助力我国养猪产业的高质量发展具有重要意义。
MAN2A1(mannosidase alpha class 2A member 1)为甘露糖苷酶2A类成员1基因，可将高甘露糖转化为N-聚糖的复杂类型结构以使膜蛋白成熟糖基化所需的高尔基酶，并在病毒感染及免疫调节过程中具有重要作用。前期研究发现，猪流感病毒H1N1和H3N2侵染猪肺泡巨噬细胞3D4/21，MAN2A1基因表达水平显著下调，表明其可能参与调控猪流感病毒对3D4/21细胞的感染及宿主细胞免疫应答反应。
单核苷酸多态性(single nucleotide polymorphism，SNP)是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。SNP具有遗传稳定性和易检测等特性，可作为遗传标记应用于基因定位、克隆、遗传育种及多样性研究。分子标记辅助选择育种是利用DNA分子标记对育种材料进行选择，综合改良畜禽重要经济性状，其中SNP标记应用最为广泛。分子育种为家畜育种开辟了一条全新的途径，随着现代分子生物技术的发展，分子标记在家畜育种中广泛应用，显著提升家畜重要经济性状和抗病性能的遗传进展，极大地促进了现代畜牧业的发展。
技术方案
发明目的：本发明要解决的技术问题是提供一种猪流感病毒抗性相关的SNP标记。
本发明还要解决的技术问题是提供上述猪流感病毒抗性相关的SNP标记在大白猪选育中的应用。
技术方案：为解决上述技术问题，本发明提供如下技术方案：
一种与猪流感病毒抗性相关的SNP标记，所述SNP标记为猪MAN2A1基因中第481位碱基发生A/T碱基突变，所述猪MAN2A1基因的核苷酸序列如SEQ ID NO.：1所示。
其中，所述的猪流感病毒包括猪流感病毒H1N1和猪流感病毒H3N2。
一种猪流感病毒抗性检测引物，所述引物的核苷酸序列如SEQ ID NO.：1～2所示。
一种猪流感病毒抗性检测试剂盒，该试剂盒中包含SEQ ID NO.：1～2所示引物。
上述与猪流感病毒抗性相关的SNP标记在大白猪选育中的应用在本发明的保护范围之内。
进一步地，以大白猪基因组DNA为模板，以SEQ ID NO.：1～2所示序列为引物，通过PCR扩增得到待测核苷酸片段，再通过测序鉴定该SNP位点是否发生突变；
所述的PCR扩增，其PCR反应体系为20μl：50ng/μl模板DNA 1μl、浓度为10μM的SEQID NO.：2～3引物各1μl，PCR mix 10μl，双蒸水7μl；
PCR反应条件为：95℃3分钟；95℃15秒，60℃15秒，72℃15秒，30个循环；72℃5分钟。
有益效果：
(1)鉴定出猪流感病毒抗性相关的SNP分子标记，为猪流感抗性选育提供可利用分子标记资源；
(2)设计了猪MAN2A1基因干扰核苷酸片段，并构建MAN2A1基因沉默的细胞系；
(3)提供了猪流感病毒及上述SNP分子标记PCR检测试剂盒。
附图说明
图1为PCR琼脂糖凝胶电泳检测图；其中，1、2、3、4、5代表不同检测个体；M：DNAmarker I。
图2为SNP标记测序峰图；其中，上图为野生型序列，下图为突变型序列。
图3为SNP标记对MAN2A1基因启动子转录活性影响分析；其中，A/T表示突变型序列；WT表示野生型序列；pGL3-basic表示空载体。不同大写字母表示差异显著(P<0.01)。
图4为MAN2A1基因shRNA干扰效率检测；其中，sh-MAN2A1表示MAN2A1基因干扰组；sh-NC表示阴性对照组；Blank control表示空白对照。不同大写字母表示差异显著(P<0.01)。
图5为H1N1和H3N2病毒M及NP基因表达量；其中，图A为H1N1病毒，H1N1+shRNA表示添加病毒的MAN2A1基因干扰细胞，H1N1+表示添加病毒的对照细胞；图B为H3N2病毒，H3N2+shRNA表示添加病毒的MAN2A1基因干扰细胞，H3N2+表示添加病毒的对照细胞；不同大写字母表示差异显著(P<0.01)。
具体实施方式
1.1提取待测大白猪耳组织基因组DNA
将来自6个家系共300头大白猪耳组织冻存于-20℃，之后采用组织核酸提取试剂盒提取耳组织基因组DNA，DNA样品质检后4℃保存备用或-20℃长期保存。
1.2扩增含SNP位点的核苷酸片段
根据NCBI(https://www.ncbi.nlm.nih.gov/)数据库收录的猪MAN2A1基因(GeneID:100514631)序列设计引物，包括正向引物F：5’-CGCTTGGAGGAAACTC-3’和反向引物R：5’-GGGAAGGAAATCAGGTC-3’，以1.1中的基因组DNA为模板，扩增出待测SNP所在的核苷酸片段。PCR扩增产物如图1所示，核苷酸如序列NO.1所示。该SNP位点位于PCR扩增片段的100bp，此处为A/T碱基突变，序列如NO.2所示。
其中，PCR反应体系20μl包括：50ng/μl模板DNA 1μl，浓度为10μM的引物F和R各1μl，PCR mix 10μl，双蒸水7μl。
PCR反应条件为：95℃3分钟；95℃15秒，60℃15秒，72℃15秒，30个循环；72℃5分钟。
1.3基因型判定
对PCR产物进行测序，根据测序信息判定个体基因型，基因型可分为野生型(无碱基突变，序列如NO.1所示)、突变型(100bp处存在A/T突变，序列如NO.2所示)。两种基因型的分型结果如图2所示。
1.4上述SNP标记对MAN2A1基因表达的影响
将含有上述SNP标记的突变序列和野生型序列分别连接pGL3-basic质粒，获得表达该SNP标记的突变型重组质粒与野生型重组质粒。将上述两种重组质粒分别转染293T细胞，24小时后检测荧光素酶活性。结果显示，表达SNP标记的突变型重组质粒显著增强MAN2A1基因启动子转录活性(图3)，表明该SNP标记可通过影响启动子转录活性而促进MAN2A1基因的表达。
其中，连接体系10μl包括：pGL3-basic质粒1μl，T4 DNA连接酶1μl，连接缓冲液1μl，DNA片段7μl。反应程序：16℃，过夜连接。
1.5MAN2A1基因对猪流感病毒(H1N1和H3N2)感染的抗性作用
根据猪MAN2A1基因编码区序列设计shRNA干扰序列片段，干扰序列为：5′-GCTCGCTGCTCAGTCCTTAGG-3’。将干扰序列连接pGPU6/GFP/Neo质粒，获得表达MAN2A1 shRNA干扰序列的重组质粒。同时，设计定量qPCR扩增引物，包括正向引物F：5’-GCCAGCTCTCATTGTTGCAA-3’和反向引物R：5’-ACCATCCTCCACAGACTCAC-3’。将shRNA重组质粒转染猪肺泡巨噬细胞，24小时后，收集细胞并使用TRIZOL方法提取细胞RNA，将之反转录为cDNA，以cNDA为模板定量qPCR检测shRNA对MAN2A1基因表达水平的影响。结果如图4所示，shRNA干扰效率为78％，MAN2A1基因表达水平显著下降。
以MOI＝1的猪流感病毒H1N1和H3N2分别侵染MAN2A1基因干扰的猪肺泡巨噬细胞及MAN2A1基因正常表达的对照组猪肺泡巨噬细胞，24小时后收集细胞样品，使用TRIZOL方法提取病毒RNA，将之反转录为cDNA，以cNDA为模板定量qPCR检测H1N1和H3N2病毒M和NP基因表达水平。病毒基因定量qPCR扩增引物，包括M基因：正向引物F：5’-GTGCCGTCGGATGGTAGT-3’和反向引物R：5’-CAGTGATGAACCGCAGGAT-3’；NP基因：正向引物F:5′-CCACAAGAGGGGTCCAGATT-3′，反向引物R:5′-GGAGATTTCGCTGCACTGAG-3′。检测结果显示(图5)，MAN2A1基因表达下降后，细胞中H1N1和H3N2病毒基因表达水平显著下降。
其中，qPCR反应体系20μl包括：cDNA 1μl，上下游引物各1μl，SYBR Green MasterMix 10μl，无酶水7μl。反应程序：95℃30秒，95℃5秒，60℃34秒，40个循环。
1.6上述SNP标记在猪流感病毒抗性大白猪选育中的应用
该SNP可作为分子遗传标记，寻找与其相关或紧密连锁的影响猪流感病毒抗性的基因位点，以对大白猪直接进行基因型选择或标记辅助选择，从而加快流感病毒抗性大白猪新品种的选育。
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 16
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cgcttggagg aaactc 16
<210> 2
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gggaaggaaa tcaggtc 17
<210> 3
<211> 203
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgcttggagg aaactcttgc ccgacccgtg agtccccgct cccggggtgc acgccggcct 60
ggtctcagcg gcggcggcgg cggcggcggc ggcagcagga aggggctcag tcccgggagg 120
cgggggctgt accgcggggg cgggcccggc tgtcccggcg ctaagttgtg cggcccggct 180
cttcccgacc tgatttcctt ccc 203
<210> 4
<211> 203
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgcttggagg aaactcttgc ccgacccgtg agtccccgct cccggggtgc acgccggcct 60
ggtctcagcg gcggcggcgg cggcggcggc ggcagcaggt aggggctcag tcccgggagg 120
cgggggctgt accgcggggg cgggcccggc tgtcccggcg ctaagttgtg cggcccggct 180
cttcccgacc tgatttcctt ccc 203
Claims (1)
1.干扰猪MAN2A1基因表达水平的shRNA在制备抗猪流感病毒感染试剂中的应用，其特征在于，所述shRNA序列为：5'-GCTCGCTGCTCAGTCCTTAGG-3’
所述猪流感病毒为猪流感病毒H1N1和猪流感病毒H3N2。
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| CN109971840A (zh) * | 2019-04-11 | 2019-07-05 | 扬州大学 | 一种用于猪抗病育种的遗传分子标记方法和应用 |
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| KR101554166B1 (ko) * | 2013-11-13 | 2015-09-22 | 건국대학교 산학협력단 | 재조합 활성 유전자 2 유전자 적중벡터, 그 벡터가 도입된 면역세포 결핍 형질전환 미니 복제돼지 생산과 그 제조방법 및 활용 |
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| CN109971840A (zh) * | 2019-04-11 | 2019-07-05 | 扬州大学 | 一种用于猪抗病育种的遗传分子标记方法和应用 |
Non-Patent Citations (1)
| Title |
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| rs1114018753.Ensembl.2021,第1-2页. * |
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