CN107653323B - 团头鲂转铁蛋白基因snp分子标记及其应用 - Google Patents
团头鲂转铁蛋白基因snp分子标记及其应用 Download PDFInfo
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Abstract
本发明属于鱼类分子标记筛选技术领域,具体涉及团头鲂转铁蛋白基因SNP分子标记及应用。所述的分子标记由团头鲂转铁蛋白基因中克隆得到,其中分子标记1的核苷酸序列如SEQ ID NO:1所示,在该序列的128位碱基处存在一个等位基因突变(A/G),(该突变位于转铁蛋白基因内含子部分)。本发明的另一个分子标记的核苷酸序列如SEQ ID NO:2所示,在该序列的169位碱基处存在一个等位基因突变(C/A),该突变导致ScaI酶酶切多态性。利用本发明筛选的两个分子标记对团头鲂抗病性状进行了关联分析,本发明为团头鲂抗病性状辅助选择提供了新的遗传资源。
Description
技术领域
本发明属于鱼类分子标记制备领域,具体涉及团头鲂转铁蛋白基因SNP分子标记及应用,所述的分子标记可应用于辅助团头鲂抗病品种的选育。
背景技术
团头鲂(Megalobrama amblycephala)又名武昌鱼,属鲤形目,鲤科,鲌亚科,鲂属,是我国重要的经济养殖鱼类。近年来人们对团头鲂需求量的提升使得养殖规模进一步扩大,已成为我国主要的经济养殖鱼类之一。但在大规模养殖的过程中,由于近亲繁殖、养殖密度高、水质恶化等导致疾病爆发频繁,其中,细菌性败血症是危害最大的疾病之一。在推广生态养殖、改善养殖环境的同时,深入分析鱼类的免疫应答机制、研究抗病相关基因、选育获得抗病品种成为不可阻挡的趋势。
鱼类是兼具特异性免疫与非特异性免疫的低等脊椎动物,与哺乳类相比,鱼类的特异免疫系统尚不发达,特异性免疫机制还不完善,非特异性免疫在抵御外界刺激及外源微生物中发挥重要作用。铁稳态对生命活动至关重要,铁代谢是通过一系列基因表达产物(蛋白)协调,主要是在肝脏中完成。这些蛋白主要通过结合和转运铁来完成调控过程,其中,转铁蛋白(Transferrin,TF)是重要的铁结合和转运蛋白。转铁蛋白主要存在于脊椎动物细胞外液和血浆中,转铁蛋白既有结合、运输铁的功能,又在抵抗细菌入侵的过程中发挥重要作用。转铁蛋白通过从病原菌隔绝铁元素起到营养免疫的作用,在病原菌入侵时,与宿主争夺铁离子,转铁蛋白与铁离子紧密结合,防止铁离子被细菌掠夺。
单核苷酸多态性(single nucleotide polymorphism,SNP)是指基因水平上单个核苷酸发生变异(单碱基插入、缺失、转换、颠换)所引起的DNA序列多态性,一般表现为二态的遗传变异,即在该位点仅存在两种不同碱基。SNP在人类基因组中分布广泛,平均500~1000个碱基对中就存在一个,占所有已知多态性的90%以上。SNP分布广泛、密度高、数量多、易于自动化分析,是继限制性片段长度多态性和微卫星多态性后的第三代DNA遗传标记。SNP技术在构建高密度遗传连锁图谱、连锁不平衡分析和关联性分析、种群进化和亲缘关系的研究中有很广阔的应用前景。在构建高密度遗传连锁图谱时,SNPs成为微卫星标记后应用最广的作图标记。
发明内容
本发明的目的在于提供团头鲂转铁蛋白基因的SNP分子标记,用于团头鲂抗病品种的选育。
本发明通过给团头鲂注射嗜水气单孢菌进行攻毒,采用引物设计、PCR扩增测序、SNP位点筛选获得SNP位点,通过高分辨率熔解曲线法和限制性内切酶酶切法对SNP位点进行分析,用SPSS软件对数据进行处理,通过卡方检验分析不同基因型与抗病性状的显著性差异。在转铁蛋白基因中发现2个与团头鲂抗病性状显著相关的SNPs位点,这两个SNPs位点分别为:序列1中128位A突变为G(1-128A/G),序列2中169位A突变为C(2-169A/C)。
申请人通过基因克隆技术,采用高分辨率溶解曲线法及限制性内切酶酶切法,首次证实2个与团头鲂抗病相关基因转铁蛋白基因(GI:633265707)多态性SNP分子标记。
128碱基位A/G位点采用高分辨率溶解曲线法制备,2-169A/C位点采用限制性内切酶酶切法制备。
申请人筛选到一个与团头鲂抗病性状相关的基因即转铁蛋白基因SNP分子标记,其核苷酸序列如下所示:
tcttctgcccatatatatttaccttttttatatttctctgcaatgataggacggagtccgtaaggatgtagtccacctaagtaaatatcttttccatctgcagttacagcatctgcctcaccattctR(A/G)tgaattaaagacaatttgatgaaaatactatagtttctactgaacaaaacaagggtgagtgaggtaaaataa taagcatgttttattacctg gatgctcttc atgcactcag ttatagaagg tcggagatga cactcaagat ctgctgattt,
该序列的128位碱基处的R是A或G的突变(该突变位于基因内含子部分)。
申请人筛选到另一个与团头鲂抗病性状相关的基因即转铁蛋白基因SNP分子标记,其核苷酸序列如下所示:
gtattttgca aaactggaag tacgtaatac ttgcctccac cattgggaca gctttctgcatggaaggagagaatacaattttaggtttca tcaaatgtaa attctgactaataggtggcttatgaactgattcataaaatatgaggtagggtttattcttacgttgatR(A/C)gtactgctcaaccatgactggaactagaccgcactttccaccaatatatacctggccaccatcaactgcaaggcatctgcttctccacgctttggtgcaa ataaaacagaatattaacct acaggtggacagtacataaa gatgactgac,
该序列的169位碱基处的R是A或C(该突变位点位于基因的外显子部分),该突变导致ScaI酶切多态性。
这两个分子标记可以单独使用且使用时无先后顺序。
申请人设计了一种检测转铁蛋白基因SNP分子标记的引物对(该引物对也是如表4所述的用于转铁蛋白基因基因型分型的引物对的序列),该引物对的序列如下所示:
正向引物F:TTACAGCATCTGCCTCACCATTC,
反向引物R:CTTGAGTGTCATCTCCGACCTTCT。
本发明筛选的团头鲂转铁蛋白基因SNP分子标记可在团头鲂抗病性状辅助选择中的应用。
附图说明
序列表SEQ ID NO:1是一个用于检测128位A/G位点的团头鲂转铁蛋白基因序列(即,与团头鲂抗病性状相关的分子标记1,该序列的128位碱基处的碱基是突变后的碱基G)。在该序列的128位碱基处存在一个等位基因突变,即由A突变为G。
序列表SEQ ID NO:2是本发明筛选的另一个用于检测169位A/C位点的团头鲂转铁蛋白基因序列(即,与团头鲂抗病性状相关的分子标记2,该序列的169位碱基处的碱基是突变后的碱基C)。在该序列的169位碱基处存在一个等位基因突变,即由A突变为C。
序列表SEQ ID NO:3是检测本发明的分子标记的引物对的正向引物序列。
序列表SEQ ID NO:4是检测本发明分子标记的引物对的反向引物序列。
序列表SEQ ID NO:5是本发明设计的用于模板扩增的引物序列(正向引物TF-s-a-F)。
序列表SEQ ID NO:6是本发明设计的用于模板扩增的引物序列(反向引物TF-s-a-R)。
序列表SEQ ID NO:7是本发明设计的用于模板扩增的引物序列(正向引物TF-s-b-F)。
序列表SEQ ID NO:8是本发明设计的用于模板扩增的引物序列(反向引物TF-s-b-R)。
图1:利用DNA提取凝胶电泳检测图。
图2:团头鲂转铁蛋白基因128碱基位A/G位点不同基因型的测序峰图。
图3:团头鲂转铁蛋白基因169碱基位A/C位点不同基因型的测序峰图。
图4:采用高利用分辨率熔解曲线法对转铁蛋白基因128碱基位A/G位点进行分型的结果统计图。
图5:利用酶切法对转铁蛋白基因169碱基位A/C位点进行分型的凝胶电泳检测图。附图标记说明:图5中的A图是AA/AC基因型酶切检测图,其中第2个泳道为AA基因型,第3个泳道为AC基因型;图5中的B图是AA/CC基因型酶切检测图,其中第1个泳道为CC基因型,第2个泳道为AA基因型。
图6:是本发明筛选的与团头鲂抗病性状相关的分子标记1的核苷酸序列,该序列的128位碱基处存在一个等位基因突变(A/G)。
图7:是本发明筛选的与团头鲂抗病性状相关的分子标记2的核苷酸序列,该序列的169位碱基处存在一个等位基因突变(A/C)。
具体实施方式
实施例1
1.试验对象(试验材料)
实验所用团头鲂均采自湖北百容水产良种有限公司,所用团头鲂为一龄团头鲂,共1500尾。从池塘打捞上来后先在桶里暂养至稳定后,对其进行嗜水气单胞菌注射感染,注射嗜水气单胞菌的浓度为1×107cfu/mL,水温保持在27-29℃。采集12h内死亡(易感组)以及5d后仍然存活的鱼(抗性组)的鳍条样本,放在无水乙醇中保存,后带回实验室-20℃保存。
2.试验方法
2.1利用酚-氯仿法提取团头鲂基因组DNA,具体步骤如下所述:
(1)取0.2g团头鲂的鳍条放入2mL离心管中,加入600μL细胞裂解液,用消过毒的剪刀将鳍条剪碎,加入6μL蛋白酶K(20mg/μL),60℃水浴消化至澄清(2-4h)。
(2)用等体积(600μL)酚:氯仿:异戊醇(体积比25:24:1)提取2次,4500r/min离心20min,取上清至1.5mL离心管中。
(3)加入2倍体积预冷的无水乙醇进行沉淀,-20℃静置30min左右。
(4)4℃,12500r/min离心20min,弃上清。
(5)用70%乙醇洗涤沉淀2次,4℃,12500r/min离心10min。
(6)将沉淀晾干,加入50-100μL ddH2O溶解沉淀,静置过夜,待其充分溶解后于-20℃保存。
2.2获取SNP位点
在易感组和抗性组中各随机选择5个样本DNA作为模板。根据转铁蛋白基因的基因组DNA序列,用Primer premier 5.0设计引物(见表1)分别对10个模板进行PCR扩增(表2),将扩增产物送武汉擎科创新生物科技有限公司进行纯化测序。用DNAstar软件对测序结果进行分析,通过序列比对和峰图分析,筛选出候选SNP位点。
表1本发明设计的用于模板扩增的引物序列
表2 PCR扩增体系
2.3SNP分型及与抗病性的关联性分析
采用高分辨率熔解曲线法对转铁蛋白基因的128A/G位点进行分型,反应体系见表3,分型所用引物见表4,用SPSS软件对分型结果进行统计分析。
采用酶切法对转铁蛋白基因169A/C位点进行分型,所用限制性核酸内切酶为ScaI,酶切识别序列为AGT↓ACT,酶切体系见表5。
表3高分辨率溶解曲线法体系
高分辨率熔解曲线法程序:预变性:95℃,5min扩增(45cycles):95℃,1min;60℃,10s;72℃,15s(搜集荧光值)高分辨率溶解曲线:95℃,1min;40℃,1min;65℃,1s;95℃连续搜集荧光值。
表4本发明设计的用于基因型分型的引物序列
表5酶切体系
3结果分析
本实施例的分析结果见表6和表7。
表6分子标记1-转铁蛋白基因128A/G位点的抗病关联分析
表6说明:该位点与团头鲂抗病性状相关,在抗病群体中,G等位基因占优势。
表7分子标记2-转铁蛋白基因169A/C位点的抗病关联分析
表7说明:在抗病群体中,AA等位基因占优势。
用SPSS软件对分型结果进行统计分析,通过卡方检验分析发现本发明中2个SNP位点等位基因频率和基因型频率在抗病组和易感组中都表现为差异极显著(P<0.01)。
参考文献
1.陈宜瑜.中国动物志硬骨鱼纲鲤形目(中卷).北京:科学出版社,1998.
2.陈婷婷.团头鲂源嗜水气单胞菌流行病调查及致病性、耐药性研究[硕士学位论文].武汉:华中农业大学,2014.
3.高明英等.黄喉拟水龟转铁蛋白重组表达及抗菌活性分析.水生生物学报,2012,36(5):892-897.
4.Ding ZJ,Zhao XH,Su LN,et al.The Megalobrama amblycephalatransferrin and transferrin receptor.Dev Comp Immunol.2015,49:290-297
5.Kucuktas H,Wang S,Li P,et al.Construction of genetic linkage mapsand comparative genome analysis of catfish using gene-associatedmarkers.Genetics.2009,181:1649-1660.
6.Lambert LA,Perri H,Halbrooks PJ,et al.Evolution of the transferrinfamily:conservation of residues associated with iron and anionbinding.Comparative Biochemistry and Physiology Part B:J Biochem MolBiol.2005,142:129-141.
7.Liu H,Takano T,Abernathy J,et al.Structure and expression oftransferrin gene of channel catfish,Ictalurus punctatus.Fish ShellfishImmunol.2010,28:159-166.
8.Matthew F B,Nels C E,Escape from bacterial iron piracy throughrapid evolution of transferrin.Science.2014,346:1362-1366。
Claims (1)
1.一种检测团头鲂转铁蛋白基因的SNP分子标记的试剂在制备团头鲂抗病性状检测产品中的的应用,所述分子标记的核苷酸序列是下列序列中之一种:
tcttctgcccatatatatttaccttttttatatttctctgcaatgataggacggagtccgtaaggatgtagtccacctaagtaaatatcttttccatctgcagttacagcatctgcctcaccattctRtgaattaaagacaatttgatgaaaatactatagtttctactgaacaaaacaagggtgagtgaggtaaaataa
taagcatgtt ttattacctg gatgctcttc atgcactcag ttatagaagg tcggagatgacactcaagat ctgctgattt,
上述序列的128位碱基处的R是A或G的突变,在抗病群体中G等位基因是优势等位基因;
或
gtattttgca aaactggaag tacgtaatac ttgcctccac cattgggaca gctttctgcatggaaggagagaatacaa
ttttaggtttca tcaaatgtaaattctgactaataggtggcttatgaactgattcataaaatatgaggtagggtttattcttacg
ttgatRgtactgctcaaccatgactggaactagaccgcactttccaccaatatatacctggccaccatcaactgcaaggca
tctgcttctccacgctttggtgcaa ataaaacaga atattaacctacaggtggacagtacataaagatgactgac,
上述序列的169位碱基处的R是A或C,该突变导致ScaI酶切多态性,在抗病群体中A等位基因是优势等位基因。
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