CN112575092A - 一种基于Cytb基因序列的飘鱼和寡鳞飘鱼的分子鉴别方法 - Google Patents
一种基于Cytb基因序列的飘鱼和寡鳞飘鱼的分子鉴别方法 Download PDFInfo
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Abstract
本发明公开了一种基于Cytb基因序列的飘鱼和寡鳞飘鱼的分子鉴别方法。该方法包含:分别提取飘鱼和寡鳞飘鱼基因组DNA;(2)PCR扩增飘鱼和寡鳞飘鱼的细胞色素b基因Ctyb;(3)扩增产物纯化后,直接测序,飘鱼的Cytb基因扩增产物如SEQ ID NO.1所示,寡鳞飘鱼的Cytb基因扩增产物如SEQ ID NO.2所示;Cytb基因第57和69位点碱基是C的为飘鱼,若该位点分别是T和A的为寡鳞飘鱼。该方法可以快速、简便、准确、有效地鉴别飘鱼和寡鳞飘鱼,结果稳定性好,重复率高,填补了目前国内分子生物学标准鉴别飘鱼和寡鳞飘鱼的空白,也有利于开展飘鱼和寡鳞飘遗传多样性研究和保护。
Description
技术领域
本发明属于分子遗传学领域,涉及一种飘鱼和寡鳞飘鱼的分子定性鉴别鉴别方法。
背景技术
飘鱼(Pseudolaubuca sinensis)和寡鳞飘鱼(Pseudolaubuca engraulis)隶属于鲤形目鲌亚科飘鱼属,是一类繁殖快、生活力强的小型鱼类,可食用,具有一定的经济价值。飘鱼和寡鳞飘鱼在我国分布广泛,从南至北诸河流及湖泊均有分布,喜成群于浅水区表层水面上来往漂游,杂食性,是淡水鱼类群落中的常见种类,数量较多,在水生态系统中具有重要的生态作用。多年来,受过度捕捞、水利工程、环境污染等不利因素的影响,飘鱼和寡鳞飘鱼种群数量下降,遗传多样性水平降低。飘鱼和寡鳞飘鱼形态相似,生态位相近,在鱼类资源调查中很难区分和鉴别。线粒DNA(mtDNA)为细胞核外遗传物质,具有母性遗传、结构简单、不发生重组、进化速度等特点。mtDNA在种间、种内群体间具有广泛的多态性,事研究动物物种间进化、进行种属鉴定的常用分子标记。线粒体细胞色素b基因(Cytb)是蛋白质编码基因之一,在鱼类遗传多样性、系统进化、种类鉴别等研究中具有重要用途,能够有效鉴别和区分近缘物种,现广泛用于分子鉴别领域。
发明内容
本发明的目的是针对形态区分鉴别的困难,提供一种飘鱼和寡鳞飘鱼的分子遗传鉴别方法。
本发明的另一目的是提供一种用于飘鱼和寡鳞飘鱼的分子遗传鉴别的引物对。
本发明的又一目的是提供该引物对的应用。
本发明的目的通过以下技术方案实现:
一种飘鱼和寡鳞飘鱼的分子遗传鉴别方法,包含以下步骤:
(1)分别提取待鉴定的飘鱼和寡鳞飘鱼基因组DNA;(2)以提取的基因组DNA为模板,PCR扩增飘鱼和寡鳞飘鱼的细胞色素b基因Ctyb,上游引物的序列如SEQ ID NO.1所示,下游引物的序列如SEQ ID NO.2所示;(3)扩增产物纯化后,直接测序,飘鱼的Cytb基因扩增产物如SEQ ID NO.3所示, 寡鳞飘鱼的Cytb基因扩增产物如SEQ ID NO.4所示; Cytb基因第57位点碱基是C的为飘鱼,若该位点是T的为寡鳞飘鱼。
SEQ ID NO.3:
ATGGCAAGCCTACGAAAGACTCACCCACTAATAAAAATCGCCAATGACGCACTAGTCGACCTCCCAACCCCATCTAATATTTCTGTGTGATGAAATTTCGGGTCCCTCCTAGGACTATGTTTAATCACTCAAATCTTAACCGGGTTATTCTTGGCCATGCACTACACCTCTGATATCTCAACCGCATTTTCATCGGTGGTCCACATCTGTCGGGACGTAAATTACGGCTGACTTATCCGCAATATTCACGCCAATGGGGCATCATTCTTTTTCATCTGTATTTACATACACATTGCTCGTGGCCTATATTATGGATCCTACCTCTACAAAGAGACCTGAAACATCGGGGTGGTTCTACTCCTCTTGGTTATAATAACGGCCTTCGTGGGCTACGTCCTCCCATGAGGACAAATGTCCTTTTGGGGGGCCACAGTAATTACAAATCTACTATCAGCAGTCCCCTATATGGGAGACACCCTTGTCCAATGAATCTGAGGCGGCTTCTCAGTAGATAATGCAACCTTGACACGATTCTTCGCATTCCACTTCCTTCTACCGTTCGTCGTAGCCGCGGCAACTATTTTACATCTACTCTTCCTCCACGAAACAGGCTCAAATAACCCAGCCGGATTAAATTCCGACGCAGATAAAATTTCATTCCACCCATACTTCTCATATAAAGACCTTCTAGGATTTGTAGTGATACTACTAGCCCTGACATCCTTAGCACTATTTTCTCCAAATCTCCTGGGAGACCCAGAAAATTTTACTCCGGCAAACCCACTAGTCACCCCTCCACATATTAAGCCAGAGTGATACTTCCTATTTGCCTACGCCATTTTACGATCCATTCCAAACAAATTGGGAGGAGTCCTTGCACTACTATTCTCTATTCTAGTACTAATAGTAGTGCCAATCCTACACACCTCAAAGCAACGAGGACTAACGTTCCGCCCAATCACCCAATTTCTATTCTGAACCCTAGTAGCAGATATGATTATCCTAACATGGATCGGGGGCATACCTGTAGAACACCCATACATTATCATCGGACAGATTGCATCGGTCCTTTACTTTGCATTATTCCTCATCCTTACCCCACTAGCAGGATGGGTAGAGAATAAAGCACTAAAATGAGCCT
SEQ ID NO.4:
ATGGCAAGCCTACGAAAGACTCACCCACTAATAAAAATCGCCAATGACGCACTAGTTGACCTCCCAACACCATCTAACATTTCTGTCTGGTGAAATTTTGGATCCCTTCTGGGGCTCTGTTTAATTACCCAGATCTTAACCGGGCTATTCCTGGCCATGCATTACACTTCTGACATCTCAACCGCGTTCTCCTCCGTTGTCCACATCTGTCGAGACGTAAATTACGGCTGACTTATTCGGAATATTCATGCCAACGGAGCATCATTCTTCTTTATCTGTATCTACATGCACATTGCTCGAGGCCTATACTATGGATCCTACCTCTACAAAGAAACCTGAAACATCGGTGTAGTCTTACTCCTGTTGGTTATAATAACAGCCTTTGTTGGCTACGTCCTTCCCTGAGGACAGATGTCCTTTTGAGGTGCCACTGTCATCACAAACCTTTTATCAGCAGTCCCCTACATAGGGGATACCCTCGTCCAATGAATCTGGGGAGGCTTCTCGGTAGACAACGCAACCTTGACGCGGTTCTTCGCATTCCACTTCCTTCTACCATTTGTCGTCGCCGCCGCAACCATCCTACACCTGCTTTTCCTCCACGAAACGGGCTCGAACAACCCGGCCGGCCTAAATTCCGACGCGGATAAGATCTCATTTCACCCATATTTCTCATATAAGGACCTTCTAGGCTTTGTAGTGATGCTATTGGCCCTCACGTCATTAGCACTGTTCTCTCCAAATCTCCTCGGGGACCCAGAGAACTTTACCCCAGCAAATCCGCTTGTCACCCCCCCGCACATCAAGCCAGAGTGATACTTCCTGTTTGCCTACGCCATCCTGCGGTCCATCCCCAACAAACTTGGTGGAGTCCTTGCGCTATTATTCTCCATTCTGGTACTAATGGTGGTACCGATTCTACACACATCAAAGCAGCGGGGACTAACGTTCCGCCCGATTACCCAATTCTTGTTCTGAACCCTTGTAGCGGACATGATCATCCTCACATGAATCGGAGGCATGCCCGTAGAGCACCCGTACATTATCATTGGACAGATTGCATCCGTCCTTTACTTCGCATTGTTCCTCATCCTTAGCCCACTAGCAGGGTGAGTAGAGAATAAAGCACTAAAATGAGCCT
所述的PCR反应体系为50 mL: 100ng/μL的模板DNA 1 mL,PCR缓冲液 5 mL,dNTP混合液4 mL,每种dNTP 0.1mmol/L, 10 μmol/L的上、下游引物各1 mL,2mL 2.5 IU的Taq酶;双蒸水36mL;所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%(g/100ml)明胶组成;PCR扩增反应程序为:94 ℃预变性4 min,94 ℃变性40s,56 ℃退火50 s,72 ℃延伸90 s,经30个循环后再72 ℃延伸10 min。
作为本发明的另一方面,本发明提供一种用于飘鱼和寡鳞飘鱼的分子鉴别的引物对,其中:上游引物的序列如SEQ ID NO.1所示,下游引物的序列如SEQ ID NO.2所示。
作为本发明的另一方面,本发明提供所述的引物对在分子鉴别飘鱼和寡鳞飘鱼中的应用。
作为本发明的另一方面,本发明提供所述的引物对在制备飘鱼和寡鳞飘鱼分子鉴别试剂中的应用。
作为本发明的另一方面,本发明提供一种飘鱼和寡鳞飘鱼分子鉴别试剂,包含本发明所述的引物对。
优选的,所述的飘鱼和寡鳞飘鱼分子鉴别试剂还包括PCR缓冲液和dNTP混合液,所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%明胶组成。
有益效果:
本发明针对飘鱼和寡鳞飘鱼细胞色素b( Cytochrome b,Cytb)基因序列差异,首次以飘鱼和寡鳞飘鱼的细胞色素b基因全长序列作为依据,从而定性鉴别飘鱼和寡鳞飘鱼。该方法可以快速、简便、准确、有效地鉴别飘鱼和寡鳞飘鱼,结果稳定性好,重复率高,填补了目前国内分子生物学标准鉴别飘鱼和寡鳞飘鱼的空白,也有利于开展飘鱼和寡鳞飘鱼物种多样性和遗传多样性研究。
附图说明
图1为本发明中Cytb基因测序序列比对结果,相同的碱基用连接号标识,差异的碱基用星号标识。星号标明的是从第57位点(Cytb-57)开始的碱基序列差异。
具体实施方式
以下结合实施例来进一步阐明本发明,但并不是对本发明做任何形式的限定,仅仅作示例说明。
实施例1
1、PCR引物
上游引物序列为:L14724:5'-GACTTGAAAAACCACCGTTG-3'(SEQ ID NO.1),下游引物为H15915:5'-CTCCGATCTCCGGATTACAAGAC-3'(SEQ ID NO.2)。
2、样本采集
从滆湖采集飘鱼和寡鳞飘鱼共60尾,其中飘鱼和寡鳞飘鱼个体各30尾。
3、基因组DNA提取
取每尾鱼的肌肉组织用于基因组DNA提取。采用Takara公司的光谱性基因组提取试剂盒提取DNA,用去离子水溶解DNA,紫外分光光度计测定DNA溶液OD值并稀释至100ng/μL,-20℃保存备用。
4、PCR扩增与检测
已提取的DNA为模板,用上述引物(L14724和H15915)进行PCR扩增。PCR反应体系为50 mL:1 mL模板DNA(100ng/μL);PCR缓冲液 5 mL(10mmol/L Tris-HCl,pH9.0, 0.5mmol/LKCl,30mmol/L MgCl2,0.01%明胶);dNTP混合液4 mL (每种dNTP 0.1mmol/L);上、下游引物(10 μmol/L)各1 mL,2mL Taq酶(2.5 IU);双蒸水36mL。扩增反应程序为:94 ℃预变性4min,94 ℃变性40 s,56℃退火50 s,72 ℃延伸90 s,经30个循环后再72 ℃延伸10 min。
PCR产物在1 %琼脂糖凝胶电泳中检测分离,120 V电压下电泳30 min之后,经凝胶成像分析系统确定 PCR 扩增产物的大小及纯度。
5、测序比对
检测后的PCR扩增产物直接送至生物公司纯化测序,获得Cytb基因全序列,长度为1141bp序列。经多重比对后可以发现Cytb基因从第57位点(Cytb-57)的碱基序列存在差异:30尾飘鱼第57和69位点(Cytb-57,Cytb-69)碱基是C,30尾寡鳞飘鱼第57和69位点(Cytb-57,Cytb-69)分别是T和A。
从序列对比图即可看出,通过对两个物种的序列进行比对,分别找出飘鱼和寡鳞飘鱼的一致性序列,在通过他们之间新型比对,发现差异序列,并排除掉其他即有共性的也有差异却无法区分的序列,最终确定第57和69位点可以直接认定相应种类。
实施例2
按照实施例1方法,从太湖、高邮湖、洪泽湖、骆马湖和庆安水库采集,将样本扩大到飘鱼和寡鳞飘鱼个体各80尾,结果依然显示飘鱼的细胞色素b基因第57和69位点(Cytb-57,Cytb-69)碱基是C,寡鳞飘鱼第57和69位点(Cytb-57,Cytb-69)分别是T和A。
序列表
<110> 江苏省淡水水产研究所
<120> 一种基于Cytb基因序列的飘鱼和寡鳞飘鱼的分子鉴别方法
<141> 2020-12-02
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<213> Artificial Sequence
<400> 4
atggcaagcc tacgaaagac tcacccacta ataaaaatcg ccaatgacgc actagttgac 60
ctcccaacac catctaacat ttctgtctgg tgaaattttg gatcccttct ggggctctgt 120
ttaattaccc agatcttaac cgggctattc ctggccatgc attacacttc tgacatctca 180
accgcgttct cctccgttgt ccacatctgt cgagacgtaa attacggctg acttattcgg 240
aatattcatg ccaacggagc atcattcttc tttatctgta tctacatgca cattgctcga 300
ggcctatact atggatccta cctctacaaa gaaacctgaa acatcggtgt agtcttactc 360
ctgttggtta taataacagc ctttgttggc tacgtccttc cctgaggaca gatgtccttt 420
tgaggtgcca ctgtcatcac aaacctttta tcagcagtcc cctacatagg ggataccctc 480
gtccaatgaa tctggggagg cttctcggta gacaacgcaa ccttgacgcg gttcttcgca 540
ttccacttcc ttctaccatt tgtcgtcgcc gccgcaacca tcctacacct gcttttcctc 600
cacgaaacgg gctcgaacaa cccggccggc ctaaattccg acgcggataa gatctcattt 660
cacccatatt tctcatataa ggaccttcta ggctttgtag tgatgctatt ggccctcacg 720
tcattagcac tgttctctcc aaatctcctc ggggacccag agaactttac cccagcaaat 780
ccgcttgtca cccccccgca catcaagcca gagtgatact tcctgtttgc ctacgccatc 840
ctgcggtcca tccccaacaa acttggtgga gtccttgcgc tattattctc cattctggta 900
ctaatggtgg taccgattct acacacatca aagcagcggg gactaacgtt ccgcccgatt 960
acccaattct tgttctgaac ccttgtagcg gacatgatca tcctcacatg aatcggaggc 1020
atgcccgtag agcacccgta cattatcatt ggacagattg catccgtcct ttacttcgca 1080
ttgttcctca tccttagccc actagcaggg tgagtagaga ataaagcact aaaatgagcc 1140
t 1141
Claims (7)
1.一种飘鱼和寡鳞飘鱼的分子鉴别方法,其特征在于:包含以下步骤,
(1)分别提取待鉴定的飘鱼和寡鳞飘鱼基因组DNA;
(2)以提取的基因组DNA为模板,PCR扩增飘鱼和寡鳞飘鱼的细胞色素b基因Ctyb,上游引物的序列如SEQ ID NO.1所示,下游引物的序列如SEQ ID NO.2所示;
(3)扩增产物纯化后,直接测序,飘鱼的Cytb基因扩增产物如SEQ ID NO.3所示, 寡鳞飘鱼的Cytb基因扩增产物如SEQ ID NO.4所示; Cytb基因第57位点碱基是C的为飘鱼,若该位点是T的为寡鳞飘鱼。
2.根据权利要求1所述的飘鱼和寡鳞飘鱼的分子鉴别方法,其特征在于:所述的PCR的反应体系为50 mL: 100ng/μL的模板DNA 1 mL,PCR缓冲液 5 mL,dNTP混合液4 mL,每种dNTP 0.1mmol/L, 10 μmol/L的上、下游引物各1 mL,2mL 2.5 IU的Taq酶;双蒸水36mL;所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%(g/100ml)明胶组成;PCR扩增反应程序为:94 ℃预变性4 min,94 ℃变性40 s,56 ℃退火50s,72 ℃延伸90 s,经30个循环后再72 ℃延伸10 min。
3.一种用于飘鱼和寡鳞飘鱼的分子鉴别的引物对,其特征在于:上游引物的序列如SEQID NO.1所示,下游引物的序列如SEQ ID NO.2所示。
4.权利要求3所述的引物对在分子遗传鉴别飘鱼和寡鳞飘鱼中的应用。
5.权利要求3所述的引物对在制备飘鱼和寡鳞飘鱼分子鉴别试剂中的应用。
6.一种飘鱼和寡鳞飘鱼分子鉴别试剂,其特征在于:包含权利要求3所述的引物对。
7.根据权利要求6所述的飘鱼和寡鳞飘鱼分子鉴别试剂,其特征在于:所述的飘鱼和寡鳞飘鱼分子鉴别试剂还包括PCR缓冲液和dNTP混合液,所述的PCR缓冲液由10mmol/L Tris-HCl,pH9.0, 0.5mmol/L KCl,30mmol/L MgCl2,0.01%明胶组成。
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GENBANK: "MH797111.1", 《GENBANK》, 31 December 2019 (2019-12-31) * |
GENBANK: "MH797113.1", 《GENBANK》, 31 December 2019 (2019-12-31) * |
张旭等: "鄱阳湖湖区及支流修水夏季鱼类系统发育群落结构分析", 《水生生物学报》, vol. 44, no. 16, 30 October 2020 (2020-10-30), pages 1 - 3 * |
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