CN110699460A - 一种基于Cytb基因序列的*和贝氏*的分子鉴别方法 - Google Patents
一种基于Cytb基因序列的*和贝氏*的分子鉴别方法 Download PDFInfo
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- 101150006264 ctb-1 gene Proteins 0.000 title claims abstract description 17
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- 238000000034 method Methods 0.000 title claims abstract description 16
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Abstract
Description
技术领域
背景技术
(Hemiculter leucisculus)和贝氏(Hemiculter bleekeri)隶属于鲤形目鲌亚科属,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。和贝氏在我国分布极广,从南至北诸河流及湖泊均有,静水、流水中都能生长、繁殖,数量较多,是鱼类生态系统的重要组成部分,具有重要的生态价值,也是群体遗传学研究的好材料。多年来,受过度捕捞、环境污染等不利因素的影响,和贝氏种群数量下降,遗传多样性水平降低。和贝氏生态位相近,形态和体色相似,在鱼类调查中很难区分和鉴别。目前用于鱼类物种鉴别的DNA序列有COI基因、Cytb基因、16S rRNA基因、微卫星等;但不同的物种适用的靶基因不同。
发明内容
本发明的目的是针对形态区分的困难,提供一种和贝氏的分子遗传鉴别方法。
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:
所述的PCR反应体系为50μL:100ng/μL的模板DNA 1μL,PCR缓冲液5μL,dNTP混合液4μL,每种dNTP 0.1mmol/L,10μmol/L的上、下游引物各1μL,2μL 2.5IU的Taq酶;双蒸水36μL;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4min,94℃变性40s,55℃退火40s,72℃延伸90s,经35个循环后再72℃延伸10min。
一种用于和贝氏的分子鉴别的引物对,上游引物为L14724:SEQ ID NO.1,下游引物为H15915:SEQ ID NO.2。
所述的和贝氏分子鉴别试剂,还优选包括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:
本发明针对和贝氏细胞色素b(Cytochrome b,Cytb)基因序列差异,首次以和贝氏的细胞色素b基因全长序列作为依据,从而定性鉴别和贝氏。该方法可以快速、简便、准确、有效地鉴别和贝氏,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和贝氏的空白,也有利于开展与贝氏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,星号标明的是从第27位点(Cytb-45)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、引物设计
设计一对特异性PCR扩增引物,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
3、基因组DNA提取
取每尾鱼的尾鳍组织约30mg,用滤纸吸干表面水分,装入1.5ml离心管,加入420μLSTE缓冲液(30mmol/L Tris-HCl,pH8.0,200mmol/L EDTA,50mmol/L NaCl)。混匀后再加入浓度为10%的SDS 80μL和20mg/mL的蛋白酶K10μL,56℃消化8-10h;加入等体积酚:氯仿:异戊醇(体积比25:24:1)抽提两次,12000r离心10min,取上清液;加入等体积氯仿:异戊醇(体积比24:1)抽提一次,12000r离心10min,取上清液;加入等体积预冷无水乙醇,12000r离心10min,弃清液;加入600mL 70%的乙醇洗涤沉淀,8000r离心10min,倒掉乙醇,干燥;加入200μL双蒸水溶解,紫外分光光度计测其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(L14321和H15634)进行PCR扩增。PCR反应体系为50μL:1μL模板DNA(100ng/μL);PCR缓冲液5μL(10mmol/L Tris-HCl,pH9.0,0.5mmol/L KCl,30mmol/L MgCl2,0.01%明胶);dNTP混合液4μL(每种dNTP 0.1mmol/L);上、下游引物(10μmol/L)各1μL,2μL Taq酶(2.5IU);双蒸水36μL。扩增反应程序为:94℃预变性4min,94℃变性40s,55℃退火40s,72℃延伸90s,经35个循环后再72℃延伸10min。
PCR产物在1%琼脂糖凝胶电泳中检测分离,120V电压下电泳30min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1141bp序列,比对后可以发现Cytb基因从第27位点(Cytb-27)的碱基序列存在差异:30尾第27位点(Cytb-27)碱基是G,30尾贝氏第27位点(Cytb-27)是A。
实施例2
序列表
<110> 江苏省淡水水产研究所
<120> 一种基于Cytb基因序列的䱗和贝氏䱗的分子鉴别方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacttgaaaa accaccgttg 20
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctccgatctc cggattacaa gac 23
<210> 3
<211> 1141
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggcaagcc tacgaaaaac ccatccgcta ataaaaatcg ccaacgacgc actggtcgac 60
ctcccaacac catctaacat ttccgtgtga tgaaacttcg ggtccctcct aggactgtgt 120
ttaatcaccc aaatcctgac tggactattc ctagccatgc actacacctc tgatatctca 180
accgcattct catcagtcgt ccatatttgt cgagatgtaa actacggctg acttattcgt 240
aatatccacg ctaatggggc gtcattcttt ttcatttgta tttatataca tattgctcgt 300
ggcctatatt atggatctta tctctacaaa gaaacctgaa acatcggagt aatcctgctc 360
ctactagtca taataacagc cttcgtcggc tacgtccttc catgaggaca aatatccttc 420
tgaggtgcca cagtaattac aaacctgtta tcagcagttc cctacatggg agacaccctt 480
gttcaatgaa tttgaggtgg cttttcagta gacaatgcaa ccctgacacg attcttcgca 540
ttccacttcc ttctaccatt cgtcgtcgct gccgcaacca tcttgcacct actcttcctt 600
cacgaaacag gatcaaacaa tccagccgga ctaaactctg acgcagacaa aatctccttc 660
cacccatact tttcatataa agaccttcta ggatttgtag tgatactact agccctcaca 720
tccttggcac tattctctcc gaacctccta ggggacccgg agaatttcac cccagcaaat 780
ccactggtca cccctccaca catcaagcca gagtgatact tcctatttgc ctacgccatc 840
ctacggtcca tcccaaataa actaggagga gtccttgcac tattattctc cattctagta 900
ctaatggtag tacccatctt acacacctca aagcaacgag gactaacatt ccgcccaatc 960
actcaatttt tattctgaac cctggtggca gatatgatta tcctgacatg aattggaggt 1020
atgcccgtag agcaccccta catcattatc ggacaaatcg catcagtcct ttacttcaca 1080
ttattcctca tccttagtcc actagcagga tgagtggaaa ataaagcact aaaatgagct 1140
t 1141
<210> 4
<211> 1141
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggcaagcc tacgaaaaac ccatccacta atgaaaatcg ccaacgacgc actagtcgat 60
ctcccaacac catctaacat ttccgtgtga tgaaacttcg ggtccctcct aggactatgt 120
ttaatcaccc aaatcttaac cgggttattc ctggccatgc actacacctc cgatatctca 180
accgcattct catcagttgt ccacatttgc cgagatgtaa actatggttg acttatccgt 240
aacatccacg ctaacggagc atcattcttt ttcatctgta tttatataca tatcgctcgt 300
ggcctgtact acggatctta tctatacaaa gagacctgaa acatcggggt agttctactt 360
ctgttagtta taatgacggc cttcgttggc tacgtccttc catgaggaca gatatcattc 420
tgaggtgcca cagtaatcac aaatctactg tcagcagttc cctacatggg agacaccctc 480
gttcaatgaa tctgaggtgg cttctcagta gacaacgcaa ccctaacacg attctttgca 540
ttccacttcc tcctaccatt tgtcgtcgct gccgcaacca tcctccacct actcttcctc 600
cacgaaacag gatcgaacaa cccggccgga ctaaactccg atgcggacaa aatctccttc 660
cacccatatt tctcatataa agatctccta gggtttgtag tgatattact agccctcaca 720
tcattagcac tattctctcc aaacttgcta ggggacccag aaaattttac gccagcaaac 780
ccactagtca cccctccaca catcaagcca gagtgatact tcctgtttgc ctacgccatt 840
ctacgatcta ttccaaacaa actagggggg gtccttgcac tactattctc gattctggtg 900
ctaatagtag taccaatctt acacacctcg aaacaacgag gactaacatt ccgcccaatc 960
actcaattct tattctgaac cctagtggca gatatgatta tcctaacatg aattggaggc 1020
atacccgtag aacacccata catcatcatt ggacagatcg catcagtcct ttacttcgca 1080
ttattcctca tccttagtcc actagcagga tgggtagaaa ataaagcact aaaatgagct 1140
t 1141
Claims (7)
2.根据权利要求1所述的分子鉴别方法,其特征在于所述的PCR的反应体系为50μL:100ng/μL的模板DNA 1μL,PCR缓冲液5μL,dNTP混合液4μL,每种dNTP 0.1mmol/L,10μmol/L的上、下游引物各1μL,2μL 2.5IU的Taq酶;双蒸水36μL;所述的PCR缓冲液由10mmol/LTris-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4min,94℃变性40s,55℃退火40s,72℃延伸90s,经35个循环后再72℃延伸10min。
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GENBANK: "KF029693.1", 《GENBANK》 * |
WEITAO CHEN等: "Phylogeographic structure, cryptic speciation and demographic history of the sharpbelly (Hemiculter leucisculus), a freshwater habitat generalist from southern China", 《BMC EVOLUTIONARY BIOLOGY》 * |
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