CN106480191A - 一种河蛏蚌f型线粒体基因组全序列扩增的方法 - Google Patents
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Abstract
一种河蛏蚌F型线粒体基因组全序列扩增的方法，包括基因组DNA提取、普通PCR扩增及测序、设计长片段引物、LA‑PCR扩增及测序、序列拼接和基因注释。本发明获得了河蛏蚌F型线粒体基因组全序列，为河蛏蚌的种类鉴定、蚌科的系统发生学、比较和进化基因组学、谱系基因组学、种质资源保护、可持续和合理地利用奠定提供重要的基础材料。
Description
技术领域
本发明涉及生物技术领域，涉及基因组序列的扩增方法。
背景技术
河蛏蚌（Solenaia rivularis）隶属于软体动物门、瓣鳃纲、蚌目、蚌科、蛏蚌属。河蛏蚌仅限于亚洲分布，是江西的新纪录种。河蛏蚌强大的足部肌肉使得身体得以稳定和抵抗强水流的冲刷，另外，强有力地足部肌肉经常保持向下挖掘，以维持壳体始终处于最佳的生活位置。蚌类具有很大的经济价值，如提取抗肿瘤成分；作为制造纽扣贝雕等工艺品的原料，珍珠层可以用来制造各种精美的螺细、器皿和家具等；蚌类软体部分可供人们食用或作为饲料；淡水蚌对水环境中Cu、Zn和Cd有去除与积累的作用，吸收富集重金属，改良水质。蚌科动物是淡水贝类最大的一个科，包括 674 个物种，淡水蚌类是淡水生态系统中一类重要的水生生物，作为滤食者，能够通过滤食营养物质、有机物和浮游生物来改良水质，对水环境还有生物监测的作用。淡水蚌类在淡水生态系统中不可或缺，其生物量和密度往往都在底栖动物中占优势，对淡水生态系统有重要作用，如果淡水湖泊或者是河流中的淡水蚌类消失，将会引起一系列的生态问题。总而言之，淡水蚌类对淡水生态系统至关重要，对水体的水质、物质循环和能量流动作用巨大，且具有较高的营养、经济和生态价值。因食用的需求大量捕捞，以及近年来由于栖息地的破坏和改变（如采沙、建坝、环境污染等），河蛏蚌分布范围狭窄、种群碎片化，已处于严重受危状态。
线粒体基因组DNA是裸露的双链超螺旋共价闭合环状分子，少数为线状分子(如原生动物草履虫与四膜虫的mtDNA)，是真核细胞的第二遗传信息系统，或称核外基因及其表达体系。相对于核DNA，mtDNA具有分子量小、多态性高、结构简单、碱基突变率高、母系遗传、进化速度快等特点，已被广泛应用于生理病理、临床诊断、遗传变异、分子标记、系统进化等多方面的研究，与线粒体和线粒体基因组相关的研究已成为进化生物学、基因组学、生物信息学和保护生物学等生命科学领域的研究热点和前沿。
蚌科动物线粒体的遗传不单单是母系遗传，还存在线粒体基因组遗传的一种特例——双单亲遗传现象（Doubly-Uniparental Inheritance, DUI）。这种遗传方式使后代中雌性mtDNA只来自母系（female-transmitted, F type），与严格的母系遗传相似；而雄性的mtDNA 来自双亲，在雄性的体细胞中存在F型mtDNA，在精巢中存在父系mtDNA（male-transmitted, M type）。因此，建立一种稳定的扩增河蛏蚌F型线粒体基因组全序列的方法在比较和进化基因组学、保护生物学和资料的可持续利用等研究中有非常重要的应用价值。
发明内容
本发明的目的是提供一种稳定、快速扩增河蛏蚌F型线粒体基因组全序列的方法。
为了实现上述目的，本发明采用以下技术方案来实现。
本发明所述的一种河蛏蚌F型线粒体基因组全序列扩增的方法，包括以下步骤：
（1）基因组DNA提取；
（2）普通PCR扩增及测序：共使用了三对通用引物如下：
COIP1: GGT CAA CAA ATC ATA AAG ATA TTG G
COIP2: TAA ACT TCA GGG TGA CCA AAA AAT CA
16SP1: CGC CTG TTT ATC AAA AAC AT
16SP2: CCG GTC TGA ACT CAG ATC ACG T
ND1P1: TGGCAGAAAAGTGCATCAGATTAAAGC
ND1P2: CCTGCTTGGAAGGCAAGTGTACT
（3）设计长片段引物：本发明设计了三对长片段引物如下：
F COI-16sP1： GCACCCAGGATAGAAGAAGCA
F COI-16sP2：CTTGGTGGTTCCGAGATTGC
F 16s-ND1 P1：GGCAATCTCGGAACCACCA
F 16s-ND1 P2：CCGTACTCGACGTTGAACCC
F ND1-COI P1：ATACCAACCGCCATACTGCTT
F ND1-COI P2： CCGCCGTTATCTGGAAATG
（4）LA-PCR扩增及测序；
（5）序列拼接；
（6）基因注释。
本发明首次公开了河蛏蚌F型线粒体基因组全序列，也是首次公开了一种扩增河蛏蚌F型线粒体基因组序列的方法，可以为蚌科的线粒体基因组学研究和种质资源保护提供重要的基础材料。
本发明的有益效果：通过设计特异性长片段引物，通过LA-PCR技术对河蛏蚌F型线粒体基因组进行扩增，再进行测序、拼接和校正，获得了河蛏蚌F型线粒体基因组全序列；本发明对实验条件要求不严格、实验具有可重复性、操作时间相对较短等优点。本发明获得的河蛏蚌F型线粒体基因组序列，可从组成和结构的角度对河蛏蚌F型线粒体基因组进行分析，可以丰富蚌科线粒体基因组信息，为蚌类的系统发育、种质资源保护、可持续和合理地利用奠定重要的分子遗传学理论基础。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述。所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一种河蛏蚌F型线粒体基因组全序列扩增的方法，包括如下步骤。
1、基因组DNA提取。
使用购自天根生化科技有限公司的海洋动物组织基因组DNA提取试剂盒，提取河蛏蚌的总DNA，包括以下步骤。
（1）取河蛏蚌的新鲜肌肉组织50mg，置于1.5ml已灭菌离心管中，用洁净的灭好菌的剪刀充分剪碎放入装有200 l GA缓冲液的离心管中，涡旋振荡15sec。
（2）加入20 l proteinase K(20mg/ml)溶液，涡旋混匀，简短离心以去除管盖内壁的水珠。在56℃放置，直至组织完全溶解，简短离心以去除管盖内壁的水珠。
（3）加入200 l 缓冲液GB，充分颠倒混匀，70℃放置10min，溶液应变清亮，简短离心以去除管盖内壁的水珠。
（4）加入200 l 无水乙醇，充分颠倒混匀，此时可能会出现絮状沉淀，简短离心以去除管盖内壁的水珠。
（5）将上一步所得溶液和絮状沉淀都加入一个吸附柱CB3中(吸附柱放入收集管中)，12000rpm(~13400*g)离心30sec，倒掉废液，将吸附柱CB3放回收集管中。
（6）向吸附柱CB3中加入500 l 缓冲液GD(GD中已加入无水乙醇)，12000rpm(~13400*g)离心30sec，倒掉废液，将吸附柱CB3放回收集管中。
（7）向吸附柱CB3中加入600 l 漂洗液PW(PW中已加入无水乙醇)，12000rpm(~13400*g)离心30sec，倒掉废液，将吸附柱CB3放回收集管中。
（8）重复操作步骤（7）。
（9）将吸附柱CB3放回收集管中，12000rpm(~13400*g)离心2min，倒掉废液，将吸附柱CB3置于室温放置数分钟，以彻底晾干吸附材料中残余的漂洗液。
（10）将吸附柱CB3转入一个干净的离心管中，向吸附膜的中间部位悬空滴加50-200μl 洗脱缓冲液TE，室温放置2-5min，12000rpm(~13400*g)离心2min，将溶液收集到离心管中。
（11）将纯化好的线粒体DNA置于-20℃保存，以备后用。
2、普通PCR扩增及测序。
对于河蛏蚌F型线粒体的cox1、16SrRNA和nad1基因部分序列的扩增分别采用如下引物扩增。扩增产物送生工生物工程(上海)股份有限公司测序。包括以下几个步骤：
（1）用于扩增河蛏蚌F型线粒体的cox1、16SrRNA和nad1基因部分序列的引物为通用引物如下：
COIP1: GGT CAA CAA ATC ATA AAG ATA TTG G
COIP2: TAA ACT TCA GGG TGA CCA AAA AAT CA
16SP1: CGC CTG TTT ATC AAA AAC AT
16SP2: CCG GTC TGA ACT CAG ATC ACG T
ND1P1: TGGCAGAAAAGTGCATCAGATTAAAGC
ND1P2: CCTGCTTGGAAGGCAAGTGTACT
（2）三个基因片段扩增的PCR反应：反应的模板为上述提取的河蛏蚌的基因组DNA100ng，反应总体系为50μl，其中10×Ex Taq PCR Buffer 5μl，dNTPs4μl、各2.5mmol/L，引物各2μl、0.4μmol/L，Ex Taq酶1U；
PCR反应条件为：98℃预变性10sec；94℃变性1min，50℃退火1min，72℃延伸2min，循环35次；最后72℃延伸7min。
3、设计长片段引物。
把cox1、16SrRNA和nad1基因的部分片段的测序结果在NCBI中进行nucleotideblast，确定都为蚌科动物线粒体DNA基因片段且均为正义链。利用Primer premier5.0将前期得到的cox1、16SrRNA和nad1基因片段三段序列为模板设计三对长PCR引物结果为：
F COI-16sP1： GCACCCAGGATAGAAGAAGCA
F COI-16sP2：CTTGGTGGTTCCGAGATTGC
F 16s-ND1 P1：GGCAATCTCGGAACCACCA
F 16s-ND1 P2：CCGTACTCGACGTTGAACCC
F ND1-COI P1：ATACCAACCGCCATACTGCTT
F ND1-COI P2： CCGCCGTTATCTGGAAATG
4、LA-PCR扩增及测序。
使用设计的三对长PCR引物及TaKaRa生物公司的LA Taq聚合酶进行扩增：反应的模板为上述提取的河蛏蚌的基因组DNA100ng，反应总体系为50μl，其中10×LA Taq PCRBuffer 5μl，dNTPs4μl、各2.5mmol/L，引物各2μl、0.4μmol/L，LA Taq酶1U；
LA-PCR反应条件为：94℃预变性2min；94℃变性30sec，54℃退火30sec，68℃延伸5min，30个循环；最后72℃延伸10min。LA-PCR产物经1%琼脂糖凝胶电泳检测，有清晰的条带；产物送生工生物工程(上海)股份有限公司测序。
5、序列拼接。
把实验所得的全部序列片段利用DNASTAR7.1软件中的SeqMan组件进行拼接，组装成一条首尾相接的序列，并仔细检查，对照测序峰图人工校正序列中相冲突的位点，校正完后再利用SeqMan把首尾的重复序列去除其中一个，最终得到完整的线粒体DNA全序列。
6、基因注释。
河蛏蚌F型线粒体DNA全序列的注释参考珠蚌亚科已测物种的F型线粒体基因组DNA，用在线工具ORF Finder (http://www.ncbi.nlm.nih.gov/projects/gorf/)和Blast查找线粒体基因组的13个蛋白质基因，利用在线工具ARWEN (http://130.235.46.10/ARWEN)和MITOS (http://mtos.biinf.unileizig.de/help. py)查找22个tRNA基因和2个rRNA基因以及控制区(Control Region，CR)。该序列总长16613bp，包括13个编码蛋白质，22个tRNA，2个rRNA和1个FORF，全基因序列还未上传GenBank。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
SEQUENCE LISTING
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<120> 一种河蛏蚌F型线粒体全基因组序列的扩增方法
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<400> 7
ctacctagac aacaacacaa ctttagcaat ttgcccgttt gaatgaaacg aagctggaaa 60
aatcccatcc tgccactcaa aagaagtcct caacgctctt ccccagacaa cagaccgctg 120
agagacaaaa gactctaaca acataaacat aaataataac acagaaacaa aagaaattaa 180
agacccccaa gaagacacca catttcactg tgcaaaccta tcagggtaat cagaatatcg 240
acgaggcatc ccagctaacc ccaaaaaatg ctgcgggaaa aaggttagat taacaccaac 300
aaacatcaag ataaaatgaa cctttcttca aacagcgtga aaagtaacac cagaaattag 360
aggataccaa tagctgaaag cactaaacaa tgcaaaaaca gcccctattc tcaaaacata 420
gtggaaatgg gccaccacat aataagtatc gtgtaacaca atatcaagag aagagttgga 480
taaaacaatc ccagttaacc caccaaccgt aaacaaaaaa ataaaaccta aagcccatat 540
aataggaggc tcagtcttat taacaaaccc atggattgta gccaaccaac taaacacctt 600
aatcccagtg ggcacagcaa taatcatagt ggcagcagta aagtaagctc gagtatccac 660
atccatccca actgtaaaca tatgatgagc ccaaacaata aaacccaaaa ccccaataga 720
gacaatagca taaaccatcc ccaaataacc aaaaacttgc tttttcccag cataatgcat 780
aacaatgtgt gaaattatcc caaatccagg caaaatcaaa atgtacactt cgggatgccc 840
aaaaaaccaa aacaaatgca tatacaaaat aggatcccca ccaccagtag ggtcaaaaaa 900
agacgtattc aaattccgat cagtcaacag cattgtaata gcacccgcca acacaggcaa 960
cgccgcaacc aacaaaaccg ctgttaccgt cacagcccac acaaataacg gaatccgctc 1020
agcaactagc ccaggagatc gcatattccc aacagttgaa ataaagttaa tagcacccag 1080
gatagaagaa gcacccgcaa ggtgcaaaga aaaaatagcc aaatcgaccg aagccccgga 1140
atgagcaaca tttccagata acggcggata cacagtccac cctgtcccaa caccactctc 1200
caccaaagaa gatctcaaca acaaaaaaag agccggcaca agtaaccaaa accttaaatt 1260
attcagacgt ggaaaagcca tatcaggtgc cccaatcata aggggaataa gtcaattccc 1320
aaaacccccg attatcattg gcatcaccaa aaagaaaatt attataaaag catgagccgt 1380
aacaatcaca ttatataatt gatcatcccc caacaatctt ccaggctgcc ccaactccgc 1440
ccggatcaaa agacttaaag ccaacccaat caaaccagat cacaaagcta acaataagta 1500
caacgtacca atatccttat gattcgtaga acacaatcac cgcaaataat taaacaagct 1560
aattaaccca ttaatcaaga ataaaaaaca tccggagaaa ccccctctac cccaataggc 1620
ataaaagagt ggttcacacc gcaaatttca ctacactgcc caaacatcac acccgaccca 1680
atcaaatgca ccgctaattg attaatacga cctggaatag catcaacctt cacccccata 1740
gaaggaagag cccaagcatg aatcacatca gcagacctga caagaactcg cctgtcaacc 1800
ccataaggca acacacatcg attatcaacc tccaacaaac gataaccccc ctccccaaca 1860
tcaccagccc tcactatata tgaatcaaac ccaactgacc tacccccatc atcatactca 1920
tactctcagt accactgatg cccaacagcc ttcatcctaa ccgcaggccc ccccacctca 1980
tctaacaaat acaacaaccg aacagacgga atagccaaaa ttagcaaaag caaccccgga 2040
atcacagtcc aagccgcctc aagcgcctga gcctccacaa gaaaccgaga agataaccta 2100
ctcttcaaca gacacaacat catgtaacca acaaacgaag acaccattac aagaacaaac 2160
atagcatgat catggaaaaa agttaactca gaactcaaag cactaaaact atcctgaaac 2220
cctaattgac ctcaaaagct catttattag cttactccgc tactccaccc tcacaaccac 2280
tcgacaaccc ccctctaaac ctaacgaaaa gatctcccca tctatagaac cacaatctat 2340
tgttttccct taaactattc gttaccttta atcaaaaaac tccgtttact actatttaaa 2400
atttttaccc taattaaact taccctatac tagagagaga gggaaagaaa caaaaaataa 2460
acaataaaca aacaccagag aagtagccga gcttatatga ggcttctaac cacataaaga 2520
gaggtttaac tcctttcact tctctttaaa ccacacccaa tgaaatcacc ccacaaattt 2580
ttattcttat tcctgataat agcaagcacc tgcatagtct tatcctcgtc caactgacta 2640
atgacatgaa taggcttaga gattaatatg ctggggttta tccccctcat atacataaaa 2700
gacactgcca acgaatcaga aacagtagta aaatacttaa tcccccaatc ccttggctcc 2760
accgtcttca ttgcctccgc cacaatctcc cttcatctcg acaacattca atcgctcata 2820
gcactagcca tatgcctaaa gctgggggcc gcaccatttc acttctgatt cccgcctgtg 2880
atagcaggtc tcccactact accagccttc attcttctga cttgacagaa aattgctccg 2940
attttcacca tcagggccat cagcccaaca cttataagca agattatgct agttgcaaga 3000
atcagggccc tatggggagg cattggcgga ataaaccaga cagacattcg ctccctacta 3060
acctactcct caatcgcaca caccggatga atactggcca gcatcaatag cgccgcccct 3120
gtgctgaggg tgtacatcat agcatatgtg ctcatcaacc tctcaatctt cacagcccta 3180
acctcaacct ccacaaaatt ccacaaacag ctacttaccc caaaagagcc ctacaactcc 3240
ctcttgctag ctgccagcat tctctccctg gggggcctcc cccctcttac agggttcatt 3300
ataaagctac tagttattag ctacacccaa accagactct tcgttatctt caccctaatt 3360
ctcggggccc taattagtct attttactac ctctctctaa ccttttcccc actgctagaa 3420
ctaaccaaaa cacgcctaaa taaatctcac atctcaaaac cctccatagt attttttctt 3480
gcccaaattc taccactatc caccctaata tttttattct aagtgggata agctaatctc 3540
ataagctgtt gggctcataa cccaaaaata gatatattct tcccaccaaa taaacaacca 3600
ccctataaca ctcccctaaa aaagctaaaa ttaaaccaaa aactgtcgtc tacctttccc 3660
actctaaaga cccctcccgc cactcatgaa taacccccac aaacaacaac accaaaaaaa 3720
acaacaatgt taaacaacca ccaacccaca ttaaaggtct cccaactacc atcacagccg 3780
gaaacagtaa aacaatctcc acatcaaaga ccacaaaaat aacagccaac agaaagaatc 3840
gcaaggaaaa aggcactcga gaagacccaa ccggatcaaa cccacattca aatggcctag 3900
acttttctcg gccaataaac aaagaaaacc ctaaaaatag accaacaacc aatagcacaa 3960
aacccaacat cgctgataat aaaacactca ctacaacttt ttccatatac cttgctaggc 4020
aacacgccac actcaccccc ccccccactc atctgaccca ccaaccccac caaatcgact 4080
taattaaaag tcacccattt attaatttcc gcaaaatctc ctttaactaa aaaattctga 4140
ttaaataagt aaaggttcta tactttaatt aaaagatctg atttgcaatc aaccattgag 4200
tgccaacttc aacactctag aactacccaa aacaaaggac ctcggagaat atttgccttg 4260
aaatcaaaca gaaagtgttc gactcactta tccttttagt ccttcgctaa ttaagtgttt 4320
actagcacat tgacttttcg cgtcaaaggg gcataccaat gcatttggct caaaatcaac 4380
tataccctta aaccccccac agtagtaaac ctgctctggc ttagctttac cgtccttacg 4440
ccgctattgc tccttaacag ctgagacacc caatcggttt tagctaacga cccaccaaca 4500
gacttccctc aaacccaaga cctaataaga agtggtggag acacaccaat cgctgcagcc 4560
tcaggggact atccagtaac caccagccaa gcacacacta atattactca accataagac 4620
taccgcgcca caaacatacc caaaccaaag tatactcgaa agatttaagt taagtaaact 4680
aatagccttc aaagctttaa ttgatctaca atcaatcttt acaacctaaa acaagaaact 4740
ctagtgttat ggtttcggcc cataaaaagg cacaagttgt gcccttgttt tcacaaatat 4800
ttcattgacg taccaagtta acttcactaa ttaaactaca tatggtagcc cacacacatt 4860
gtgccttacg gttagtccgc ttggggattt ttaatagacc cccaaacaac ttaatcgagg 4920
acttcacccg ttaaagtcca aggtttatat cccacaacac caatgtccta tattaggtta 4980
gctaggagac taggccctag aaaacaagta tttaggggtg gcaaaaatgg tgccagcagt 5040
cgcggttata ccattacctc aagctaacta acacaaagtg ggaccaacca caagactaaa 5100
agattaactt cctacgtaaa aaataaatta caaccaaacc caaccaagtc atccactaaa 5160
agtcccaaca aaccacaacc tcaaaacccg gattagatac ccgactattg tgtggcccaa 5220
caaaaagaat actacaagcg aaagctcaaa cctcaaacaa tgtggcggtg cttaactcct 5280
catcagggga tcctgtggct taaaccgata atccacgaat accttagctc ttctagctct 5340
cagcttatgt atggccgttt acgcttgctc aaaaaagatc acaaaaggaa gcaatggggc 5400
cattccccgc caattcaggt gacatatagc caacgaaaag ctgactcatg ggatacaaat 5460
aaaccaacta ccagacttaa aagttaaacc ttttaataaa ggaggacttg aaagtaaggc 5520
tcacaccaaa taaagccagc ctgaacaaga acttaagcgc gcacaaatcg cccgtcactc 5580
cggcagtaaa gtcggataag tcgtaacata gtaggggtaa tggaaattgc ccctatcacg 5640
tccgaggtgg ccgagacccc aggcatcgag cttttaactc gaccacagta attactccag 5700
gacagctttg agaagctaat tagcatcggt cttgtaaacc gaagacaggc tataaatctc 5760
cccaaagctt agcaaagtgg ccgagagcag gcgaaagatt gtagctcttt acacggatca 5820
tcccctttgc aaacaacact accaacaata aaacagaact caagaaaata accacctcgc 5880
tcagtacccc atcaaaatag ctattgcctc tagacccgca agggtaaacc atccgtacac 5940
cacagaaaag cttacaactt gtcccttttg catcatggag aagcaacaca aacctaacaa 6000
cttaagctcc cgaataacta tgagctacta aaccttaaaa atcaatgtcg cacaattgat 6060
aaattaacct ttagtagaag taacaagcct ttcatatagt tagatagctg gctttcccct 6120
caaagcatca aagtgctgcc ccgtagacta actcctctgg gacactaaat aactacgcgg 6180
gttaactttt cgaggattag ctcgaaaaga caacaaaaat atagaaaacc ccaccaccaa 6240
aagtaggctt aaaaacagcc acctaacagc gttctagtta atggccccac acaccaacaa 6300
tattaaaacc ccaatttacc aaaatagggg aacttggagc caaacacaaa gcaccactga 6360
accggcattc tattagtatt aacatcacca agccccaaga actccagact aattgggcca 6420
ccacaaagcg actaaagcta aaactataaa aagtgaactc ggcaacaaag ttctctgcct 6480
gtttaccaaa aacatcgtct tttgagccat cgcataaaag ataatgcctg cccagtgaaa 6540
tacttaaacg gccgcgttag cgtgagcgtg ctaaggtagc gtaataaata gcctctcaat 6600
tggaggccag tgaatggcaa gactaagaac acctttacct tttatattag aaaaactttt 6660
cgtctaagtg aaaagactta gataatgaag gaagacgaaa agaccccgcg gaactttact 6720
ttttcttacg cttacaccca aaagtacaaa gcgccaaaag tttgattggg gcaatctcgg 6780
aaccaccaag cttccgatca ctacaataaa acatctaaaa tatggtatgg acaaaacaga 6840
agttaccccg gggataacag cgttatccaa cttaagagta catatcgaaa gttgggtttg 6900
cgacctcgat gttggcttaa ggacatccac attagtgcaa ccgctatgga aggatagtct 6960
gttcgactat tataccctta cacgagctga gttaagaccg gcgtaagcta ggttggtttc 7020
tatctccttt acaaacacac ctcttgattg tacgaaagga ccccaaaaga tgcacaaaaa 7080
agcacacctt aaacaattat ccaattagct caagcataaa caaaacccca atgtgagtta 7140
gtataataat accactgact taggatcagt aaataagtaa ccacttactc accaaaaggg 7200
aaagaagcta caattagcaa ttagctgtta cctaatggat agtgagcaac tcacctaccc 7260
taaccaacca ccagaaaata gtttaacaaa aacaaaaact ttgggagttt tagacttagg 7320
cactctaatt ttctgaatca aaccatcaat acgaaaaact caccccattg ccaaaatctt 7380
aaataactcc ctctacgatc tcccagcccc aactaactta tcaacctgat gaaatttcgg 7440
atctctctta ggcctatgtc taggaacaca aattgtcaca ggcctcttcc tggccataca 7500
ctacacatca agcgtagacc ttgcttttta ctcagtctcc cacatctcac gagacgtaaa 7560
ctttggctga ctaatacgag ccattcatgc aaacggcgcc tcatttttct tcgtttgcct 7620
ctatatgcac tcaggacgag gaatgtatta cggctcatac ttagccactg aaacttgaaa 7680
catcggggtt attctaatac tcctaaccat agcaacagct ttcctcggat atgttcttcc 7740
atggggacaa atgtcttttt ggggcgccac cgtaattacc aacctactat cagcaattcc 7800
atacgtggga aaaaccctgg tttactgact gtggggtggc ttctcagtgt ctaatgcaac 7860
cctaaatcga ttttttgtgt tgcactttgt gctcccattt gctatcgcag catttgctgt 7920
aatccaccta ttgtttctac acgaaagcgg gtctaacaac ccactaggta tctcctcaaa 7980
cgcaagcctt atccccttcc acatctacta cacagtaaaa gatgtggtgg gattcatctt 8040
tctcctgggc ctgctaacaa gcatttgctt cttctcacca aacctgctaa cagaccccga 8100
aaacttcatc ccagcaaacc ccctaagaac tcctgtacac attcaaccag agtgatattt 8160
cttatttgca tacgcaatcc tgcgatccat ccctaataag ttagggggtg ttcttgcact 8220
actactgtct attctaatct taactattat accactaacc cactttaata ccatacgctc 8280
tattagattc tacccgctta accaaatctt attctgaggg tttatcagaa ccttcctaat 8340
tctaacctga attggtaaac aaccagtcga agacccattt atttgggttg gacaaacttt 8400
ctccacccta tactttatat actttttgct agccccctta atcgccaaac tctgggacac 8460
cctggtattc taccaaccac gctaaatcgc cctcaactcg ctggacaagt agtttaacca 8520
taaaacataa cgctgaaaat gttaaaatga catccgtcct tttccaaaaa ccttatttct 8580
aatcatctaa caaaaaacaa ttatacatac tataaaaaag ctatccctac cagtgaaaaa 8640
acaacaaaaa taataaaaac ccgaacaaac actaacaacc tattcctctg cagcataaaa 8700
gaaaacctaa cccccttccc caaaacctta aacataccct gcccgccaaa aacctctatt 8760
cacccctgat ccaaatgcat atgagccaac acaccacccc tcaaaccagt acctgccatt 8820
aaccttccag aaaccaaatt cataaatcac attctggata aaaattgcct aaaccaacta 8880
acacgcttta gaccaaattt ccaaactctc acatacctaa cagccccata gattagccca 8940
gcaaacgtaa caagcacaat aatcgccttc ccaaaagacc caaccacatc aaacccattc 9000
acagacaccc aaaccccctg caaaagtcac ccacccaaaa tcgcccctac tgctaacacc 9060
aaaatagaag ccagcatata tttactctca gagccgcaag agagcaaaga accgccaaaa 9120
ctctgcccaa acacagccac caacagaata cgcaagctat acaccaaact caaccccgcc 9180
cccatcaata caactacaat ctccaaccca ccactcaacc cgctaaaagc cccctccaga 9240
atcaaatcct tagagtaaaa ccccctaaga aaaggcatac cgcataaaga cgcactcctc 9300
atgattaaac acccccttct aaatggcaac agtaaactaa caccccccaa aattcgacca 9360
tcctgcgtgt ccccagtaga atgaataata gaaccagcac atagaaacaa caaagcctta 9420
aataaagcgt gtgtaaacaa gtgaaaaaca gccacagtcg gatagccaac accaaccgta 9480
aacattataa gccccaactg cctcaaagta gacaaggcaa tgatcttctt caaatcaacc 9540
tcaaaacaag cccttaaccc cgccattaac atagtcaaac cacccacttt ccttaaagcc 9600
cacaaaacct caggtctctc tagcaaagat ctgtaaaaac gaatcactaa gtaaacacct 9660
gccgtaacca aagtagacga atggacaaga gctgataccg gagtgggagc agccattgct 9720
gccggtaacc aggcagaaaa gggaatttgc gcccttttag ttatagcccc aactaccacc 9780
aaaaaacaaa tcaacacccc aaacccacca aacaaaccaa aaccaaaacg ccactccccc 9840
caattaacta gaaaacaaat agctaaaatt aacaaagcat caccaatacg attaaccaaa 9900
acagttaaca tcccagcagc caaagacttt ttattctggt aataaataac caaggcaaaa 9960
gaaacaatgc ctaggccatc ccaacccaaa agaaccgtaa ctagcctagg aacaaaaata 10020
agtaaattta tagacccaac aaaccccata atcaacaaca tgaaccgtcg aataaacacc 10080
tctctttcca tataggacac gctaaacaag gacaccgacc cagaaattaa acaaacaaaa 10140
caacaaaaga tgaccctaac ataatcaaca ataatcggca aagtccattc tcttctacat 10200
aacgaaaaaa actgccactc aattaaataa cctcccccac tgtagacaac ccctcaaacc 10260
ccaaccaccc acataaacac cctaacaaaa aaaaggaaaa tcgacgagaa caagggagcc 10320
cccaaaactt ttaaaatttt cacatagcac cacgaactcg ctcgcaaagc aaccacaaca 10380
agccactcct taacctctac ctatgctact tactcctata caagtatact agagacctat 10440
aatccctact ctttatgtat tcttaccccc ctctaccagg aacactcctt aacctctacc 10500
tatgctactt actcctatac aagtatacta gagacctata atccctactc tttatgtatt 10560
cttacccccc tctaccagga acactcctta acctctacct atgctactta ctcctataca 10620
agtatactag agacctataa tccctactct ttatgtattc ttacccccct ctaccaggaa 10680
cactccttaa cctctaccta tgctacttac tcctatacaa gtatactaga gacctataat 10740
ccctactctt tatgtattct tacccccctc taccaggaac actccttaac ctctacctat 10800
gctacttact cctatacaag tatactagag acctataatc cctactcttt atgtattctt 10860
acccccctct accaggaaca ctccttaacc tctacctatg ctacttactc ctatacaagt 10920
atactagaga cctataatcc ctactcttta tgtattctta cccccctcta ccaggaacac 10980
tccttaacct ctacctatgc tacttactcc tatacaagta tactagagac ctataatccc 11040
tactctttat gtattcttac ccccctctac caggaacact ccttaacctc tacctatgct 11100
acttactcct atacaagtat actagagacc tataatccct actctttatg tattcttacc 11160
cccctctacc ttctattact atcttcacta tacctatact actatcattc cacttctact 11220
aagcttaacc cagcttttcc acttcctcac ctattatatt aaaatcaatt attataaggc 11280
acaactaatt aagtaaacga aataattacg atcagagaga gagagagaga gaaactgaag 11340
gctagtgttt tcacgaatga gtttggatca ttagatggag ttaaacctcc gcctccaagc 11400
cactcaaact ataacaactt gtcttgtagt atataaaaca cattactgta aactgcaact 11460
ttaccaaagc aatagccaag acatccaacc ccagtatcac gccggaagaa cggactaccc 11520
tgatgaggca gaacataggc ctgtgccttg atacttactc aaaaggtagt atattcatta 11580
caacttgctt acaccaagta aaaggtccct ggcccctctt gaagtaaagt ggcagaaaag 11640
tgcctcaggt ttaagccctg atcagggaga ttactccctt tactaatcgt tcagcacatt 11700
atttcaaccc ttatcacata tctcttaatc ctactcggtg tggccttctt caccctgttg 11760
gagcgcaaag ccctagggta tttccaaatc cgaaaaggcc cgaataaagt cggaatcatt 11820
gggattccac aaccactagc cgatgcccta aaactcttcg taaaggaatg agtcataccc 11880
ctatcctcta actaccttcc atttatttta accccaacaa ccatattaat tttagccctt 11940
agtttatggc aactattccc ctcatttatg acctcctctt acataacctt aggcatactt 12000
ctctttctgt gcatctcctc cctagccgtt tacacgaccc taatagcggg atgatcctcc 12060
aactctaagt atgccttatt gggtgccatc cgagcaatag cccagactat ctcatacgaa 12120
gtaaccataa cactaatcat tctcttttac ttattcttaa gaataaaaat agacctggta 12180
acaattcgcc taattaatca atcaatacca accgccatac tacttctccc actaacaatt 12240
atatgaacag cggtcatctt agctgagaca aaccgagccc cattcgactt tgctgaaggc 12300
gaatcagaat tagtgtctgg gttcaacgtc gagtacgggg gagccggttt cgctttttta 12360
tttatagccg aatacagaaa catcctcata atgagactta tcacatcctg cctcataaca 12420
ggcactctaa ctgcagccat cccagtagca gtgatatttc tctgggctcg agccaccctc 12480
ccacggtatc ggtatgatct ccttatagca atggcctgaa aatctttcct accagtaagc 12540
ctaacaattt taacctgcct aaccccactc ctgtttctat aggacccaac acagatagta 12600
tacaagtaca gctgccttcc aagcagaaag cccaaacctg gtcagtgtaa ttacacttct 12660
aattttatca actttgtgat tatactccat actcacaata gccctcccaa tacacccatt 12720
atccctaggc ataataatct tagtgctagc cttcatcaac tgcaccctca tcgcctcgat 12780
aagaccatga tacgcctata tactgttttt tatctttatc ggcggaatac tggtaatatt 12840
tgcctatatt gcatcactct caccaaatac cacattttca ctaaacagac aagcaatacc 12900
aattattata acagctaccc ttcttgcctt ctccaccagc ttaggcccga gctcaaacta 12960
cctcactaac ccagaacttg accttagcct gaaagcctca agtcaggcct taagatccct 13020
atacctatac aatagaggca actcctgcgt aatcctttta gcctgtgtgc tgcttttcac 13080
catggtcgcc agagtgaaga tttgtaaacc aaaaatagga gcactacgcc cgtacctgta 13140
aggctactca ttaatccagc aaataattca aaactaaaca ccttatacaa aagctattag 13200
tgacacccca actccgccca aaggcaacac tagtacaaac ctaactaaaa gaccgataaa 13260
ctcctccacc acaatacccc tacgcaccca aatggggcac tgaccatgtt gagtggcaga 13320
ataaacatac caggagtaca agccacttaa gaaggttata gccccggtaa aaacagcaaa 13380
aaccgtcgaa taagataaaa cagagatacc taacatcaac tccccccaca aattcaaaga 13440
cggcggagca gccatattaa tagcacacat taaaaaccaa cacaaagcaa ccccagggac 13500
caaaactagc ccacctttca ccaagtacaa cctccgagtt ttgtagcacc tgtaagtctc 13560
cctggccaaa aaaaacatcc cagaagagca cagcccatga gcaaccatta ttagcaaaca 13620
gcctaatcac ccccaatcag tatttgaaaa tactccgcct aaaaccaatc tcatgtgccc 13680
cacagaagaa taagccacta aggcttttac atcattctgg gcaaaacaca ccatcctagc 13740
aacaaccccc ccacctaaac ctaagcaaaa caccaaagta gtagtcaaag acccaaacac 13800
ccccaaccca caaaaaacac ggattaaacc atacccaccc aacttcaaca aaacaccggc 13860
taagatcata gacccggcca caggggcttc tacatgagcc tttggcagcc acaaatgaaa 13920
cccgtaaatg ggcaacttaa ccaaaaaggc caatgaacca cataaagtaa ctaaccaacc 13980
ccatctaaag cccccataac tccaccccca aaagacagac cccccctcaa acaaaaccag 14040
taaaatcaat accagcaagg gaagagaagc actcacagta tataacatca tgtacttgcc 14100
cgcctgcaat cgctcaggct gatagcctca ccctaaaatg attaacaaaa ttggaatcag 14160
cctgaactcg aacatgacgt agaagttgag taaagaccta aacctaaaac aaaaaataag 14220
caccaaagtt agtaccaaaa cccaagccac aaacccaaca ctcctattcc cagatttctg 14280
aatgtcccgc acccttgcta acaccctcaa cctagagacc aagatagtca atgacacaag 14340
accaaaagaa aaattatcaa caaccaaccc ctgaccccaa cacccagcca gcccaatcga 14400
cctaaaccac aactcaaacc tcaacaataa cataacaacg caaccccaaa caaaccccca 14460
ccaaaataca gctggtctaa acaaaccaac caaagccaca atcatccctg taaccaacaa 14520
cctaaaaatg cccagaaacc aacctaccca cgtaatcact accagtgcta cgaaccaaag 14580
aaactaacac cctgagcccc aacgctgcct cacatacccc aaacctcaaa aataacagac 14640
aagcacttgt caaccttccc gaaaatcaaa ccacactaaa aatcataata taaaccctca 14700
aagtaaaaac ctccattctc aagagagccc caaaaagcct ttttcgctga gacattaaac 14760
acaccccgcc aatcaacacc ccaataaccc ccacacccac agctgggaaa aacctcatac 14820
caactacttc ttcagcccta aaccccccct aaaacctcac ctaaccccct tcttactagt 14880
tgcacccact ttaacgcctt tacaaaccct atactctttg cgagcccttc accaaaaaac 14940
taccataatt gaaattcaac aaaccaaaaa aagtaaaaac actatcaccc atgacatagg 15000
acttaattga ggcactaaag aatgccgcac aggcaatcca agcttgacaa gcttaagtta 15060
ttagttaact aattctttat agcgtcaaca ctaatgccct gcccccattg ggtgatcagt 15120
agagtacaac ccaactaaca aaacaaaaac ataggcctgc aaaaatctaa cagcaaactc 15180
aaacaacaca taaccccaca ttaccaaact ccctagcacc cccccagtaa atgacgcact 15240
aaataaaaac ccaaccaaat actcaccaat aacatgaagc ataaggtgcc ccatagtaat 15300
gtttgcagcc aatcgaaccc ccaaggtaat cggccggatt aaaatcctaa ccctttcaat 15360
tagtactagc aaaggaatta acccaaccgg cctcccccta ggaaccaaat gagccgccct 15420
ttgacctcaa gaataactcc atcccctaaa aatcaaacaa aaccacacaa ctctagccaa 15480
cacaaaagtc aaagacaaat gactagtagg actaaaaaca tcaggtacca tcccagaaat 15540
atttacaata aaaagcacta taaataaaga cacttgaccc aaagcaaatc cgccaaaaaa 15600
cttaccagaa caatttttaa ctaatcccct aaccaactca attaacagag aggaaacaac 15660
cccaacccca gaaaccccaa cccacagctg gaccagagaa actaccaaac caagaaaccc 15720
gcttagccaa ataaagcccc aaaggctaaa actagaatgc ataccagcgt ctaaagacga 15780
aaaaatatct atcaacatta cctcagccta cctaagaccc tcaccaataa atgcacaaat 15840
aaagaaaaat ccaaacaaca tcaacaaaat gccagtacca cgcagcagcc tcaaaaccaa 15900
aatggtgagt gacagaaaaa tgatgcaaat aacaacgaac agtgcaaaca accagaaaag 15960
ccctcccgat taaaacatgc aacccatgaa acccagttat aacaaaaaaa gtagagccgt 16020
aaaccctatc agcaacacta aaagaggcct cctcatactc cccccactga agcacagtaa 16080
aatacacccc caacaacaca gtcaacctca aaccatataa agcctcctca cgattccccg 16140
ccatcagtcc atgatgcgcc caagtaaccc tcacacccga ccccaacaac acagccgtgt 16200
tcaacaacgg aaccttaaaa gcatttaacg gcatgatacc aacaggtggc caaacacaac 16260
ctaattcaac tgtcggaaca agtctcctgt gaaaaaaccc ccaaaaaaaa gcaaaaaaaa 16320
agcatacctc agaaaaaata aacaaaacca tcccccaacg aagatttata ccaacccaac 16380
tagtatgata accttgataa gtcccctccc gaaccacatc ccgtcatcac tgcaccatag 16440
ttaaaacaat taccaaaata cctaagacca tcaaactcaa gcccttgcca tgaaatcacc 16500
taaccaaacc aaccgttaaa aataaagccc ctctcgctgc agtaaatggc caaggactaa 16560
gctccactaa atgaaaagga ttacgtaaca tttactttct caacaaccag ttt 16613
Claims (1)
1.一种河蛏蚌F型线粒体基因组全序列扩增的方法，其特征是包括以下步骤：
（1）基因组DNA提取；
（2）普通PCR扩增及测序：共使用了三对通用引物如下：
COIP1: GGT CAA CAA ATC ATA AAG ATA TTG G
COIP2: TAA ACT TCA GGG TGA CCA AAA AAT CA
16SP1: CGC CTG TTT ATC AAA AAC AT
16SP2: CCG GTC TGA ACT CAG ATC ACG T
ND1P1: TGGCAGAAAAGTGCATCAGATTAAAGC
ND1P2: CCTGCTTGGAAGGCAAGTGTACT
（3）设计长片段引物：设计了三对长片段引物如下：
F COI-16sP1： GCACCCAGGATAGAAGAAGCA
F COI-16sP2：CTTGGTGGTTCCGAGATTGC
F 16s-ND1 P1：GGCAATCTCGGAACCACCA
F 16s-ND1 P2：CCGTACTCGACGTTGAACCC
F ND1-COI P1：ATACCAACCGCCATACTGCTT
F ND1-COI P2： CCGCCGTTATCTGGAAATG
（4）LA-PCR扩增及测序；
（5）序列拼接；
（6）基因注释。
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|---|---|---|---|---|
| CN113512593A (zh) * | 2021-05-21 | 2021-10-19 | 南昌大学 | 一种蚌类环境dna宏条形码引物、鉴定方法及应用 |
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| EP2302070A2 (en) * | 2005-06-23 | 2011-03-30 | Keygene N.V. | Strategies for high throughput identification and detection fo polymorpisms |
| CN104988135A (zh) * | 2015-06-16 | 2015-10-21 | 南昌大学 | 一种扩增天津尖丽蚌f型线粒体基因组序列的方法 |
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| EP2302070A2 (en) * | 2005-06-23 | 2011-03-30 | Keygene N.V. | Strategies for high throughput identification and detection fo polymorpisms |
| CN104988135A (zh) * | 2015-06-16 | 2015-10-21 | 南昌大学 | 一种扩增天津尖丽蚌f型线粒体基因组序列的方法 |
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| CN113512593A (zh) * | 2021-05-21 | 2021-10-19 | 南昌大学 | 一种蚌类环境dna宏条形码引物、鉴定方法及应用 |
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