CN109234409A - A kind of Portumidae mitochondrial COI gene universal primer and design and amplification method - Google Patents
A kind of Portumidae mitochondrial COI gene universal primer and design and amplification method Download PDFInfo
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- CN109234409A CN109234409A CN201811296203.4A CN201811296203A CN109234409A CN 109234409 A CN109234409 A CN 109234409A CN 201811296203 A CN201811296203 A CN 201811296203A CN 109234409 A CN109234409 A CN 109234409A
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Abstract
The present invention relates to a kind of siphonopods mitochondrial genomes research fields, more particularly to a kind of Portumidae mitochondrial COI gene universal primer and design and amplification method, the general forward primer of Portumidae mitochondrial COI gene is SEQ ID NO.1:5 '-TCNACAAAYCATAAAGAYATYGG-3 ', and reverse primer is SEQ ID NO.2:5 '-TANACYTCWGGRTGHCCRAARAAYCA-3 '.The present invention solves the method that do not identified in the prior art using DNA bar code technology Portumidae, with 1) Portumidae mitochondrial COI gene universal primer provided by the invention, disposably efficiently a variety of Portumidaes can be expanded in batches, great convenience can be provided for Portumidae species identification；2) present invention designs universal primer in two sections of conservative regions of Portumidae mitochondrial COI gene, amplify intermediate series of variation, amplification length is in 600-700 bp or so, existing enough informative sites are distinguished for material evidence identification, it can guarantee that a sequencing reaction can be completed again, it is easy to operate, the advantages of save the cost.
Description
Technical field
The present invention relates to a kind of siphonopods mitochondrial genomes research field more particularly to a kind of Portumidae mitochondrias
COI gene universal primer and design and amplification method.
Background technique
One emerging skill of the species identification that DNA bar code (DNA barcoding) technology is got up as developed recently
Art has received more and more attention.Its principle is usually to utilize one section of short mitochondriaCOI(cytochrome c
Oxidase subunit I) gene order progress species identification, it is widely used to fish, birds, reptiles, lactation at present
The biological groups such as class, but be not very common in crab class.Portumidae crab resources are abundant, and type is more, have high warp
Ji value；However, traditional form and ethological technological means are based primarily upon to the classification of crab class, and phenotypic variation is by environment
Effects of Factors is significant, leads to the uncertainty of phenotypic character.Species identification is carried out based on molecular biology method, utilizes species sheet
The DNA information of body, is not interfered by environmental factor, can effectively supplement conventional sorting methods deficiency.
Patent Office of the People's Republic of China disclosed a kind of spider mitochondrial genome complete sequence amplimer and expansion on September 29th, 2017
The invention authorization of increasing method, authorizes publication number: CN104531688B, which is passed through using the total DNA for extracting spider genome
The long PCR (L-PCR) of mitochondrial genomes DNA is expanded and spider is identified in sequencing, but it is only capable of identification spider mitochondria
DNA cannot identify siphonopods mitochondrial COI gene, and need to design multipair primer pair genom sequence and expanded, time-consuming
Longer inadequate convenience and high-efficiency.
Summary of the invention
To solve the above-mentioned method that do not identified in the prior art using DNA bar code technology Portumidae,
The present invention provides one kind the universal primer of a variety of Portumidae mitochondrial COI genes is expanded with efficient quick, and provides it
Design and amplification method.
To achieve the above object, the invention adopts the following technical scheme:
A kind of Portumidae mitochondrial COI gene universal primer, the Portumidae mitochondrial COI gene universal primer can
Disposable batch amplification Portumidae mitochondrial COI gene, forward primer are SEQ ID NO.1: 5 '-
TCNACAAAYCATAAAGAYATYGG-3 ', reverse primer are SEQ ID NO.2: 5 '-
TANACYTCWGGRTGHCCRAARAAYCA-3’。
Preferably, the Portumidae includes Dun Chi Charybdis, Jing Ying Charybdis, Ban Charybdis, Shuan Ban Charybdis, Shan Yong Charybdis, blue crab, saw
Edge mud crab, Scylla paramamosain, Portunus pelagicus and P.sanguinolentus.
A kind of design of foregoing Portumidae mitochondrial COI gene universal primer, under in Genbank database
All Portumidae COI gene orders announced at present are carried, multiple alignment is carried out to these sequences, is found out than more conservative
Region and the biggish region of variation design universal primer in two sections of conservative regions, amplify intermediate series of variation, general to draw
Object amplification length is 600-700 bp.
The amplification method of the Portumidae mitochondrial COI gene universal primer, the Portumidae line grain before a kind of
The amplification method of body COI gene universal primer the following steps are included:
(1) DNA extraction agent box method extracts Portumidae mitochondrial COI gene to be measured；
(2) using Portumidae mitochondrial COI gene universal primer described in claim 1；
(3) using Portumidae mitochondrial COI gene as template, PCR amplification is carried out；
(4) amplified production is tapped and recovered amplified fragments with 1.0% agarose gel electrophoresis, RNA isolation kit, is sequenced.
Preferably, the reaction system of step (3) described PCR amplification forms are as follows: dNTP2 μ L, the 10 × TaqDNA of 2.5mM
Polymerase Buffer2.5 μ L, 10 μM of forward primer SEQ ID NO.1 and each 1 μ L of reverse primer SEQ ID NO.2,5U/ μ L
0.2 μ L of Taq DNA polymerase, the 1 μ L of DNA template solution of 100g/ μ L and sterilizing 17.3 μ L of distilled water.
Preferably, the PCR amplification condition in the step (3) are as follows: 95 DEG C of initial denaturation 5min, subsequent 95 DEG C of denaturation
30s, 56 DEG C of annealing 30s, 72 DEG C of extension 45s carry out 35 circulations altogether, finally carry out 72 DEG C of extension 5min, protect under the conditions of 4 DEG C
It deposits.
Preferably, the Portumidae to be measured includes Dun Chi Charybdis, Jing Ying Charybdis, Ban Charybdis, Shuan Ban Charybdis, Shan Yong Charybdis, indigo plant
Crab, mud crab, Scylla paramamosain, Portunus pelagicus and P.sanguinolentus.
The beneficial effects of the present invention are:
1) Portumidae mitochondrial COI gene universal primer provided by the invention, can be disposably efficiently in batches to a variety of shuttles
Crab section is expanded, and can provide great convenience for Portumidae species identification；
2) present invention designs universal primer in two sections of conservative regions of Portumidae mitochondrial COI gene, amplifies intermediate variation
Sequence, amplification length in 600-700 bp or so, distinguish for material evidence identification, and can guarantee one by existing enough informative sites
Sequencing reaction can be completed, easy to operate, save the cost.
Detailed description of the invention
Fig. 1 is Portumidae COI expanding effect figure provided by the present invention.
Wherein: M:DL2000；1: Dun Chi Charybdis (Charybdis hellerii)；2 Jing Ying Charybdis (Charybdis
Lucifera)；3 Xiu Ban Charybdis (Charybdis feriatus)；4 Shuan Ban Charybdis (Charybdis bimaculata)；5 Shan Yong Charybdis
(Charybdis natator)；6: blue crab (Callinectes sapidus)；7Mud crab (Scylla serrata)；8
Scylla paramamosain (Scylla paramamosain)；9 Portunus pelagicus (Portunus pelagicus)；10 P.sanguinolentus
(Portunus sanguinolentus).
Specific embodiment
Main material, reagent and instrument and equipment:
Software and sequence resources: Premier Primer5.0 primer-design software (downloading is from biosoftware net http: //
Www.bio-soft.net/), seawater fish mitochondrial genomes sequence resources (under be downloaded from Genbank database in NCBI
Http:// www.ncbi.nlm.nih.gov/), sequence analysis software MEGA5.0(is downloaded from biosoftware net http: //
Www.bio-soft.net/).
PCR amplification detects related reagent instrument: marine animal genome DNA extracting reagent kit (TIANGEN, Beijing), fine jade
Sepharose DNA QIAquick Gel Extraction Kit (TIANGEN, Beijing), conventional desktop centrifuge (Thermo), Bio-Rad C1000TM
Thermal Cycler amplification instrument (Bio-Rad, the U.S.), micropipettor (Enppdorf, Germany), 96 hole PCR plates
(Axygen), dNTP(TIANGEN, Beijing), 10 × Taq DNA polymerase Buffer(TIANGEN, Beijing), Taq DNA polymerization
Enzyme (TIANGEN, Beijing), sterilize distilled water, DL2000 DNA marker(TIANGEN, Beijing), and agarose (Biowest, it is fragrant
Port), electrophoresis apparatus (DYY-6C type, Beijing 6 one), gel imager (Bio-Rad GD2000, the U.S.).
Cloning and sequencing related reagent instrument: high-pressure steam sterilizing pot (SANYO, Japan), ammonification benzyl (Amp) antibiotic goes out
Bacterium LB culture medium, LB solid culture plate (the raw work in Shanghai), superclean bench (SW-CJ -1G type, the star of famous brand, China) are permanent
Warm water bath (upper Nereid is macro), DH5 α competent cell (TIANGEN), Amp antibiotic (the raw work in Shanghai), the chloro- 3- Yin of the bromo- 4- of 5-
Diindyl-β-D- galactoside (the raw work in Shanghai), isopropylthio-β-D-galactoside (the raw work in Shanghai), pGEM-T carrier
(Promega, the U.S.), constant incubator (PH070A type, Shanghai Yiheng Scientific Instruments Co., Ltd), (training of constant temperature oscillation shaking table
English, Taicang experimental facilities factory) automated DNA sequenator (3730 type of ABI, the U.S.).
Test method without specific conditions in embodiment is such as authors such as Sambrook according to normal conditions
Described in Molecular Cloning: A Laboratory room handbook (New York:Cold Spring Harbor Laboratory Press, 1989)
Condition, or the condition according to manufacturer's specification suggestion.
Embodiment 1
1, Portumidae mitochondrial COI gene universal primer and design and amplification method
(1) acquisition, identification and preservation of Portumidae sample
Portumidae sample used picks up from the natural environment of field in embodiment, and adopted sample takes back laboratory and carries out information
Registration, is identified with stereomicroscope, to determine type.The good Portumidae sample of Identification of Species is impregnated with 100% absolute alcohol
And it is stored in spare in 4 DEG C of refrigerator.Portumidae type collected includes: Dun Chi Charybdis, Jing Ying Charybdis, Ban Charybdis, Shuan Ban Charybdis, is apt to
Yong Charybdis, blue crab, mud crab, Scylla paramamosain, Portunus pelagicus and P.sanguinolentus.
(2) extraction of Portumidae mitochondrial COI gene
Siphonopods sample mitochondrial COI gene to be measured is extracted using DNA extraction agent box method, the mitochondrial COI gene of extraction is protected
It is spare under the conditions of being stored in -20 DEG C.
(3) design and synthesis of the universal primer of Portumidae mitochondrial COI gene
From Genbank database download at present all siphonopods species COI gene orders announced, to these sequences into
Row multiple alignment finds out region more biggish than more conservative region and variation, and designs universal primer in two sections of conservative regions,
Amplify intermediate series of variation.Synthesized universal primer are as follows:
Shown in forward primer SEQ ID NO.1: 5 '-TCNACAAAYCATAAAGAYATYGG-3 '
Shown in reverse primer SEQ ID NO.2: 5 '-TANACYTCWGGRTGHCCRAARAAYCA-3 '.
(4) PCR amplification mitochondrial COI gene
The reaction system of PCR amplification forms are as follows: dNTP2 μ L of 2.5mM, 10 × Taq DNA polymerase Buffer2.5 μ L, 10 μM
0.2 μ L of Taq DNA polymerase, the 100g/ μ of forward primer SEQ ID NO.1 and each 1 μ L of reverse primer SEQ ID NO.2,5U/ μ L
The 1 μ L of DNA template solution and sterilizing 17.3 μ L of distilled water of L；
PCR amplification condition are as follows: 95 DEG C of initial denaturation 5min, subsequent 95 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 45s, altogether into
Row 35 circulations finally carry out 72 DEG C of extension 5min, save under the conditions of 4 DEG C.
(5) sequencing of PCR product
1.0% agarose gel electrophoresis of PCR product, gel electrophoresis spectrum is as shown in Figure 1, obtain size in 600~700bp
Between segment, the segment expanded is all made of RNA isolation kit and is tapped and recovered, recovery product is sequenced, sequencing result with
The upper homologous sequence of GenBank is compared, it was demonstrated that is the amplified production of head Portumidae plastochondria COI gene.
It is as follows that acquired results are sequenced:
The sequencing result of Dun Chi Charybdis (Charybdis hellerii) is as shown in SEQ ID NO.3:
5’-ATAGTTGGAACATCATTAAGATTAATTATTCGAGCAGAATTAGGTCAACCAGGTACTTTAATTGGCAAT
GATCAAATTTACAATGTCGTAGTTACAGCTCATGCATTTGTTATAATTTTCTTTATAGTTATGCCAATTATGATTG
GAGGATTTGGTAATTGACTCGTACCTTTAATATTAGGAGCCCCTGATATAGCATTCCCTCGAATAAATAATATAAG
ATTCTGACTCCTTCCTCCCTCACTAACTTTACTATTAATAAGAGGAATAGTTGAAAGAGGTGTTGGTACCGGTTGA
ACAGTATACCCTCCCTTAGCGGCTGCCATCGCCCATGCAGGTGCCTCTGTAGATCTAGGAATTTTCTCTCTTCATT
TAGCAGGTGTTTCTTCTATTTTAGGGGCCGTTAATTTTATAACAACTGTTATTAATATACGCTCCTTTGGTATAAG
TATAGACCAAATACCTTTATTTGTTTGGTCAGTTTTCATTACTGCAATCCTTCTACTTCTGTCACTTCCTGTATTA
GCTGGCGCTATTACTATACTA TTAACAGACCGAAATTTAAATACTTCATTCTTTGATCCTGCTGGAGGAGGAGAC
CCCGTTCTTTACCAACATTTATTT TGATTTTTTGGCACCC-3’。
The sequencing result of Jing Ying Charybdis (Charybdis lucifera) is as shown in SEQ ID NO.4:
5’-ATAGTTGGAACATCATTAAGACTAATTATTCGAGCAGAGTTAGGTCAGCCAGGAACGTTAATTGGTAAT
GACCAGATTTATAATGTTGTAGTCACAGCTCATGCATTTGTCATAATTTTTTTCATAGTTATACCAATTATAATTG
GAGGGTTTGGTAATTGACTTGTACCTTTAATATTAGGAGCTCCTGATATAGCCTTTCCCCGTATAAACAATATAAG
ATTCTGACTCCTTCCTCCTTCATTAACTTTACTATTAATAAGTGGTATAGTCGAAAGAGGTGTAGGTACTGGTTGG
ACAGTTTATCCTCCTCTAGCAGCTGCTATTGCCCACGCAGGGGCCTCTGTAGATTTGGGAATTTTCTCTCTTCACT
TAGCAGGGGTTTCTTCTATTTTAGGAGCCGTTAATTTCATAACAACTGTTATTAATATACGTTCCTTCGGTATAAG
TATAGATCAAATACCTTTATTTGTTTGATCAGTCTTCATTACTGCAATTCTCCTTTTACTTTCACTTCCTGTTCTA
GCCGGAGCTATTACTATATTACTAACAGATCGAAACTTAAATACTTCATTTTTTGATCCTGCTGGGGGAGGGGACC
CTGTTCTTTATCAACATTTATTCTGATTTTTTGGCCCCC-3’。
The sequencing result of Xiu Ban Charybdis (Charybdis feriatus) is as shown in SEQ ID NO.5:
5’-ATGGTTGGGACATCATTAAGACTAATTATTCGAGCCGAACTAGGTCAACCAGGTACCCTAATTGGGAAT
GATCAAATTTATAATGTTGTTGTTACTGCCCATGCATTTGTTATAATTTTCTTTATAGTTATACCAATTATAATTG
GAGGATTTGGTAACTGACTTGTACCTTTAATATTAGGAGCTCCTGATATAGCATTTCCTCGTATAAATAATATAAG
ATTTTGACTTCTTCCTCCTTCTTTAACATTACTCCTAATAAGAGGGATAGTTGAAAGAGGTGTCGGTACTGGATGA
ACCGTGTACCCTCCTTTAGCAGCCGCTATTGCCCACGCAGGTGCTTCTGTTGATCTTGGTATTTTCTCTCTTCACC
TGGCCGGTGTTTCCTCTATTTTAGGAGCTGTAAATTTTATAACTACTGTTATTAACATACGCTCTTTTGGTATAAG
AATAGATCAAATACCTCTATTTGTATGATCAGTATTTATTACCGCAATTCTCCTTTTATTATCTCTCCCTGTCCTG
GCTGGAGCTATTACTATATTATTGACAGACCGTAATTTAAATACTTCATTTTTTGATCCTGCAGGAGGAGGAGATC
CTGTTCTCTACCAACACTTATTTTGATTTTTTGGTCCCC-3’。
The sequencing result of Shuan Ban Charybdis (Charybdis bimaculata) is as shown in SEQ ID NO.6:
5’-ATAGTTGGAACTTCTTTAAGATTAATTATTCGAGCTGAACTTGGTCAACCCGGAACCTTAATTGGTAAT
GATCAAATTTATAATGTTGTAGTTACAGCACACGCATTTGTTATAATTTTCTTCATAGTTATACCAATTATAATTG
GAGGATTTGGAAACTGACTTGTTCCTTTAATACTTGGGGCTCCTGATATAGCATTCCCTCGTATAAATAATATAAG
ATTTTGACTTCTACCTCCTTCTTTAACTTTATTATTAATAAGAGGAATGGTTGAAAGAGGTGTAGGTACCGGATGA
ACAGTTTACCCTCCTTTGGCAGCTGCCATTGCTCATGCAGGTGCCTCTGTAGATTTAGGTATTTTTTCTCTTCATC
TTGCAGGTGTTTCTTCTATTTTAGGAGCTGTAAATTTTATAACTACTGTTATTAATATACGATCTTATGGTATAAC
GATGGATCAAATACCCTTATTTGTCTGATCAGTTTTCATTACCGCTATTCTTCTTCTTTTATCATTACCCGTTTTA
GCTGGAGCTATTACAATACTATTAACTGATCGTAATTTAAACACTTCATTTTTTGATCCTGCCGGAGGTGGAGACC
CTGTTCTTTATCAACATTTATTCTGATTTTTTGGTCATC-3’。
Shan Yong Charybdis (Charybdis natator)Sequencing result as shown in SEQ ID NO.7:
5’-ATAGTAGGGACATCACTAAGATTAATCATTCGAGCAGAACTCGGACAGCCCGGAACTCTAATTGGAAAT
GATCAAATTTATAATGTTGTAGTTACAGCACACGCATTTGTTATAATTTTCTTTATAGTTATACCAATTATAATTG
GAGGATTTGGTAATTGACTAGTACCCTTAATATTAGGAGCCCCCGATATAGCATTTCCTCGAATAAATAATATAAG
ATTCTGACTTTTACCTCCTTCCTTAACTCTATTATTAATAAGAGGTATAGTTGAAAGAGGTGTAGGTACAGGATGA
ACTGTTTACCCTCCTTTAGCTGCTGCTATTGCCCATGCAGGAGCTTCTGTAGATTTGGGAATTTTTTCTCTCCATC
TAGCAGGTGTATCTTCTATTTTAGGGGCCGTTAATTTCATAACAACTGTTATTAATATACGTTCTTTCGGAATAAC
TATAGACCAAATACCTTTATTCGTTTGATCTGTGTTTATTACTGCTATTCTCCTTCTTTTATCCCTTCCCGTCTTA
GCCGGAGCAATTACTATACTATTAACTGATCGTAATTTAAATACTTCTTTCTTTGATCCTGCTGGAGGAGGAGATC
CTGTTCTCTATCAACATTTATTTTGATTTTTTGGTCACC-3’。
Blue crab (Callinectes sapidus)Sequencing result as shown in SEQ ID NO.8:
5’-ATAGTAGGTACATCACTTAGTTTAATCATTCGAGCTGAACTAGGACAACCTGGAACCCTTATTGGAAAC
GACCAAATTTATAACGTTGTAGTCACAGCTCACGCCTTTGTTATAATTTTCTTCATGGTTATACCTATTATAATTG
GAGGATTTGGTAATTGACTAGTTCCCCTTATACTAGGAGCTCCTGATATAGCCTTCCCACGAATAAATAACATAAG
ATTCTGACTCCTACCTCCATCACTAACTCTATTACTAATAAGAGGTATGGTCGAAAGTGGAGTTGGTACAGGATGA
ACTGTTTACCCTCCCCTTGCTGCTGCTATTGCTCACGCAGGGGCCTCAGTTGATCTTGGTATTTTCTCTCTCCACT
TAGCTGGTGTATCATCAATTCTAGGGGCTGTTAACTTTATAACTACCGTTATTAATATACGTTCATTTGGTATAAG
AATAGACCAAATACCTTTATTCGTTTGATCTGTATTTATTACCGCTATTCTTCTACTTCTTTCTCTACCTGTATTA
GCAGGTGCTATTACTATACTTCTCACTGATCGAAACTTAAATACCTCATTCTTCGACCCAGCTGGAGGAGGCGACC
CTGTTCTCTACCAGCATCTATTCTGATTTTTCGGACATC-3’。
The sequencing result of mud crab (Scylla serrata) is as shown in SEQ ID NO.9:
5’-ATAGTAGGGACTTCATTAAGTCTAATTATCCGTGCTGAATTAGGACAGCCAGGTACACTTATTGGCAAC
GATCAAATCTATAATGTTGTTGTTACCGCTCATGCTTTTGTTATAATCTTTTTCATAGTTATACCAATTATAATTG
GAGGATTTGGTAATTGATTAGTTCCACTTATACTAGGAGCTCCTGATATAGCTTTTCCTCGTATAAATAATATAAG
ATTCTGACTTTTACCTCCATCTCTAACTCTATTATTAATAAGAGGTATAGTAGAAAGAGGAGTTGGTACAGGTTGA
ACTGTTTATCCACCTTTAGCAGCAGCTATTGCCCATGCAGGTGCTTCAGTCGACCTTGGTATTTTTTCGCTCCATC
TTGCAGGTGTCTCTTCAATCCTTGGTGCAGTTAATTTTATAACTACTGTAATTAATATACGATCTTTCGGTATAAG
AATAGACCAAATACCTTTATTCGTTTGATCTGTTTTCATTACGGCAATTCTTCTTCTTTTATCCCTACCAGTTCTA
GCAGGAGCAATCACTATACTTTTAACCGACCGAAATCTTAATACATCATTTTTTGACCCTGCTGGTGGTGGAGACC
CTGTCTTATATCAACACTTATTCTGATTTTTTGGTCACC-3’。
Scylla paramamosain (Scylla paramamosain) sequencing result as shown in SEQ ID NO.10:
5’-ATAGTAGGAACCTCATTAAGTTTAATTATTCGTGCTGAACTAGGACAACCAGGAACACTTATTGGTAAT
GATCAAATTTATAATGTTGTTGTTACCGCTCATGCTTTCGTTATAATCTTTTTTATAGTTATACCAATTATAATTG
GAGGATTTGGTAATTGACTAGTCCCCCTTATATTAGGAGCCCCTGACATAGCATTCCCTCGTATAAACAATATAAG
ATTTTGACTCTTACCCCCATCTCTGACTCTATTATTAATAAGAGGTATAGTAGAAAGAGGTGTCGGCACAGGTTGA
ACTGTCTATCCCCCACTAGCAGCAGCTATTGCCCATGCAGGGGCTTCAGTCGATCTAGGTATTTTCTCACTTCATC
TTGCAGGTGTCTCTTCTATTCTTGGTGCAGTTAATTTTATAACAACTGTAATTAATATACGATCTTTTGGTATAAG
AATAGACCAAATACCTTTATTTGTTTGATCTGTATTTATTACTGCAATTCTCTTACTTTTATCCTTACCAGTTCTA
GCAGGAGCAATTACTATACTTCTAACCGATCGAAACCTTAATACATCATTTTTTGACCCTGCTGGCGGTGGAGACC
CTGTTTTGTACCAGCATTTATTCTGATTCTTTGGTCACC-3’。
The sequencing result of Portunus pelagicus (Portunus pelagicus) is as shown in SEQ ID NO.11:
5’-ATAGTAGGGACTTCACTTAGTCTGATTATTCGAGCAGAACTAGGTCAACCTGGTACTCTTATTGGTAAT
GACCAAATTTACAATGTTGTAGTTACAGCTCATGCTTTTGTGATAATTTTCTTTATAGTTATACCAATTATGATTG
GAGGATTTGGTAACTGACTAGTACCATTAATGCTAGGAGCCCCTGATATGGCTTTTCCTCGTATGAACAACATAAG
ATTTTGACTTCTCCCTCCTTCTTTAACTCTACTTCTTATAAGAGGTATGGTAGAAAGAGGTGTTGGTACAGGCTGA
ACCGTCTATCCTCCTCTTTCGGCAGCGATCGCTCACGCAGGAGCTTCTGTAGATCTGGGTATTTTCTCTTTACATC
TAGCAGGTGTTTCCTCTATTTTAGGTGCAGTAAATTTCATAACCACCGTTATTAATATGCGATCTTTTGGGATAAG
AATAGACCAAATACCATTATTCGTTTGATCAGTATTTATCACTGCTATTCTTCTCCTTTTATCTCTCCCTGTTCTT
GCTGGAGCTATTACTATGCTTCTTACAGATCGAAATCTCAATACTTCATTCTTTGACCCTGCCGGTGGTGGAGACC
CTGTACTCTACCAACACTTATTTTGATTTTTTGGTCCCC-3’。
The sequencing result of P.sanguinolentus (Portunus sanguinolentus) is as shown in SEQ ID NO.12:
5’-ATAGTAGGAACCTCACTAAGTCTGATCATTCGAGCTGAGTTAGGGCAGCCAGGAACTCTTATTGGTAAC
GATCAAATTTATAACGTTGTTGTCACCGCTCATGCCTTTGTTATAATTTTCTTTATAGTTATACCAATTATAATTG
GTGGATTTGGTAACTGACTTGTACCCTTAATGCTTGGGGCCCCTGATATAGCATTCCCTCGTATAAATAATATAAG
ATTTTGACTCCTTCCTCCCTCACTCACCCTCCTTCTTATAAGAGGTATGGTTGAAAGTGGTGTAGGTACTGGTTGA
ACTGTCTACCCTCCTCTTGCTGCCGCTATTGCCCACGCTGGAGCTTCAGTCGATTTGGGGATTTTCTCCTTGCATT
TAGCTGGTGTGTCCTCTATTCTAGGTGCCGTAAACTTTATAACTACTGTAATTAACATACGATCCTTCGGCATAAG
AATGGACCAGATACCGTTATTTGTGTGATCAGTCTTCATTACCGCTATTCTGCTCCTTCTCTCCCTACCTGTTCTT
GCAGGAGCTATTACTATGCTTTTAACAGATCGTAACCTCAACACCTCCTTCTTTGATCCTGCAGGTGGTGGAGACC
CTGTTTTATATCAACACTTATTCTGATTTTTTGGTCCCC-3’。
Sequence table
<110>Zhejiang Ocean university
<120>a kind of Portumidae mitochondrial COI gene universal primer and design and amplification method
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>forward primer (Primer-F)
<400> 1
tcnacaaayc ataaagayat ygg 23
<210> 2
<211> 26
<212> DNA
<213>reverse primer (Primer-R)
<400> 2
tanacytcwg grtghccraa raayca 26
<210> 3
<211> 640
<212> DNA
<213>Dun Chi Charybdis (Charybdis hellerii)
<400> 3
atagttggaa catcattaag attaattatt cgagcagaat taggtcaacc aggtacttta 60
attggcaatg atcaaattta caatgtcgta gttacagctc atgcatttgt tataattttc 120
tttatagtta tgccaattat gattggagga tttggtaatt gactcgtacc tttaatatta 180
ggagcccctg atatagcatt ccctcgaata aataatataa gattctgact ccttcctccc 240
tcactaactt tactattaat aagaggaata gttgaaagag gtgttggtac cggttgaaca 300
gtataccctc ccttagcggc tgccatcgcc catgcaggtg cctctgtaga tctaggaatt 360
ttctctcttc atttagcagg tgtttcttct attttagggg ccgttaattt tataacaact 420
gttattaata tacgctcctt tggtataagt atagaccaaa tacctttatt tgtttggtca 480
gttttcatta ctgcaatcct tctacttctg tcacttcctg tattagctgg cgctattact 540
atactattaa cagaccgaaa tttaaatact tcattctttg atcctgctgg aggaggagac 600
cccgttcttt accaacattt attttgattt tttggcaccc 640
<210> 4
<211> 640
<212> DNA
<213>Jing Ying Charybdis (Charybdis lucifera)
<400> 4
atagttggaa catcattaag actaattatt cgagcagagt taggtcagcc aggaacgtta 60
attggtaatg accagattta taatgttgta gtcacagctc atgcatttgt cataattttt 120
ttcatagtta taccaattat aattggaggg tttggtaatt gacttgtacc tttaatatta 180
ggagctcctg atatagcctt tccccgtata aacaatataa gattctgact ccttcctcct 240
tcattaactt tactattaat aagtggtata gtcgaaagag gtgtaggtac tggttggaca 300
gtttatcctc ctctagcagc tgctattgcc cacgcagggg cctctgtaga tttgggaatt 360
ttctctcttc acttagcagg ggtttcttct attttaggag ccgttaattt cataacaact 420
gttattaata tacgttcctt cggtataagt atagatcaaa tacctttatt tgtttgatca 480
gtcttcatta ctgcaattct ccttttactt tcacttcctg ttctagccgg agctattact 540
atattactaa cagatcgaaa cttaaatact tcattttttg atcctgctgg gggaggggac 600
cctgttcttt atcaacattt attctgattt tttggccccc 640
<210> 5
<211> 640
<212> DNA
<213>Xiu Ban Charybdis (Charybdis feriatus)
<400> 5
atggttggga catcattaag actaattatt cgagccgaac taggtcaacc aggtacccta 60
attgggaatg atcaaattta taatgttgtt gttactgccc atgcatttgt tataattttc 120
tttatagtta taccaattat aattggagga tttggtaact gacttgtacc tttaatatta 180
ggagctcctg atatagcatt tcctcgtata aataatataa gattttgact tcttcctcct 240
tctttaacat tactcctaat aagagggata gttgaaagag gtgtcggtac tggatgaacc 300
gtgtaccctc ctttagcagc cgctattgcc cacgcaggtg cttctgttga tcttggtatt 360
ttctctcttc acctggccgg tgtttcctct attttaggag ctgtaaattt tataactact 420
gttattaaca tacgctcttt tggtataaga atagatcaaa tacctctatt tgtatgatca 480
gtatttatta ccgcaattct ccttttatta tctctccctg tcctggctgg agctattact 540
atattattga cagaccgtaa tttaaatact tcattttttg atcctgcagg aggaggagat 600
cctgttctct accaacactt attttgattt tttggtcccc 640
<210> 6
<211> 640
<212> DNA
<213>Shuan Ban Charybdis (Charybdis bimaculata)
<400> 6
atagttggaa cttctttaag attaattatt cgagctgaac ttggtcaacc cggaacctta 60
attggtaatg atcaaattta taatgttgta gttacagcac acgcatttgt tataattttc 120
ttcatagtta taccaattat aattggagga tttggaaact gacttgttcc tttaatactt 180
ggggctcctg atatagcatt ccctcgtata aataatataa gattttgact tctacctcct 240
tctttaactt tattattaat aagaggaatg gttgaaagag gtgtaggtac cggatgaaca 300
gtttaccctc ctttggcagc tgccattgct catgcaggtg cctctgtaga tttaggtatt 360
ttttctcttc atcttgcagg tgtttcttct attttaggag ctgtaaattt tataactact 420
gttattaata tacgatctta tggtataacg atggatcaaa tacccttatt tgtctgatca 480
gttttcatta ccgctattct tcttctttta tcattacccg ttttagctgg agctattaca 540
atactattaa ctgatcgtaa tttaaacact tcattttttg atcctgccgg aggtggagac 600
cctgttcttt atcaacattt attctgattt tttggtcatc 640
<210> 7
<211> 640
<212> DNA
<213>Shan Yong Charybdis (Charybdis natator)
<400> 7
atagtaggga catcactaag attaatcatt cgagcagaac tcggacagcc cggaactcta 60
attggaaatg atcaaattta taatgttgta gttacagcac acgcatttgt tataattttc 120
tttatagtta taccaattat aattggagga tttggtaatt gactagtacc cttaatatta 180
ggagcccccg atatagcatt tcctcgaata aataatataa gattctgact tttacctcct 240
tccttaactc tattattaat aagaggtata gttgaaagag gtgtaggtac aggatgaact 300
gtttaccctc ctttagctgc tgctattgcc catgcaggag cttctgtaga tttgggaatt 360
ttttctctcc atctagcagg tgtatcttct attttagggg ccgttaattt cataacaact 420
gttattaata tacgttcttt cggaataact atagaccaaa tacctttatt cgtttgatct 480
gtgtttatta ctgctattct ccttctttta tcccttcccg tcttagccgg agcaattact 540
atactattaa ctgatcgtaa tttaaatact tctttctttg atcctgctgg aggaggagat 600
cctgttctct atcaacattt attttgattt tttggtcacc 640
<210> 8
<211> 640
<212> DNA
<213>blue crab (Callinectes sapidus)
<400> 8
atagtaggta catcacttag tttaatcatt cgagctgaac taggacaacc tggaaccctt 60
attggaaacg accaaattta taacgttgta gtcacagctc acgcctttgt tataattttc 120
ttcatggtta tacctattat aattggagga tttggtaatt gactagttcc ccttatacta 180
ggagctcctg atatagcctt cccacgaata aataacataa gattctgact cctacctcca 240
tcactaactc tattactaat aagaggtatg gtcgaaagtg gagttggtac aggatgaact 300
gtttaccctc cccttgctgc tgctattgct cacgcagggg cctcagttga tcttggtatt 360
ttctctctcc acttagctgg tgtatcatca attctagggg ctgttaactt tataactacc 420
gttattaata tacgttcatt tggtataaga atagaccaaa tacctttatt cgtttgatct 480
gtatttatta ccgctattct tctacttctt tctctacctg tattagcagg tgctattact 540
atacttctca ctgatcgaaa cttaaatacc tcattcttcg acccagctgg aggaggcgac 600
cctgttctct accagcatct attctgattt ttcggacatc 640
<210> 9
<211> 640
<212> DNA
<213>mud crab (Scylla serrata)
<400> 9
atagtaggga cttcattaag tctaattatc cgtgctgaat taggacagcc aggtacactt 60
attggcaacg atcaaatcta taatgttgtt gttaccgctc atgcttttgt tataatcttt 120
ttcatagtta taccaattat aattggagga tttggtaatt gattagttcc acttatacta 180
ggagctcctg atatagcttt tcctcgtata aataatataa gattctgact tttacctcca 240
tctctaactc tattattaat aagaggtata gtagaaagag gagttggtac aggttgaact 300
gtttatccac ctttagcagc agctattgcc catgcaggtg cttcagtcga ccttggtatt 360
ttttcgctcc atcttgcagg tgtctcttca atccttggtg cagttaattt tataactact 420
gtaattaata tacgatcttt cggtataaga atagaccaaa tacctttatt cgtttgatct 480
gttttcatta cggcaattct tcttctttta tccctaccag ttctagcagg agcaatcact 540
atacttttaa ccgaccgaaa tcttaataca tcattttttg accctgctgg tggtggagac 600
cctgtcttat atcaacactt attctgattt tttggtcacc 640
<210> 10
<211> 640
<212> DNA
<213>Scylla paramamosain (Scylla paramamosain)
<400> 10
atagtaggaa cctcattaag tttaattatt cgtgctgaac taggacaacc aggaacactt 60
attggtaatg atcaaattta taatgttgtt gttaccgctc atgctttcgt tataatcttt 120
tttatagtta taccaattat aattggagga tttggtaatt gactagtccc ccttatatta 180
ggagcccctg acatagcatt ccctcgtata aacaatataa gattttgact cttaccccca 240
tctctgactc tattattaat aagaggtata gtagaaagag gtgtcggcac aggttgaact 300
gtctatcccc cactagcagc agctattgcc catgcagggg cttcagtcga tctaggtatt 360
ttctcacttc atcttgcagg tgtctcttct attcttggtg cagttaattt tataacaact 420
gtaattaata tacgatcttt tggtataaga atagaccaaa tacctttatt tgtttgatct 480
gtatttatta ctgcaattct cttactttta tccttaccag ttctagcagg agcaattact 540
atacttctaa ccgatcgaaa ccttaataca tcattttttg accctgctgg cggtggagac 600
cctgttttgt accagcattt attctgattc tttggtcacc 640
<210> 11
<211> 640
<212> DNA
<213>Portunus pelagicus (Portunus pelagicus)
<400> 11
atagtaggga cttcacttag tctgattatt cgagcagaac taggtcaacc tggtactctt 60
attggtaatg accaaattta caatgttgta gttacagctc atgcttttgt gataattttc 120
tttatagtta taccaattat gattggagga tttggtaact gactagtacc attaatgcta 180
ggagcccctg atatggcttt tcctcgtatg aacaacataa gattttgact tctccctcct 240
tctttaactc tacttcttat aagaggtatg gtagaaagag gtgttggtac aggctgaacc 300
gtctatcctc ctctttcggc agcgatcgct cacgcaggag cttctgtaga tctgggtatt 360
ttctctttac atctagcagg tgtttcctct attttaggtg cagtaaattt cataaccacc 420
gttattaata tgcgatcttt tgggataaga atagaccaaa taccattatt cgtttgatca 480
gtatttatca ctgctattct tctcctttta tctctccctg ttcttgctgg agctattact 540
atgcttctta cagatcgaaa tctcaatact tcattctttg accctgccgg tggtggagac 600
cctgtactct accaacactt attttgattt tttggtcccc 640
<210> 12
<211> 640
<212> DNA
<213>P.sanguinolentus (Portunus sanguinolentus)
<400> 12
atagtaggaa cctcactaag tctgatcatt cgagctgagt tagggcagcc aggaactctt 60
attggtaacg atcaaattta taacgttgtt gtcaccgctc atgcctttgt tataattttc 120
tttatagtta taccaattat aattggtgga tttggtaact gacttgtacc cttaatgctt 180
ggggcccctg atatagcatt ccctcgtata aataatataa gattttgact ccttcctccc 240
tcactcaccc tccttcttat aagaggtatg gttgaaagtg gtgtaggtac tggttgaact 300
gtctaccctc ctcttgctgc cgctattgcc cacgctggag cttcagtcga tttggggatt 360
ttctccttgc atttagctgg tgtgtcctct attctaggtg ccgtaaactt tataactact 420
gtaattaaca tacgatcctt cggcataaga atggaccaga taccgttatt tgtgtgatca 480
gtcttcatta ccgctattct gctccttctc tccctacctg ttcttgcagg agctattact 540
atgcttttaa cagatcgtaa cctcaacacc tccttctttg atcctgcagg tggtggagac 600
cctgttttat atcaacactt attctgattt tttggtcccc 640
Claims (7)
1. a kind of Portumidae mitochondrial COI gene universal primer, which is characterized in that the Portumidae mitochondrial COI base
Because universal primer can disposably expand Portumidae mitochondrial COI gene in batches, forward primer is SEQ ID NO.1:
5 '-TCNACAAAYCATAAAGAYATYGG-3 ', reverse primer are SEQ ID NO.2: 5 '-
TANACYTCWGGRTGHCCRAARAAYCA-3’。
2. a kind of Portumidae mitochondrial COI gene universal primer according to claim 1, which is characterized in that described
Portumidae includes Dun Chi Charybdis, Jing Ying Charybdis, Ban Charybdis, Shuan Ban Charybdis, Shan Yong Charybdis, blue crab, mud crab, Scylla paramamosain, off-lying sea shuttle
Sub- crab and P.sanguinolentus.
3. a kind of design of Portumidae mitochondrial COI gene universal primer as described in claim 1, which is characterized in that from
All Portumidae COI gene orders announced at present are downloaded in Genbank database, and multiple ratio is carried out to these sequences
It is right, region more biggish than more conservative region and variation is found out, universal primer is designed in two sections of conservative regions, amplifies centre
Series of variation, universal primer amplification length be 600-700 bp.
4. a kind of amplification method of Portumidae mitochondrial COI gene universal primer as described in claim 1, feature exist
In, the Portumidae mitochondrial COI gene universal primer amplification method the following steps are included:
(1) DNA extraction agent box method extracts Portumidae mitochondrial COI gene to be measured；
(2) using Portumidae mitochondrial COI gene universal primer described in claim 1；
(3) using Portumidae mitochondrial COI gene as template, PCR amplification is carried out；
(4) amplified production is tapped and recovered amplified fragments with 1.0% agarose gel electrophoresis, RNA isolation kit, is sequenced.
5. a kind of amplification method of Portumidae mitochondrial COI gene universal primer according to claim 4, feature exist
In the reaction system composition of step (3) described PCR amplification are as follows: dNTP2 μ L of 2.5mM, 10 × Taq DNA polymerase
The TaqDNA of Buffer2.5 μ L, 10 μM of forward primer SEQ ID NO.1 and each 1 μ L of reverse primer SEQ ID NO.2,5U/ μ L
0.2 μ L of polymerase, the 1 μ L of DNA template solution of 100g/ μ L and sterilizing 17.3 μ L of distilled water.
6. a kind of amplification method of Portumidae mitochondrial COI gene universal primer according to claim 4 or 5, special
Sign is, the PCR amplification condition in the step (3) are as follows: 95 DEG C of initial denaturations 5min, subsequent 95 DEG C of denaturation 30s, 56 DEG C of annealing
30s, 72 DEG C of extension 45s carry out 35 circulations altogether, finally carry out 72 DEG C of extension 5min, save under the conditions of 4 DEG C.
7. a kind of amplification method of Portumidae mitochondrial COI gene universal primer according to claim 4, feature exist
In the Portumidae to be measured includes Dun Chi Charybdis, Jing Ying Charybdis, Ban Charybdis, Shuan Ban Charybdis, Shan Yong Charybdis, blue crab, mud crab, intends
Cave mud crab, Portunus pelagicus and P.sanguinolentus.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114350823A (en) * | 2022-01-24 | 2022-04-15 | 山东省海洋资源与环境研究院(山东省海洋环境监测中心、山东省水产品质量检验中心) | Method for rapidly detecting dynamic change of portunus trituberculatus resource quantity |
| CN116064834A (en) * | 2022-09-23 | 2023-05-05 | 中国水产科学研究院黄海水产研究所 | Method for evaluating genetic diversity of Chinese prawn population by using environmental DNA |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630244A (en) * | 2015-01-26 | 2015-05-20 | 中国科学院海洋研究所 | COI gene sequence of species of coral reef benthos and species identification method utilizing COI gene sequence |
| CN107937560A (en) * | 2017-11-20 | 2018-04-20 | 浙江海洋大学 | A kind of siphonopods mitochondrial COI gene universal primer and design and amplification method |
-
2018
- 2018-11-01 CN CN201811296203.4A patent/CN109234409A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630244A (en) * | 2015-01-26 | 2015-05-20 | 中国科学院海洋研究所 | COI gene sequence of species of coral reef benthos and species identification method utilizing COI gene sequence |
| CN107937560A (en) * | 2017-11-20 | 2018-04-20 | 浙江海洋大学 | A kind of siphonopods mitochondrial COI gene universal primer and design and amplification method |
Non-Patent Citations (4)
| Title |
|---|
| J GELLER等: "Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all‐taxa biotic surveys", 《MOL ECOL RESOUR》 * |
| S UMAMAHESWARI等: "Discrimination of four marine crabs and one freshwater crab through mt-COI gene", 《JOURNAL OF ENTOMOLOGY AND ZOOLOGY STUDIES》 * |
| 常抗美等: "中国沿海7个长蛸（Octopus variabilis）群体COI基因的遗传变异研究", 《海洋与湖沼》 * |
| 王秀亮等: "舟山海域梭子蟹科重要种类DNA条形码建立及其系统发育研究", 《浙江海洋学院学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114350823A (en) * | 2022-01-24 | 2022-04-15 | 山东省海洋资源与环境研究院(山东省海洋环境监测中心、山东省水产品质量检验中心) | Method for rapidly detecting dynamic change of portunus trituberculatus resource quantity |
| CN114350823B (en) * | 2022-01-24 | 2023-09-19 | 山东省海洋资源与环境研究院(山东省海洋环境监测中心、山东省水产品质量检验中心) | Method for rapidly detecting dynamic change of portunus trituberculatus resource |
| CN116064834A (en) * | 2022-09-23 | 2023-05-05 | 中国水产科学研究院黄海水产研究所 | Method for evaluating genetic diversity of Chinese prawn population by using environmental DNA |
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Application publication date: 20190118 |