CN105462995A - Barnea dilatata mitochondrial COI gene amplification primer - Google Patents

Barnea dilatata mitochondrial COI gene amplification primer Download PDF

Info

Publication number
CN105462995A
CN105462995A CN201610021309.8A CN201610021309A CN105462995A CN 105462995 A CN105462995 A CN 105462995A CN 201610021309 A CN201610021309 A CN 201610021309A CN 105462995 A CN105462995 A CN 105462995A
Authority
CN
China
Prior art keywords
dilatata
barnea
primer
coi gene
seqidno
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610021309.8A
Other languages
Chinese (zh)
Inventor
张婷婷
李莉
王英俊
邹琰
刘童
刘洪军
王慧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong Marine Biology Institute
Original Assignee
Shandong Marine Biology Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong Marine Biology Institute filed Critical Shandong Marine Biology Institute
Priority to CN201610021309.8A priority Critical patent/CN105462995A/en
Publication of CN105462995A publication Critical patent/CN105462995A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0053—Oxidoreductases (1.) acting on a heme group of donors (1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y109/00—Oxidoreductases acting on a heme group of donors (1.9)
    • C12Y109/03—Oxidoreductases acting on a heme group of donors (1.9) with oxygen as acceptor (1.9.3)
    • C12Y109/03001—Cytochrome-c oxidase (1.9.3.1)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a barnea dilatata mitochondrial COI gene. The nucleotide sequence of the gene is SEQ ID NO: 1. The base sequence is used for designing an amplification primer for detecting barnea dilatata. The primer is designed for barnea dilatata mitochondrial COI gene segments. Compared with a COI gene primer universal for mollusks, the amplification primer has the advantage of absolute specificity. The amplification efficiency of the primer reaches as high as 90% or above, amplification efficiency is high, and time and cost for amplification are greatly reduced.

Description

A kind of Barnea dilatata mitochondrion COI gene amplification primers
Technical field
The invention belongs to molecular marker screening technical field, be specifically related to a kind of Barnea dilatata mitochondrion COI gene amplification primers.
Background technology
Barnea dilatata (Barneadilatata) is under the jurisdiction of Mollusca (Mollusca), Bivalvia (Bivalvia), MYOIDA (Myoida), Pholadidae (Pholadidae), full piddock belongs to (GenreBarneaLeach), is distributed in Philippines, Japan and China coast; To hide at the bottom of many ooze near river mouth life, can dive in mud very dark.Barnea dilatata is as the shellfish of distribution coastal area of china, and rich in proteins and VITAMIN, meat flavour is delicious.All propagate artificially at China Fujian, zhejiang and other places.Research in Barnea dilatata artificial propagation, reproductive growth, Physiology and biochemistry a lot, but is carried out less based on the research of the genetic diversity of molecule marker, and this present situation is extremely unfavorable to the development and protection of Barnea dilatata germ plasm resource.Mitochondrial DNA (MitochondrialDNA and mtDNA) has monolepsis, without characteristics such as restructuring, Neutral Evolutions, plays an important role in molecule genetics research.COI gene in mtDNA is the relatively fast region of evolutionary rate, differentiates, is widely used in population genetic diversity clam molecular evolution in invertebrates phylogenetic systematics, kind.The amplification of universal primer COIL1490 and COIL2198 in Barnea dilatata designed due to Folmer (1994) is undesirable, and agarose gel electrophoresis detected result display major part is individual without amplified band.It is less that current Pholadidae COI Gene increases out, only has 12 sequences at US National Biotechnology Information center, and Barnea dilatata only 2 sequences.Exploitation practicality good COI gene amplification primer can effectively address these problems, and also can be the diversity evaluation of Barnea dilatata, conservation of resources and the aspect such as utilization, genetic breeding and lays the foundation.
Summary of the invention
The invention provides the good primer of a kind of Barnea dilatata mitochondrial COI gene suitability and discrimination method thereof, thus make up the deficiencies in the prior art.
First the present invention provides a kind of Barnea dilatata COI gene, and its nucleotides sequence is classified as SEQIDNO:1;
Above-mentioned gene order detects the amplimer of Barnea dilatata for designing;
Another aspect of the present invention provides a kind of Barnea dilatata mitochondrion COI gene amplification primers, and the sequence information of primer is as follows:
Upstream primer COIBF:5`-CGACGAATCATAAGGACAT-3` (SEQIDNO:2),
Downstream primer COIBR:5`-GCCCGAGGGATCAAAG-3` (SEQIDNO:5);
Above-mentioned upstream primer, its sequence can also be SEQIDNO:3 or SEQIDNO:4;
Above-mentioned downstream primer, its sequence can also be SEQIDNO:6 or SEQIDNO:7;
Above-mentioned primer is used for the multifarious detection of population genetic of Barnea dilatata.
Compared with prior art, tool of the present invention has the following advantages:
(1) high specificity, this primer is for the design of Barnea dilatata COI Gene, and the COI gene primer that relative mollusk is general, has absolute specificity advantage.
(2) efficiency is high, and the amplification efficiency of this primer is up to more than 90%, and amplification efficiency is high, greatly reduces time and the expense of amplification.
(3) easy and simple to handle, and cost is not high.Relative cloning and sequencing, utilizes Auele Specific Primer to carry out the method increased, simple to operate, saves time and expense.Be applicable to the experimental study of needs extensive amplification COI gene.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1: the amplification of Barnea dilatata COI gene fragment and other several shellfish genome C OI gene fragments and sequential analysis
Amplification to Barnea dilatata COI gene:
(1) by CTAB method, DNA extraction is carried out to the Barnea dilatata (number is 16, totally 32) picking up from Ningbo of Zhejiang and Qinhuangdao, add distilled water dilution for the working fluid of 100ng/ μ l for subsequent use.
(2) utilize above-mentioned primer pair DNA working fluid to carry out pcr amplification, the obtain solution of this reaction system comprises: 1 μ l template DNA, 0.05 μ lTaq polysaccharase, 1 μ l10 × PCRb μ ffer, 0.8 μ ldNTPs, each 1 μ l of upstream and downstream primer, 5.15 μ l distilled waters.Pcr amplification condition is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 45s, 46-56 DEG C of annealing temperature 45s, and 72 DEG C extend 45s, totally 35 circulations, is finally 72 DEG C and extends 10min.
(3) detect amplified production with 1.5% biological agarose gel electrophoresis, amplified production transfers to Hua Da gene to carry out two-way order-checking.After the sequence BioEdit software splicing recorded, carry out multiple compare of analysis with the COI sequence that NCBI downloads, determine that gained sequence is Barnea dilatata COI sequence.
Applicant analyzes Barnea dilatata COI Gene Partial sequence (SEQIDNO:1) that amplification obtains; Blast comparison is carried out in ncbi database, the COI gene fragment of DNASTAR software to Barnea dilatata and other several shellfishes is utilized to be analyzed, the COI gene nucleotide consistence of discovery Barnea dilatata and the full piddock of crisp shell is 84%, be 87% with the consistence of the COI gene nucleotide of short tube callistin shellfish, be 79% with the COI gene nucleotide consistence of decorative pattern dish clam, result shows that the COI gene nucleotide series of Barnea dilatata and the COI gene nucleotide series of other several shellfishes exist obvious difference.
Embodiment 2: the design of primer
(1) by CTAB method, DNA extraction is carried out to the Barnea dilatata (number is 16, totally 32) picking up from Ningbo of Zhejiang and Qinhuangdao, add distilled water dilution for the working fluid of 100ng/ μ l for subsequent use.
(2) COI universal primer (LCO1490F and HCO2198R, Folmer1994) is utilized to amplify the partial sequence of Barnea dilatata.
(3) from the part Barnea dilatata sequence of above-mentioned gained, by software BioEdit (http://www.Mbio.Ncsu.edu/BioEdit/bioedit.html), multi-contrast's analysis is carried out to above-mentioned sequence, the conservative region before and after determining.
(4) through comparison, each 150 base pairs in front and back end, at conservative region, devise 3 pairs of primers with primer-design software PrimerPremier5, all do not occur primer dimer, hairpin structure and mismatching phenomenon during the primer of design as far as possible.The primer of design transfers to Shanghai Sheng Gong Bioisystech Co., Ltd to synthesize, and the sequence information of primer is as follows:
Upstream primer: COIBF:5`-CGACGAATCATAAGGACAT-3` (SEQIDNO:2);
COIBF-1：5`-CGACGAATCATAAGGAC-3`(SEQIDNO:3)；
COIBF-2：5`-CGACGAATCATAAGGACAT-3`(SEQIDNO:4)。
Downstream primer: COIBR:5`-GCCCGAGGGATCAAAG-3` (SEQIDNO:5);
COIBR-1：5`-GCCGAAGAATCAAAA-3`(SEQIDNO:6)；
COIBR-2：5`-GGATGGCCGAAGAATC-3`(SEQIDNO:7)。
(5) in 10 μ lPCR reaction systems, to increase respectively the individual COI gene fragment of Barnea dilatata 32 with 3 pairs of primers of synthesis, the obtain solution of this reaction system comprises: 1 μ l template DNA, 0.05 μ lTaq polysaccharase, 1 μ l10 × PCRbuffer, 0.8 μ ldNTPs, each 1 μ l of upstream and downstream primer, 5.15 μ l distilled waters.Pcr amplification condition is: 94 DEG C of denaturation 3min, 94 DEG C of sex change 45s, 46-56 DEG C of annealing temperature 45s, and 72 DEG C extend 45s, totally 38 circulations, is finally 72 DEG C and extends 10min.
(6) by product that each primer amplification goes out, detect with 1.5% biological agarose gel electrophoresis, by the result observation analysis of gel electrophoresis, three pairs of primers can amplify single band, but the band that COIBF and COIBR amplifies is the most clear, the more other two pairs of primers of amplification efficiency are high, and the individual amount of amplification is far above other two pairs of primers, amplify 29 individualities, amplification efficiency is up to 90%; Show that the amplification efficiency of COIBF and COIBR amplimer is higher.
(7) amplified production transfers to Hua Da gene to carry out two-way order-checking.After the sequence BioEdit software splicing recorded, carry out multiple compare of analysis with the COI sequence that NCBI downloads, determine that gained sequence is Barnea dilatata COI sequence, thus show that COIBF and COIBR amplimer has the specificity of amplification.

Claims (6)

1. a Barnea dilatata COI gene, is characterized in that, the nucleotides sequence of described Barnea dilatata COI gene is classified as SEQIDNO:1.
2. Barnea dilatata COI gene according to claim 1 detects the application in the amplimer of Barnea dilatata in design.
3. detect a primer for Barnea dilatata, it is characterized in that, the sequence information of described primer is as follows:
The sequence of upstream primer be SEQIDNO:2,
The sequence of downstream primer is SEQIDNO:5.
4. primer as claimed in claim 3, it is characterized in that, the sequence of described upstream primer is SEQIDNO:3 or SEQIDNO:4.
5. primer as claimed in claim 3, it is characterized in that, the sequence of described downstream primer is SEQIDNO:6 or SEQIDNO:7.
6. the application of the primer described in any one of claim 3-5 in the population genetic diversity detecting Barnea dilatata.
CN201610021309.8A 2016-01-14 2016-01-14 Barnea dilatata mitochondrial COI gene amplification primer Pending CN105462995A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610021309.8A CN105462995A (en) 2016-01-14 2016-01-14 Barnea dilatata mitochondrial COI gene amplification primer

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610021309.8A CN105462995A (en) 2016-01-14 2016-01-14 Barnea dilatata mitochondrial COI gene amplification primer

Publications (1)

Publication Number Publication Date
CN105462995A true CN105462995A (en) 2016-04-06

Family

ID=55601154

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610021309.8A Pending CN105462995A (en) 2016-01-14 2016-01-14 Barnea dilatata mitochondrial COI gene amplification primer

Country Status (1)

Country Link
CN (1) CN105462995A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110438062A (en) * 2019-08-01 2019-11-12 北京林业大学 A kind of extraction purification and identification method of moso bamboo shoot high activity mitochondria
CN114698580A (en) * 2022-04-12 2022-07-05 世倍(厦门)海洋科技有限公司 Artificial breeding method for large-size seeds of Meretrix Linnaeus

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010040901A1 (en) * 2008-10-07 2010-04-15 Pierre Rustin Alternative oxidase and uses thereof
CN101942432A (en) * 2010-08-12 2011-01-12 中国海洋大学 Method for screening tegillarca granosa mitochondria COI gene amplification primers
CN101979536A (en) * 2010-09-26 2011-02-23 中国海洋大学 Method for screening veneridae mitochondria COI gene amplification primers
CN102776181A (en) * 2012-06-07 2012-11-14 中国海洋大学 Amplification primer of corbiculidae shellfish mitochondria COI (Cytochrome Oxidase I) gene
CN102776182A (en) * 2012-06-07 2012-11-14 中国海洋大学 Amplification primer of psammobiidae shellfish mitochondria COI (Cytochrome Oxidase I) gene

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010040901A1 (en) * 2008-10-07 2010-04-15 Pierre Rustin Alternative oxidase and uses thereof
CN101942432A (en) * 2010-08-12 2011-01-12 中国海洋大学 Method for screening tegillarca granosa mitochondria COI gene amplification primers
CN101979536A (en) * 2010-09-26 2011-02-23 中国海洋大学 Method for screening veneridae mitochondria COI gene amplification primers
CN102776181A (en) * 2012-06-07 2012-11-14 中国海洋大学 Amplification primer of corbiculidae shellfish mitochondria COI (Cytochrome Oxidase I) gene
CN102776182A (en) * 2012-06-07 2012-11-14 中国海洋大学 Amplification primer of psammobiidae shellfish mitochondria COI (Cytochrome Oxidase I) gene

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
LIU,M. 等: "Barnea dilatata isolate KKQHS-5 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial", 《GENBANK DATABASE》 *
NT MIKKELSEN 等: "Local scale DNA barcoding of bivalves (Mollusca): a case study", 《ZOOLOGICA SCRIPTA》 *
O. FOLMER 等: "DNA primers for amplification of mitochondrial cytochrome C oxidase subunit I from diverse metazoan invertebrates", 《MOL MAR BIOL BIOTECHNOL.》 *
刘亮伟 等: "《基因工程原理与实验指导》", 30 September 2010, 中国轻工业出版社 *
李琪 等: "DNA条形码及其在海洋生物中的引用", 《中国海洋大学学报》 *
管成林 等: "基于线粒体COI基因比较分析野生与养殖厚壳贻贝种群的遗传多样性", 《安徽农业科学》 *
郑文娟 等: "基于线粒体COI基因序列探讨泥蚶的遗传分化", 《动物学研究》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110438062A (en) * 2019-08-01 2019-11-12 北京林业大学 A kind of extraction purification and identification method of moso bamboo shoot high activity mitochondria
CN114698580A (en) * 2022-04-12 2022-07-05 世倍(厦门)海洋科技有限公司 Artificial breeding method for large-size seeds of Meretrix Linnaeus

Similar Documents

Publication Publication Date Title
Liu et al. Complete mitochondrial DNA sequence of the ark shell Scapharca broughtonii: an ultra-large metazoan mitochondrial genome
Wen et al. Genetic identification of global commercial sea cucumber species on the basis of mitochondrial DNA sequences
Wen et al. The application of PCR–RFLP and FINS for species identification used in sea cucumbers (Aspidochirotida: Stichopodidae) products from the market
CN108950017B (en) Universal amplification primer for mitochondria 16S rRNA gene of tridacnidae species in southern China and screening method thereof
CN103740729A (en) SNP locus related to growth characteristics of patinopecten yessoensis and detection and application thereof
CN102140522A (en) Detection method for Apostichopus japonicas AjE101 micro-satellite DNA label
CN103583470A (en) Molecular-assisted-selection cultivation method for green-shin recessive-white-feather chicken line
Wakabayashi et al. Phylogenetic relationships among the family Ommastrephidae (Mollusca: Cephalopoda) inferred from two mitochondrial DNA gene sequences
Xu et al. Polymorphisms of heat shock protein 90 (Hsp90) in the sea cucumber Apostichopus japonicus and their association with heat-resistance
Qiu et al. Characterization of novel EST-SSR markers and their correlations with growth and nacreous secretion traits in the pearl oyster Pinctada martensii (Dunker)
Chan et al. Application of FINS and multiplex PCR for detecting genuine abalone products
Lv et al. Comparative analyses of the complete mitochondrial genomes of Dosinia clams and their phylogenetic position within Veneridae
CN103609527A (en) Green-shin recessive white-feather chicken strain breeding method
AlMomin et al. Draft genome sequence of the silver pomfret fish, Pampus argenteus
CN105462995A (en) Barnea dilatata mitochondrial COI gene amplification primer
CN105349691B (en) It is a kind of identify Crustin genetic sex DNA sequence tag and its application
CN112626238B (en) Characteristic sequence for identifying crassostrea sikamea, specificity identification primer and identification method
CN101942432B (en) Tegillarca granosa mitochondria COI gene amplification primers
Chi et al. High genetic diversities between isolates of the fish parasite Cryptocaryon irritans (Ciliophora) suggest multiple cryptic species
Ma et al. Development of 12 polymorphic microsatellite markers in Coilia ectenes Jordan and Seale, 1905 (Clupeiformes: Engraulidae) and cross-species amplification in Coilia mystus Linnaeus, 1758
Thanh et al. Genetic diversity of cultured populations of giant freshwater prawn (Macrobrachium rosenbergii) in China using mtDNA COI and 16S rDNA markers
Gharsallah et al. Insights into the genetic diversity and the phylogenetic analysis of Tunisian isolates of Tomato chlorosis virus
Xia et al. Complete mitochondrial genome of spined sleeper Eleotris oxycephala (Perciformes, Eleotridae) and phylogenetic consideration
Jiang et al. A microsatellite genetic linkage map of half smooth tongue sole (Cynoglossus semilaevis)
Yuan et al. Mitogenome evidence for the existence of cryptic species in Coelomactra antiquata

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20160406

RJ01 Rejection of invention patent application after publication