CN103074335A - Goby mitochondrion COIII and ND3 gene amplimer, design and amplification method - Google Patents
Goby mitochondrion COIII and ND3 gene amplimer, design and amplification method Download PDFInfo
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Abstract
The invention belongs to the fish mitochondrial genome research field, and concretely relates to a COIII and ND3 gene amplimer, the gene amplimer is composed of two strips of single chain oligonucleotide chains, wherein a light chain primer is a nucleotide sequence shown in SEQ ID NO.1, and a heavy chain primer is a nucleotide sequence shown in SEQ ID NO.2. The invention also provides a design and an amplification method of the amplimer. The invention can specifically amplify a plurality of goby mitochondrion COIII and ND3 gene sequences with high efficiency, and can be used in goby different classification category systems evolution and classification research.
Description
Technical field
The invention belongs to fish Mitochondrial Genome Overview research field, be specifically related to a kind of efficient amplification multiple Gobioidei mitochondrial cytochrome c (Cytc) oxidase subunit III (cytochrome c oxidase subunit III; The CO III) the most of sequence of gene and NADH reductase enzyme complex subunit 3(NADH dehydrogenase subunits 3; ND3) amplimer of gene complete sequence and design thereof and amplification method.
Background technology
The fish Mitochondrial Genome Overview by 13 proteins encoded plasmagenes, 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA) totally 37 encoding genes and one section main non-coding region (control region) form, have matrilinear inheritance simple in structure, that copy number is many, strict, recombinate hardly, coding efficiency is high, rate of evolution is fast and the different zones rate of evolution such as there are differences at the characteristics.Because these characteristics that the fish Mitochondrial Genome Overview has make it become the important molecular markers of the research fields such as fish molecular population genetics, phylogenetics and conservation biology.
Gobioidei is under the jurisdiction of Perciformes, goby suborder, is most species in the world, and one of the widest fish distribute.Estimate that global goby kind has kind more than 2000, they form a huge biological group, occupy an important position in the water surrounding especially marine eco-environment.Goby mostly is carnivorous small fish, is found everywhere through the world, and especially take the torrid zone as many, is mainly seawater fish, the life of dwelling at the end of most of battalion, and principal character is that its abdomeinal fin heals into a sucker shape.In recent years, owing to reasons such as overfishing, climatope variations, many traditional tuna fisheries resources can not form fishing news, some in addition exhausted, but the reasons such as Gobioidei is adaptable because of it, life cycle is short, reproductivity is strong, stock number but increase year by year.Most of Gobioidei economic worth is not high, and relatively less relevant for the research of Gobioidei both at home and abroad, of a great variety because of it, the formalness feature is very similar, causes being difficult for differentiating and assert.At present, the classification of Gobioidei is also very not clear, and the classification position of many types is also relatively more chaotic.
Genes involved on the full genome of plastosome and the Mitochondrial Genome Overview has been widely used in the research fields such as goby population genetics, phylogenetics and conservation biology as molecule marker.CO III and ND3 are as 3 Codocyte pigment c (Cytc) oxidase subunits and 7 one of genes of NADH reductase enzyme complex subunits of encoding in the vertebrates encoding mitochondrial protein gene, its contained phylogeny information is arranged in the better-than-average of 13 encoding egg white genes, can be applicable to vertebrate phyletic evolution and sort research.Yet the universal primer of reporting increase plastosome CO III and ND3 gene order had not been arranged, especially for Gobioidei.Therefore design the universal primer of specific amplification Gobioidei plastosome CO III and ND3 gene order, thereby lay the foundation for efficiently obtaining goby plastosome CO III and ND3 gene order and mitochondrial genome complete sequence.
Summary of the invention
Vacancy for above-mentioned prior art existence, the present invention aims to provide the single stranded oligonucleotide primer of a pair of efficiently specific amplified multiple Gobioidei plastosome CO III and ND3 gene order, thereby provides a powerful for effectively obtaining fast Gobioidei plastosome CO III and ND3 gene order to carry out phyletic evolution and sort research.The present invention also aims to provide the method for design of described Gobioidei plastosome CO III and ND3 gene amplification primer, and the method for utilizing described Gobioidei plastosome CO III and ND3 gene amplification primer prawn tiger fish plastosome CO III and ND3 gene to increase.
For realizing goal of the invention of the present invention, the contriver provides following technical scheme:
Gobioidei plastosome CO III and ND3 gene amplification primer are comprised of two single stranded oligonucleotide chains, and wherein the light chain primer is the nucleotide sequence shown in the SEQ ID No.1; The heavy chain primer is the nucleotide sequence shown in the SEQ ID No.2.
Shown in light chain primer Gobies-CO III of the present invention-ND3-F(SEQ ID No.1) 21 base: ATACCACATAGTAGACCCCAG are arranged, be positioned on the CO III gene; Heavy chain primer Gobies-CO III-ND3-R (shown in the SEQ ID No.2) has 21 base: GACTTTAACCACGAGCTTTTG, is positioned on the tRNA-Arg gene.
Gobioidei plastosome CO III of the present invention and the ND3 gene amplification primer 17 seed shrimps tiger fish to gathering at Area of The East China Sea, all can obtain fragment length and be the specific amplification products about 1150 bp, through order-checking and with GenBank in the comparison of homologous sequence, confirm as and comprise the chondriosome ND 3 gene complete sequence, tRNA-Gly gene complete sequence, and length is the amplified production of the CO III gene overwhelming majority sequence about 720 bp, embody the wider amplification scope of the present invention and stronger amplification ability, thereby provide a powerful for effectively obtaining fast Gobioidei line grain CO III and ND3 gene order to carry out phyletic evolution and sort research.
As preferably, according to Gobioidei plastosome CO III of the present invention and ND3 gene amplification primer, wherein said Gobioidei includes but not limited to following: Bostrichthys sinensis, blue or green mudskipper, speckle tongue goby, moustache thin white silk used in ancient China goby, hole goby, the thin sour jujube goby of green statin, red wolf's fang goby, the thin sour jujube goby of Pu Shi, spot acanthogobius, mudskipper, lance tail goby, Zhoushan reins goby, tack pole goby, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth, Taiwan ditch goby, eyeball tail tadpole goby.
The present invention also provides the method for design of above-mentioned Gobioidei plastosome CO III and ND3 gene amplification primer, namely login CO III gene and the tRNA-Arg gene order of the Mitochondrial Genome Overview of other species of Gobioidei that GenBank database (http://www.ncbi.nlm.nih.gov/) search in the NCBI website measured and nearly edge species, through homology relatively, find conserved sequence, and utilize Premier Primer5.0 software design to go out Gobioidei plastosome CO III and ND3 gene amplification primer.Used Premier Primer5.0 software is downloaded at biosoftware net http://www.bio-soft.net/.
The present invention also provides a kind of method that Gobioidei plastosome CO III and ND3 gene are increased, and it is that the dna profiling solution that adopts above-mentioned Gobioidei plastosome CO III and ND3 gene amplification primer to treat the test sample product carries out pcr amplification.
As preferably, according to a kind of method that Gobioidei plastosome CO III and ND3 gene are increased of the present invention, wherein the condition of pcr amplification is: 95 ℃ of denaturation 5min, then 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min20s, totally 35 circulations, and last 72 ℃ are extended 5min.
As preferably, according to a kind of method that Gobioidei plastosome CO III and ND3 gene are increased of the present invention, wherein the reaction composition of pcr amplification is: the PCR reaction system is 25 μ L, includes dNTP(2.5mM) light chain primer and heavy chain primer each 1 μ L, Taq archaeal dna polymerase (5U/ul) 0.2 μ L of 2 μ L, 10 * Taq archaeal dna polymerase Buffer, 2.5 μ L, 10 μ M, contain dna profiling solution 1 μ L and the ddH of 100ng
2O 17.3 μ L.
As preferably, according to a kind of method that Gobioidei plastosome CO III and ND3 gene are increased of the present invention, wherein said testing sample is from including but not limited to following Gobioidei: Bostrichthys sinensis, blue or green mudskipper, speckle tongue goby, moustache thin white silk used in ancient China goby, the hole goby, the thin sour jujube goby of green statin, red wolf's fang goby, the thin sour jujube goby of Pu Shi, the spot acanthogobius, mudskipper, lance tail goby, Zhoushan reins goby, tack pole goby, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth, Taiwan ditch goby, eyeball tail tadpole goby.
Compared with prior art, the present invention has following advantage:
Gobioidei plastosome CO III provided by the invention and ND3 gene amplification primer, can efficiently increase specifically multiple Gobioidei plastosome CO III and ND3 gene order can be applied to the different taxonomic category phyletic evolutions of shrimp tiger and sort research.Universal primer of the present invention and method of design thereof also can be used as the specific examples of universal primer PCR amplification principle and key parameter research simultaneously, promote the progressive development of the field that the invention relates to, thereby possess important invention value and theory significance.
Description of drawings
Fig. 1 is that Gobioidei plastosome CO III of the present invention and ND3 gene amplification primer are in order to the electrophoretogram of increase different Gobioidei plastosome CO III and ND3 gene order.
M:DNA molecular weight standard (DL2000) wherein, 1-17 is followed successively by: Bostrichthys sinensis, blue or green mudskipper, speckle tongue goby, moustache thin white silk used in ancient China goby, hole goby, the thin sour jujube goby of green statin, red wolf's fang goby, the thin sour jujube goby of Pu Shi, spot acanthogobius, mudskipper, lance tail goby, Zhoushan reins goby, tack pole goby, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth, Taiwan ditch goby, eyeball tail tadpole goby.
Embodiment
Below in conjunction with embodiment and Figure of description, be described more specifically content of the present invention.Should be appreciated that enforcement of the present invention is not limited to the following examples, any pro forma accommodation and/or change that the present invention is made all will fall into protection domain of the present invention.
In the present invention, if not refer in particular to, all part, per-cents are weight unit, and all equipment and raw material etc. all can be buied from market or the industry is commonly used.If without specializing, the method that embodiment adopts is this area current techique.
Main raw, reagent and plant and instrument:
Software and sequence resource: Premier Primer5.0 primer-design software (downloading from biological software net http://www.bio-soft.net/), Gobioidei Mitochondrial Genome Overview sequence resource (downloading Genbank database http://www.ncbi.nlm.nih.gov/ in NCBI), sequence analysis software MEGA5.0(downloads from biological software net http://www.bio-soft.net/).
Pcr amplification detects the related reagent instrument: marine animal genome DNA extracting reagent kit (TIANGEN, Beijing), and sepharose DNA reclaims test kit (TIANGEN, Beijing), conventional desk centrifuge (Thermo), the Bio-Rad C1000TM Thermal Cycler instrument (Bio-Rad, the U.S.) that increases, micropipet (Enppdorf, Germany), 96 hole PCR plates (Axygen), dNTP(TIANGEN, Beijing), 10 * Taq archaeal dna polymerase Buffer(TIANGEN, Beijing), Taq archaeal dna polymerase (TIANGEN, Beijing), the sterilization distilled water, DL2000 DNA marker(TIANGEN, Beijing), agarose (Biowest, Hong Kong), electrophoresis apparatus (DYY-6C type, Beijing 6 1), gel imaging instrument (Bio-Rad GD2000, the U.S.).
Cloning and sequencing related reagent instrument: high-pressure steam sterilizing pot (SANYO, Japan), the antibiotic sterilization LB of ammonification benzyl (Amp) substratum, LB solid culture plate (worker is given birth in Shanghai), Bechtop (SW-CJ-1G type, the star of famous brand, China), thermostat water bath (upper Nereid is grand), DH5 α competent cell (TIANGEN), Amp microbiotic (worker is given birth in Shanghai), 5-bromo-4-chloro-3-indoles-β-D-galactoside (worker is given birth in Shanghai), isopropylthio-β-D-galactoside (worker is given birth in Shanghai), pGEM-T carrier (Promega, the U.S.), constant incubator (PH070A type, Shanghai Yiheng Scientific Instruments Co., Ltd), constant temperature oscillation shaking table (training English, Taicang experimental installation factory) automated DNA sequenator (ABI 3730 types, the U.S.).
The experimental technique of unreceipted actual conditions among the embodiment, according to normal condition, molecular cloning laboratory manual (New York:Cold Spring Harbor Laboratory Press such as authors such as Sambrook, 1989) condition described in, or according to the condition of manufacturer specification sheets suggestion.
1, the design of Gobioidei plastosome CO III and ND3 gene amplification primer and synthetic:
CO III gene and the tRNA-Arg gene order of other species of Gobioidei that the GenBank database search in the login NCBI website has been measured and the Mitochondrial Genome Overview of nearly edge species, sequence is loaded among the analysis software MEGA5.0, utilize the ClustalW algorithm to carry out multisequencing contraposition compare of analysis, find conserved sequence, the conserved sequence that finds is loaded into Premier Primer5.0 software (namely under the manual option, is selecting Low under the manual designs pattern, design parameter is default setting) design Gobioidei plastosome CO III and ND3 gene amplification primer: wherein shown in light chain primer Gobies-CO III-ND3-F(SEQ ID No.1) and 21 base: ATACCACATAGTAGACCCCAG are arranged, be positioned on the CO III gene; Heavy chain primer Gobies-CO III-ND3-R (shown in the SEQ ID No.2) has 21 base: GACTTTAACCACGAGCTTTTG, is positioned on the tRNA-Arg gene.
Synthesize the primer that meets nucleotide sequence shown in SEQ ID No.1 and the SEQ ID No.1 by Nanjing Genscript Biotechnology Co., Ltd. according to contriver's requirement.
2, Gobioidei plastosome CO III and ND3 gene are increased
17 samples to be tested among the present invention all are collected in the Area of The East China Sea of China.Wherein the Gobioidei of amplification is following kind: Bostrichthys sinensis, blue or green mudskipper, speckle tongue goby, moustache thin white silk used in ancient China goby, hole goby, the thin sour jujube goby of green statin, red wolf's fang goby, the thin sour jujube goby of Pu Shi, spot acanthogobius, mudskipper, lance tail goby, Zhoushan reins goby, tack pole goby, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth, Taiwan ditch goby, eyeball tail tadpole goby.
Utilize the marine animal genome DNA extracting reagent kit to extract the genomic dna of sample to be tested, method is carried out according to the test kit specification sheets, the genomic dna of agarose electrophoresis Detection and Extraction.
PCR reaction system and reaction conditions for increase multiple Gobioidei plastosome CO III and ND3 gene are as follows: the PCR reaction system is 25 μ L, include dNTP(TIANGEN), 10 * TaqDNA polysaccharase Buffer(TIANGEN), heavy chain primer Gobies-CO III-ND3-R of light chain primer Gobies-CO III-ND3-F of 10 μ M and 10 μ M, Taq DNA polysaccharase (TIANGEN), the genomic dna template solution that contains the said extracted of 100ng, ddH
2O, wherein:
10 * TaqDNA polysaccharase Buffer, 2.5 μ L
dNTP 2μL
Gobies- COⅢ-ND3-F 1μL
Gobies- COⅢ-ND3-R 1μL
Template DNA 1μL
ddH
2O 17.3μL
Taq DNA polysaccharase 0.2 μ L.
Amplified reaction is at Bio-Rad C1000
TMCarry out on the Thermal Cycler instrument, amplification condition is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 30s then, 56 ℃ of annealing 30s, 72 ℃ are extended 1min20s, totally 35 circulations, last 72 ℃ are extended 5min.Amplified production detects its size and purity with 1% agarose gel electrophoresis.The electrophoretogram of different Gobioidei plastosome CO III and ND3 gene amplification product is seen Fig. 1.
Use the most Gobioidei line grain CO III of primer amplification and the ND3 gene order of design, all obtained single purpose fragment, the product size is about 1150 bp, and pcr amplification product directly checked order, after the steps include: that pcr amplification product tentatively quantitatively, concentration reaches the sample of about 100ng/ μ l, sends Nanjing Genscript Biotechnology Co., Ltd., entrust it to carry out two-way order-checking, sequencing primer is Gobies-CO III-ND3-F(10 μ M) and Gobies-CO III-ND3-R(10 μ M).Comprise chondriosome ND 3 gene complete sequence, tRNA-Gly gene complete sequence through checking order and compare with the homologous sequence among the GenBank, confirming as, reach the amplified production that length is the CO III gene overwhelming majority partial sequences about 720 bp.
Use the thin sour jujube goby of primer amplification Pu Shi and blue or green mudskipper plastosome CO III and the ND3 gene of design, all obtained single purpose fragment, the product size is about 1150 bp, and use sepharose DNA to reclaim test kit (TIANGEN, Shanghai) pcr amplification product is carried out glue and reclaim purifying, purified product connects with pGEM-T carrier (Promega company, the U.S.), transform, identify positive colony and order-checking.The steps include: that the pcr amplification product that reclaims purifying through glue carries out ligation with the pGEM-T carrier under the effect of T4 dna ligase.The ligation system is carrier 1 μ l, and T4 dna ligase 1 μ l connects Buffer 5ul, PCR purified product 3 μ l.Ligation is in operation on ice, and reaction solution places 4 ℃ of refrigerators to connect spend the night (at least 12 hours).Get DH5 α competent cell (TIANGEN, Beijing), the heat shock method transforms, carry out the white screening of indigo plant with containing ammonia benzyl microbiotic (Amp) LB dull and stereotyped (evenly being coated with in advance the isopropylthio-β-D-galactoside (IPTG) of 5-bromo-4-chloro-3-indoles-β of 40ul 20mg/ml-D-galactoside (X-Gal) and 30ul 0.2mol/L before the use), and sub by the positive colony of bacterium liquid pcr amplification affirmation picking.The bacterium liquid of positive colony after enlarged culturing is sent Nanjing Genscript Biotechnology Co., Ltd. and is carried out two-way direct Sequencing.Sequencing primer is SP6/T7.Sequenator is ABI 3730 full-automatic DNA sequencers.Comprise chondriosome ND 3 gene complete sequence, tRNA-Gly gene complete sequence through checking order and compare with the homologous sequence among the GenBank, confirming as, reach the amplified production that length is the CO III gene overwhelming majority sequences about 720 bp.
Practicality
Gobioidei plastosome CO III of the present invention and ND3 gene amplification primer carry out pcr amplification to the dna profiling solution at 17 kinds of ocean Gobioideis of Area of The East China Sea collection, all can obtain the specific amplification products of fragment about 1150 bp, through order-checking and with GenBank in the comparison of homologous sequence, confirm as and comprise the chondriosome ND 3 gene complete sequence, tRNA-Gly gene complete sequence, and length is the amplified production of the CO III gene overwhelming majority sequence about 720 bp, embody the wider amplification scope of the present invention and stronger amplification ability, therefore, Gobioidei plastosome CO III of the present invention and ND3 gene amplification primer can be used in the different hierarchies evolution of Gobioidei and sort research field.
Although the contriver has done comparatively detailed elaboration to technical scheme of the present invention and has enumerated, be to be understood that, for the those skilled in the art in this area, above-described embodiment is modified and/or flexible or to adopt the replacement scheme that is equal to be obvious, the essence that all can not break away from spirit of the present invention, the term that occurs among the present invention is used for elaboration and the understanding to technical solution of the present invention, can not be construed as limiting the invention.
SEQUENCE LISTING
＜110〉Oceanography Institute Of Zhejiang
＜120〉Gobioidei plastosome CO III and ND3 gene amplification primer, design and amplification method
<130> Z122339
<160> 2
<170> PatentIn version 3.3
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ataccacata gtagacccca g 21
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gactttaacc acgagctttt g 21
Claims (7)
1. Gobioidei plastosome CO III and ND3 gene amplification primer is characterized in that being comprised of two single stranded oligonucleotide chains, and wherein the light chain primer is the nucleotide sequence shown in the SEQ ID No.1; The heavy chain primer is the nucleotide sequence shown in the SEQ ID No.2.
2. Gobioidei plastosome CO III as claimed in claim 1 and ND3 gene amplification primer is characterized in that described Gobioidei comprises Bostrichthys sinensis, blue or green mudskipper, speckle tongue goby, moustache thin white silk used in ancient China goby, hole goby, the thin sour jujube goby of green statin, red wolf's fang goby, the thin sour jujube goby of Pu Shi, spot acanthogobius, mudskipper, lance tail goby, Zhoushan reins goby, tack pole goby, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth, Taiwan ditch goby, eyeball tail tadpole goby.
3. the method for design of a Gobioidei plastosome CO III as claimed in claim 1 and ND3 gene amplification primer, it is characterized in that logining CO III gene and the tRNA-Arg gene order of the Mitochondrial Genome Overview of other species of Gobioidei that the GenBank database search in the NCBI website measured and nearly edge species, through homology relatively, find conserved sequence, and utilize Premier Primer5.0 software design to go out Gobioidei plastosome CO III and ND3 gene amplification primer.
4. the method that Gobioidei plastosome CO III and ND3 gene are increased is characterized in that the dna profiling solution that adopts Gobioidei plastosome CO III as claimed in claim 1 and ND3 gene amplification primer to treat the test sample product carries out pcr amplification.
5. a kind of method that Gobioidei plastosome CO III and ND3 gene are increased as claimed in claim 4, the condition that it is characterized in that pcr amplification is: 95 ℃ of denaturation 5min, then 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min20s, totally 35 circulations, last 72 ℃ are extended 5min.
6. a kind of method that Gobioidei plastosome CO III and ND3 gene are increased as claimed in claim 4, the reaction composition that it is characterized in that pcr amplification is: the PCR reaction system is 25 μ L, include the light chain primer of dNTP 2 μ L, 10 * Taq archaeal dna polymerase Buffer, 2.5 μ L, 10 μ M of 2.5mM and heavy chain primer each 1 μ L, 5U/ul Taq archaeal dna polymerase 0.2 μ L, contain dna profiling solution 1 μ L and the ddH of 100ng
2O 17.3 μ L.
7. a kind of method that Gobioidei plastosome CO III and ND3 gene are increased as claimed in claim 4 is characterized in that described testing sample is from Bostrichthys sinensis, blue or green mudskipper, speckle tongue goby, moustache thin white silk used in ancient China goby, hole goby, the thin sour jujube goby of green statin, red wolf's fang goby, the thin sour jujube goby of Pu Shi, spot acanthogobius, mudskipper, lance tail goby, Zhoushan reins goby, tack pole goby, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth, Taiwan ditch goby, eyeball tail tadpole goby.
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| CN117418023A (en) * | 2023-12-18 | 2024-01-19 | 中国科学院海洋研究所 | Double-band onyx and imogolix species-specific markers, specific primers, application and identification method and kit |
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| CN111961733A (en) * | 2020-08-28 | 2020-11-20 | 海南热带海洋学院 | DNA bar code for identifying goby and goby of China comb and identification method and application thereof |
| CN117418023A (en) * | 2023-12-18 | 2024-01-19 | 中国科学院海洋研究所 | Double-band onyx and imogolix species-specific markers, specific primers, application and identification method and kit |
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Address after: Ocean Science College, No. 105 Wenhua Road, Dinghai District, Zhoushan City, Zhejiang Province, 316000 Patentee after: Zhejiang Ocean University Address before: Ocean Science College, No. 105 Wenhua Road, Dinghai District, Zhoushan City, Zhejiang Province, 316000 Patentee before: ZHEJIANG OCEAN University |
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Application publication date: 20130501 Assignee: Zhejiang Xinjiahe Biotechnology Co.,Ltd. Assignor: Zhejiang Ocean University Contract record no.: X2023980043657 Denomination of invention: Primers, design, and amplification methods for mitochondrial CO III and ND3 genes in shrimp, tiger, and fish Granted publication date: 20140709 License type: Common License Record date: 20231019 |
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