CN103074336B - Goby mitochondrion ND1 gene complete sequence amplimer, design and amplification method - Google Patents
Goby mitochondrion ND1 gene complete sequence 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 ND1 gene complete sequence 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 ND1 gene total 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 the multiple Gobioidei plastosome of a kind of efficient amplification NADH reductase enzyme complex subunit 1(NADH dehydrogenase subunits 1; ND1) gene complete sequence amplimer and design thereof and amplification method.
Background technology
Fish Mitochondrial Genome Overview 37 encoding genes and one section of main non-coding region (control region) is comprised of 13 proteins encoded plasmagenes, 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA) totally, has matrilinear inheritance simple in structure, copy number is many, strict, recombinates hardly, coding efficiency is high, rate of evolution is fast and different zones rate of evolution such as there are differences at the feature.Due to these features that fish Mitochondrial Genome Overview has, 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 assemblage distributes.Global goby kind estimates at kind more than 2000, and they form a huge biological group, in the water surrounding especially marine eco-environment, occupy an important position.Goby mostly is carnivorous small fish, is found everywhere through the world, and especially take the torrid zone as many, is mainly sea water 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, due to reasons such as overfishing, climatope variations, many traditional tuna fisheries resources can not form fishing news, and some is even exhausted, but Gobioidei is because of reasons such as it are adaptable, life cycle is short, reproductivity is strong, and stock number but increases year by year.Because Gobioidei economic worth is not high, relatively less relevant for the research of Gobioidei both at home and abroad, of a great variety because of it, 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 more chaotic.
Genes involved in the full genome of plastosome and Mitochondrial Genome Overview has been widely used in the research fields such as population genetics, phylogenetics and conservation biology of fish as molecule marker.ND1 is as 7 one of genes of NADH reductase enzyme complex subunits of encoding in vertebrates encoding mitochondrial protein gene, and it is comparatively conservative on evolving, and can be applicable to phyletic evolution and the sort research of animal.Yet there is not the universal primer of reporting the mitochondrial ND1 gene complete sequence that increases, the Gobioidei especially extensive for species distribution, kind is numerous.Therefore design the universal primer of specific amplification Gobioidei mitochondrial ND1 gene complete sequence, thereby lay the foundation for efficiently obtaining goby mitochondrial ND1 gene complete sequence and mitochondrial genome complete sequence.
Summary of the invention
The vacancy existing for above-mentioned prior art, the present invention aims to provide the nucleotide primer of the multiple Gobioidei mitochondrial ND1 gene of a pair of efficiently specific amplified complete sequence, thereby to carry out phyletic evolution and sort research, provides a powerful for effectively obtaining fast Gobioidei mitochondrial ND1 gene complete sequence.The present invention also aims to provide the method for design of described Gobioidei mitochondrial ND1 gene complete sequence amplimer, and the method for utilizing described Gobioidei mitochondrial ND1 gene complete sequence amplimer to increase to Gobioidei mitochondrial ND1 gene complete sequence.
For realizing goal of the invention of the present invention, contriver provides following technical scheme:
Gobioidei mitochondrial ND1 gene complete sequence amplimer, is comprised of two single stranded oligonucleotide chains, and wherein light chain primer is the nucleotide sequence shown in SEQ ID No.1; Heavy chain primer is the nucleotide sequence shown in SEQ ID No.2.
Shown in light chain primer Gobies-ND1-F(SEQ ID No.1 of the present invention) there are 19 base: CGGAGAAATCCAGGTCAGT, be positioned on 16S rRNA gene; Heavy chain primer Gobies-ND1-R (shown in SEQ ID No.2) has 19 base: CCAACATGTTTGGGGTATG, is positioned on tRNA-Met gene.
Gobioidei mitochondrial ND1 gene complete sequence amplimer of the present invention is to 15 kinds of Gobioideis that gather at Area of The East China Sea, all can obtain fragment length is the specific amplification products of 1300 bp left and right, through order-checking and with GenBank in the comparison of homologous sequence, confirm as and comprise mitochondrial ND1 gene complete sequence, tRNA-Val gene complete sequence, tRNA-Ile gene complete sequence, tRNA-Gln gene complete sequence and length are the amplified production of 16S rRNA gene 3 ' the end sub-sequence of 115 bp left and right, embody the wider amplification scope of the present invention and stronger amplification ability, thereby to carry out phyletic evolution and sort research, provide a powerful for effectively obtaining fast Gobioidei line grain ND1 gene complete sequence.
As preferably, according to Gobioidei mitochondrial ND1 gene complete sequence amplimer of the present invention, 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, Zhoushan reins goby, tack pole goby, lance acanthogobius, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth.
The present invention also provides the method for design of above-mentioned Gobioidei mitochondrial ND1 gene complete sequence amplimer, login the Gobioidei of other species that the search of GenBank database (http://www.ncbi.nlm.nih.gov/) in NCBI website announced and 16S rRNA gene and the tRNA-Met gene order of the Mitochondrial Genome Overview of nearly edge fish species, through homology comparison, find conserved sequence, and utilize Premier Primer5.0 software design to go out Gobioidei mitochondrial ND1 gene complete sequence amplimer.Premier Primer5.0 software used is downloaded at biosoftware net http://www.bio-soft.net/.
The present invention also provides a kind of method that Gobioidei mitochondrial ND1 gene complete sequence is increased, and comprises and adopts above-mentioned Gobioidei mitochondrial ND1 gene complete sequence amplimer to carry out pcr amplification to the DNA profiling solution of testing sample.
As preferably, according to a kind of method that Gobioidei mitochondrial ND1 gene complete sequence is 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 1min30s, totally 35 circulations, and last 72 ℃ are extended 5min.
As preferably, according to a kind of method that Gobioidei mitochondrial ND1 gene is increased of the present invention, wherein the reaction composition of pcr amplification is: PCR reaction system is 25 μ L, includes dNTP(2.5mM) the light chain primer of 2 μ L, 10 * Taq archaeal dna polymerase Buffer, 2.5 μ L, 10 μ M and heavy chain primer each 1 μ L, Taq archaeal dna polymerase (5U/ul) 0.2 μ L, containing 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 mitochondrial ND1 gene is increased of the present invention, 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, Zhoushan reins goby, tack pole goby, lance acanthogobius, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth.
Compared with prior art, tool of the present invention has the following advantages:
Gobioidei mitochondrial ND1 gene complete sequence amplimer provided by the invention, multiple Gobioidei mitochondrial ND1 gene complete sequence specifically can efficiently increase, rather than the Partial Fragment sequence of the ND1 that only increases, 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.
Accompanying drawing explanation
Fig. 1 is that Gobioidei mitochondrial ND1 gene complete sequence amplimer of the present invention is in order to the electrophoretogram of the different Gobioidei mitochondrial ND1 gene complete sequences that increase.
M:DNA molecular weight standard (DL2000) wherein, 1-15 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, Zhoushan reins goby, tack pole goby, lance acanthogobius, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth.
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 that the present invention is made and/or change 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 conventional.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 biosoftware 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 biosoftware net http://www.bio-soft.net/).
Pcr amplification detects related reagent instrument: marine animal genome DNA extracting reagent kit (TIANGEN, Beijing), sepharose DNA reclaims test kit (TIANGEN, Beijing), conventional desk centrifuge (Thermo), Bio-Rad C1000TM Thermal Cycler amplification instrument (Bio-Rad, the U.S.), micropipet (Eenppdorf, Germany), 96 hole PCR plates (Axygen), dNTP(TIANGEN, Beijing), 10 * Taq archaeal dna polymerase Buffer(TIANGEN, Beijing), Taq archaeal dna polymerase (TIANGEN, Beijing), sterilizing 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 sterilizing LB of ammonification benzyl (Amp) substratum, LB solid culture plate (the raw work 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 (the raw work in Shanghai), the chloro-3-indoles-β-D-of the bromo-4-of 5-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), 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 in 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's specification sheets suggestion.
1, the design of Gobioidei mitochondrial ND1 gene complete sequence amplimer and synthetic:
16S rRNA gene and the tRNA-Met gene order of the Gobioidei of other species that the GenBank database search in login NCBI website has been measured and the Mitochondrial Genome Overview of nearly edge fish species, sequence is loaded in analysis software MEGA5.0, utilize ClustalW algorithm to carry out multisequencing contraposition compare of analysis, find conserved sequence, found conserved sequence is loaded into Premier Primer5.0 software and under manual designs pattern, (under manual option, selects Low, design parameter is default setting) design Gobioidei mitochondrial ND1 gene complete sequence amplimer: wherein shown in light chain primer Gobies-ND1-F(SEQ ID No.1) there are 19 base: CGGAGAAATCCAGGTCAGT, be positioned on 16S rRNA gene, heavy chain primer Gobies-ND1-R (shown in SEQ ID No.2) has 19 base: CCAACATGTTTGGGGTATG, is positioned on tRNA-Met gene.
By Nanjing Genscript Biotechnology Co., Ltd., according to contriver's requirement, synthesized the primer that meets nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.1.
2, Gobioidei mitochondrial ND1 gene is increased
15 samples to be tested in the present invention are all 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, Zhoushan reins goby, tack pole goby, lance acanthogobius, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth.
Utilize marine animal genome DNA extracting reagent kit to extract the genomic dna of sample to be tested, method is carried out according to test kit specification sheets, the genomic dna of agarose electrophoresis Detection and Extraction.
PCR reaction system and reaction conditions for the multiple Gobioidei mitochondrial ND1 gene complete sequence that increases are as follows: PCR reaction system is 25 μ L, include dNTP(TIANGEN), 10 * TaqDNA polysaccharase Buffer(TIANGEN), the heavy chain primer Gobies-ND1-R of the light chain primer Gobies-ND1-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- ND1-F 1μL
Gobies- ND1-R 1μL
Template DNA 1μL
ddH
2O 17.3μL
Taq DNA polysaccharase 0.2 μ L.
Amplified reaction is at Bio-Rad C1000
tMon Thermal Cycler instrument, carry out, amplification condition is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 30s then, 56 ℃ of annealing 30s, 72 ℃ are extended 1min30s, 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 mitochondrial ND1 gene complete sequence amplified productions is shown in Fig. 1.
Use the most Gobioidei line of the primer amplification grain ND1 gene complete sequence of design, all obtained single object fragment, product size is about 1300 bp, and pcr amplification product is 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-ND1-F(10 μ M) and Gobies-ND1-R(10 μ M).Through checking order and compare with the homologous sequence in GenBank, confirming as, comprising mitochondrial ND1 gene complete sequence, tRNA-Val gene complete sequence, tRNA-Ile gene complete sequence, tRNA-Gln gene complete sequence and length is the amplified production of 16S rRNA gene 3 ' the end sub-sequence of 115 bp left and right.
In addition, use the thin sour jujube goby of primer amplification Pu Shi and the blue or green mudskipper mitochondrial ND1 gene of design, all obtained single object fragment, product size is about 1300 bp, and uses sepharose DNA to reclaim test kit (TIANGEN, Shanghai) pcr amplification product is carried out to glue recovery purifying, purified product and pGEM-T carrier (Promega company, the U.S.) connect, transform, identify positive colony order-checking.The steps include: that the pcr amplification product that reclaims purifying through glue carries out ligation with pGEM-T carrier under the effect of T4 DNA ligase.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 is placed in 4 ℃ of refrigerators and connects spend the night (at least 12 hours).Get DH5 α competent cell (TIANGEN, Beijing), heat shock method transforms, with containing ammonia benzyl microbiotic (Amp) LB dull and stereotyped (being evenly coated with in advance the bromo-4-of 5-chloro-3-indoles-β-D-galactoside (X-Gal) of 40ul 20mg/ml and the isopropylthio-β-D-galactoside (IPTG) of 30ul 0.2mol/L before use), carry out the white screening of indigo plant, and by bacterium liquid pcr amplification, confirm positive colony of 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 the full-automatic DNA sequencer of ABI 3730.Through checking order and compare with the homologous sequence in GenBank, confirming as, comprising mitochondrial ND1 gene complete sequence, tRNA-Val gene complete sequence, tRNA-Ile gene complete sequence, tRNA-Gln gene complete sequence and length is the amplified production of 16S rRNA gene 3 ' the end sub-sequence of 115 bp left and right.
practicality
Gobioidei mitochondrial ND1 gene complete sequence amplimer of the present invention carries out pcr amplification to the DNA profiling solution of 15 kinds of ocean Gobioideis in Area of The East China Sea collection, all can obtain fragment at the specific amplification products of 1300 bp left and right, through order-checking and with GenBank in the comparison of homologous sequence, confirm as and comprise mitochondrial ND1 gene complete sequence, tRNA-Val gene complete sequence, tRNA-Ile gene complete sequence, tRNA-Gln gene complete sequence and length are the amplified production of 16S rRNA gene 3 ' the end sub-sequence of 115 bp left and right, embody the wider amplification scope of the present invention and stronger amplification ability, because 15 kinds of selected Gobioideis of the present invention belong to respectively below goby suborder the not species of equal genus, therefore, Gobioidei mitochondrial ND1 gene complete sequence amplimer of the present invention all can be applied at the different taxonomic category phyletic evolutions of Gobioidei and sort research field.
Although contriver has done comparatively detailed elaboration and has enumerated technical scheme of the present invention, be to be understood that, for one of this area those skilled in the art, above-described embodiment is modified and/or flexible or to adopt the replacement scheme being equal to be obvious, all can not depart from the essence of spirit of the present invention, the term occurring in the present invention, for to the elaboration of technical solution of the present invention and understanding, can not be construed as limiting the invention.
SEQUENCE LISTING
<110> Oceanography Institute Of Zhejiang
<120> Gobioidei mitochondrial ND1 gene complete sequence amplimer and design and amplification method
<130> Z122340
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<400> 1
cggagaaatc caggtcagt 19
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
ccaacatgtt tggggtatg 19
Claims (5)
1. Gobioidei mitochondrial ND1 gene complete sequence amplimer, is characterized in that being comprised of two single stranded oligonucleotide chains, and wherein light chain primer is the nucleotide sequence shown in SEQ ID No.1; Heavy chain primer is the nucleotide sequence shown in SEQ ID No.2.
2. Gobioidei mitochondrial ND1 gene complete sequence amplimer as claimed in claim 1, 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, Zhoushan reins goby, tack pole goby, lance acanthogobius, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth.
3. the method for design of a Gobioidei mitochondrial ND1 gene complete sequence amplimer as claimed in claim 1, it is characterized in that logining 16S rRNA gene and the tRNA-Met gene order of the Gobioidei of other species that the GenBank database search in NCBI website measured and the Mitochondrial Genome Overview of nearly edge fish species, through homology comparison, find conserved sequence, and utilize Premier Primer5.0 software design to go out Gobioidei mitochondrial ND1 gene complete sequence amplimer.
4. the method to the amplification of Gobioidei mitochondrial ND1 gene complete sequence, is characterized in that adopting Gobioidei mitochondrial ND1 gene complete sequence amplimer as claimed in claim 1 to carry out pcr amplification to the DNA profiling solution of testing sample,
The condition of pcr amplification is: 95 ℃ of denaturation 5min, and 95 ℃ of sex change 30s then, 56 ℃ of annealing 30s, 72 ℃ are extended 1min30s, totally 35 circulations, last 72 ℃ are extended 5min,
The reaction composition of pcr amplification is: PCR reaction system is 25 μ L, include the dNTP 2 μ L of 2.5mM, the Taq archaeal dna polymerase 0.2 μ L of each 1 μ L, 5U/ul of the light chain primer of 10 * Taq archaeal dna polymerase Buffer, 2.5 μ L, 10 μ M and heavy chain primer, containing DNA profiling solution 1 μ L and the ddH of 100ng
2o 17.3 μ L.
5. a kind of method that Gobioidei mitochondrial ND1 gene total order is 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, Zhoushan reins goby, tack pole goby, lance acanthogobius, biobelt thin white silk used in ancient China goby, the not easily seen goby of canine tooth.
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