CN105713973B - A kind of labeled primer and method using Mitochondrial D-loop Region specific regions identification soft-shelled turtle - Google Patents
A kind of labeled primer and method using Mitochondrial D-loop Region specific regions identification soft-shelled turtle Download PDFInfo
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Abstract
The present invention provides a kind of labeled primers using Mitochondrial D-loop Region specific regions identification soft-shelled turtle, are following primer sets: D1F:5 '-ATTAATTCATGCTTGTAGGAC-3 ', D1R:5 '-CGGGGTAGGGGGTTTAG-3 '.The present invention also provides the methods using Mitochondrial D-loop Region specific regions identification soft-shelled turtle, PCR amplification is carried out using complete genome DNA of the primer sets to sample to be tested, then electrophoresis is carried out, the band of two soft-shelled turtle specific polymorphic segments can such as be obtained, the species for judging sample to be tested are soft-shelled turtles, on the contrary then obtain adverse consequences.
Description
Technical field
The present invention relates to a kind of labeled primers and method using Mitochondrial D-loop Region specific regions identification soft-shelled turtle.
Background technique
Soft-shelled turtle (Pelochelys cantorii) it is that soft-shelled turtle belongs to one of only three kinds, be otherwise known as " giant panda in water ".Soft-shelled turtle
It lives in fresh water, is maximum one kind of figure in Trionychidae.Soft-shelled turtle is widely distributed in Southeast Asian countries, SOUTHERN CHINA, India and bar
The seashore of Ji Sitan.However, the destruction of habitat and the pollution of environment, the distribution and quantity of soft-shelled turtle are sharply because hunting
It reduces.In 1996, soft-shelled turtle was put into World Conservation Union (IUCN) world's endangered animal Red List susceptible species, and
It is promoted to endangered species within 2000.Because of the critically endangered state of soft-shelled turtle, it is also classified as first class of protection animal by China.
The rareness of soft-shelled turtle makes people lack the understanding to it, and morphological feature can not reflect separately as the standard accurately judged
It is not accurate enough to determine method.Soft-shelled turtle and soft-shelled turtle are closely similar in juvenile stage, and the individual in field cannot get effective protection.Because without profession
Knowledge, people it is wrong sometimes field is caught sold at soft-shelled turtle as wild big soft-shelled turtle.Soft-shelled turtle has been further aggravated in this problem
The reduction of germ plasm resource, to affect the validity of safeguard measure.
Mitochondrial genomes are the cyclic DNAs of 16-18kb size, encode 13 albumen, 22 tRNA, 2 rRNA.Line grain
Body DNA has structure simple, and high mutation rate lacks the characteristic of genetic recombination and stringent matrocliny.Because of this feature,
Mitochondrial DNA is widely used in species identification, phyletic evolution, the research of population diversity etc..
Summary of the invention
One of the object of the invention is to provide a kind of labeled primer using Mitochondrial D-loop Region specific regions identification soft-shelled turtle.
The present invention is achieved the object of the present invention using following technical scheme: a kind of to utilize the special area in Mitochondrial D-loop Region
The labeled primer of soft-shelled turtle is identified in domain, is following primer sets:
D1F:5 '-ATTAATTCATGCTTGTAGGAC-3 '
D1R:5 '-CGGGGTAGGGGGTTTAG-3 '.
The control zone mitochondrial DNA (mtDNA), the also known as area D-loop are one section of noncoding regions, be usually located at tRNAPro and
Between tRNAPhe gene.Structure is complex, is divided into 3 sections: termination sequence area, central conserved region and conserved sequence area.By
In the pressure for lacking coding, the evolutionary rate of control zone is relatively fast, and the variation of sequence not only has the replacement between nucleotide,
There are also the tandem sequence repeats of the missing of different length nucleotide sequence, insertion or different copy numbers, are mtdna sequence and length
Make a variation maximum region, the genetic diversity being usually used in kind, evolutionary analysis etc..Although mtDNA aberration rate is high, there is also one
A little conserved sequences, these conserved sequences can undertake the effect of marker gene.The present inventor uses the brilliant and Zhaoqing in Guangdong Province
The mitochondrial DNA of Xiamen soft-shelled turtle and Yongjia soft-shelled turtle compares on the dead young soft-shelled turtle sample extraction mitochondrial DNA and NCBI of Guangning, finds soft-shelled turtle
Mitochondrion DNA control area there are the special construction of repeated fragment, and the special construction in the testudinate species being currently known
Do not find.As it can be seen that mitochondrion DNA control area sequence is almost identical in kind, in the widely different of inter-species, can be used for planting
Between identify.Thus, the present invention designs specific primer according to the special construction and is used to realize the identification to soft-shelled turtle.The present inventor is also
A possibility that being verified using multiple soft-shelled turtle individuals, eliminated the special construction as produced by single idiovariation, ensure that and draw
The popularity and reliability of object application.Repeated fragment design primer pair existing for mitochondrion DNA control area of the inventor for soft-shelled turtle,
And verify the specificity of involved primer.The place that dotted line marks in Fig. 1 is the design position of upstream primer, the ground of solid line mark
Side is the design position of downstream primer, and the expection clip size of each primer sets amplification is shown in Table 1.By verifying, the D1 of design draws
Object is most strong to specificity.
The second object of the present invention is to provide a kind of method for identifying soft-shelled turtle.Specifically, a kind of utilize Mitochondrial D-loop Region
The method that soft-shelled turtle is identified in specific regions carries out PCR amplification to the complete genome DNA of sample to be tested using above-mentioned primer sets, then into
Row electrophoresis can such as obtain the band of two soft-shelled turtle specific polymorphic segments, that is, judge that the species of sample to be tested are soft-shelled turtles, it is on the contrary then
Obtain adverse consequences.
Alternatively, after electrophoresis, gel extraction soft-shelled turtle specific polymorphic segment is attached carrier sequencing, acquired results with
There is the comparison of soft-shelled turtle mitochondrial DNA, thus can more definitely judge whether the species of sample to be tested are soft-shelled turtles.
The loop parameter of pcr amplification reaction of the present invention are as follows: 94 DEG C of unwinding 4min, 94 DEG C of denaturation 30s, 59 DEG C are annealed
30s, 72 DEG C of extension 30s, 30 circulations, 72 DEG C of extension 10min.
The beneficial effects of the present invention are:
(1) there are the special constructions of repeated fragment to design specific primer according to soft-shelled turtle mitochondrion DNA control area by the present invention,
It is expanded using complete genome DNA of the designed specific primer to sample to be tested, whether is gone out according to the electrophoresis after amplification
Whether the specific polymorphic segment of existing soft-shelled turtle is soft-shelled turtle come the species for judging sample to be tested.
(2) present invention is marked using mtDNA, but does not need individually to extract mtDNA for identifying, is only needed according to a conventional method
Full-length genome is extracted, it can be by primer by being identified after PCR amplification and electrophoresis using mtDNA a small amount of in full-length genome
As a result, easy to operate, cycle time is short, can in high volume be identified, qualification result can be obtained within one day.
Detailed description of the invention
Fig. 1 is the comparison of the amino acid sequence of control zone in the mitochondrial DNA of different soft-shelled turtles,
The place of dotted line mark is the design position in the area primer D1 and D3 upstream primer D-loop；Black line mark is D1 and D3
Downstream primer design position, there are two binding site in sequence.
Fig. 2 is special primer screening amplified fragments electrophoretogram,
1 is primer D1 amplification；2 and 3 be primer D2 amplification；4 be primer D3 amplification；5 be DL5000
mark。
Fig. 3 is special primer amplified fragments electrophoretogram,
1-4 is Guangning soft-shelled turtle amplified production；5-8 is brilliant soft-shelled turtle amplified production；9 be DL2000 mark；10-15 is that yellow larynx is quasi-
Terrapin amplified production；16-22 is Shelled Turtle Trionyx Sinensis amplified production.
Specific embodiment
Following embodiment is used merely to explain the present invention, and protection scope of the present invention is not intended to be limited to following implementation
Example.The purpose of the present invention can be achieved according to the above present disclosure in the those of ordinary skill of the technical field.
Material and method
1.1 material
In brilliant and Zhaoqing Guangning in Guangdong Province, discovery has the living materials of soft-shelled turtle respectively.They be protected in pond into
Row cultivation, female soft-shelled turtle sexal maturity oviposition, successfully hatches young soft-shelled turtle.The tissue of experiment is derived from dead young soft-shelled turtle sample, with utilizing group
Knit E.Z.N.A.TM Tissue DNA Kit (Omega Bio-Tek, Guangzhou, China) extracts the soft-shelled turtle of distribution two places
Complete genome DNA, -20 degree save backup.
Extraction step is specific as follows:
1) go bail for the 30 μ g of musculature or so being stored in dehydrated alcohol, and the alcohol on nail is washed with deionized, and
It is placed on filter paper dry.
2) it is transferred in 1.5ml centrifuge tube the tissue dried is cleaned, 200 μ L Buffer TL is added, shred.
3) 20 μ L OB protease are added, mixes, is placed in shaking table, 55 DEG C, 180r/min clears up 1-3 h.
4) after digesting, 1000g is centrifuged 5min, and Aspirate supernatant is transferred in 1.5ml centrifuge tube, abandons precipitating.
5) 220ul Buffer BL is added, mixes, places 10min in 70 DEG C of water-baths, during which mixes 2-3 times.
6) 220 ul dehydrated alcohols are added after taking out, mix.
7) mixed liquor in previous step is transferred to and is covered in 2ml collecting pipe in HiBind DNA column, 8000g in room temperature
It is centrifuged 1min, DNA is integrated on filter membrane, abandons filtrate.
8) by posts transfer into another group of 2ml collecting pipe, 500 μ L HB Buffer, room temperature 8000g centrifugations are added
1min abandons filtrate.
9) again by posts transfer into another group of 2ml collecting pipe, the 650 diluted DNA Wash of μ L ethyl alcohol are added
Buffer, 8000g is centrifuged 1min in room temperature, outwells filtrate
10) it is repeated the above steps 9) with same group of collecting pipe.
11) pillar is relay in collecting pipe, 15000g is centrifuged 3min in room temperature.
12) pillar is mounted on 1.5ml centrifuge tube, opening pillar lid, in dry 3min, adds 70 DEG C of 50ul preheatings
Sterile water.3min is placed at room temperature.At room temperature, 10000g is centrifuged 2min, and obtained filtrate is DNA solution.
13) purity and integrality of agarose gel electrophoresis detection DNA, NanoQTM microspectrophotometer detect DNA
Concentration, and it is diluted to final concentration 20ng/ μ L with deionized water, it is placed in -20 DEG C and saves backup.
The design and screening of specific primer
In the area D-loop, there are duplicate part (Fig. 1), devise 2 pairs of primers (table 1) according to repeating part.Because in the presence of
Length polymorphism will be presented by PCR electrophoresis primer amplification result in repeated fragment.
PCR amplification is carried out respectively with the complete genome DNA of the primer pair soft-shelled turtle of design, with reference to expected polymorphic bands size
It is compared with electrophoresis result, screening reaches expected primer.
PCR use 20 μ l reaction systems, the template DNA (20 ng) of 1 μ l, 10 μ l'sPremix Taq TM (Takara
Biotechnology, Dalian, China), the upstream and downstream primer (10 mM) of 0.5 μ l, 8 μ l water are mended to 20 μ l.
The loop parameter of pcr amplification reaction are as follows: 94 DEG C of unwinding 4min, 94 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 30s, 30
Circulation, 72 DEG C of extension 10min.
As shown in Fig. 2, the primer of design carries out primary dcreening operation using soft-shelled turtle genomic DNA amplification, primer D1-3 is in electrophoresis result
Polymorphic bands are showed.But D2-3, since specificity is insufficient, the segment in addition to obtaining expected size also occurs expected big
Segment except small.And primer D1 does not have miscellaneous band, while having obtained the purpose band of expected size, selectes and is used as main screening
Primer.
The verifying of primer amplification sequencing fragment
Pcr amplification product is utilized into E.Z.N.A plastic recovery kit (Omega) purification and recovery:
(1) with the Ago-Gel (containing 0.5 μ g/mL DNAgreen) of tbe buffer liquid preparation 1.2%(w/v).
(2) loading wells, 220V voltage is added in DNA sample, room temperature carries out 13 min of electrophoresis.
(3) observation DNA band migration position is cut with clean blade and is contained when the separation of purpose band understands in the UV lamp
There is the gel of purpose DNA, is transferred in 1.5mL centrifuge tube.
(4) the method recovery purifying PCR product introduced according to E.Z.N.A plastic recovery kit operating procedure (Omega).
(5) weight for weighing sky centrifuge tube cuts the gel with target fragment in 1.5mL centrifuge tube and claims its heavy
Amount, determines approximately its volume.Under normal circumstances, the density of gel is 1g/mL, and then the volume of gel and the relationship of weight can
By following conversion: the weight of gel slice is that then its volume is 0.2mL to 0.2g；The Binding of equimultiple gel volume is added
Mixture is placed in 55 DEG C of -65 DEG C of water-baths warm bath 7min and melted completely to gel by Buffer, mixes one every 2-3min therebetween
It is secondary.
(6) the DNA- agarose solution of 700 μ L is shifted to a HiBindTM DNA pillar, and pillar is dry mounted in one
In net 2mL collecting pipe, at room temperature, 10,000 × g is centrifuged 1min, discards liquid.
(7) HiBind DNA pillars can at most accommodate the solution of 700 μ L, if the volume of DNA- agarose mix
Greater than 700 μ L, 700 μ L solution can be first shifted to pillar, after being centrifuged, remaining solution is continued plus on pillar.But it is every
One HiBind TM pillar can at most combine 25-30 μ g DNA.If it is expected that yield is larger, then sample is added separately to close
In the column of suitable number.
(8) pillar is recovered in collecting pipe again, is added in 300 μ L Binding Buffer to HiBind DNA pillars；
At room temperature, 10,000 × g are centrifuged 1min, completely remove filter liquor.
(9) pillar is recovered in collecting pipe again, 700 μ L SPW Wash buffer to HiBind DNA pillars is added
In, at room temperature, 10,000 × g is centrifuged 1min, goes to abandon filter liquor.
(10) pillar is recovered in collecting pipe again, repeats that 700 μ L SPW Wash buffer to HiBind DNA are added
In pillar, at room temperature, 10,000 × g is centrifuged 1min, discards filter liquor；
(11) after discarding filter liquor, pillar is recovered in collecting pipe again, 10,000 × g is centrifuged 1min to dry base for post
The liquid of matter remnants.
(12) pillar on a clean 1.5mL centrifuge tube, 30 ~ 50 μ L eluents or aqua sterilisa upper prop is added
On sub- film, 10,000 × g is centrifuged 1min, and the solution in centrifuge tube is exactly the DNA product purified, is stored in -20 DEG C.
The DNA of recycling is connected in pMD18-T Vector carrier, competent cell DH5a, 37 DEG C of culture 8h are transformed into
Afterwards, it is sequenced after selecting positive colony detection.Two clip size results of primer D1 amplification are 107bp and 370bp respectively, with
Expected results are identical.By the sequence of sequencing result with NCBI(National Center for Biotechnology
Information the mitochondrial DNA for the soft-shelled turtle having determined on) is compared, and the similitude of comparison result is 99%, therefore determination is drawn
The DNA fragmentation that object D1 and D3 is obtained by PCR amplification is the DNA fragmentation of soft-shelled turtle.
Multiple bodies and negative control verifying
After filtering out primer, 6 samples are taken to carry out PCR amplification using primer D1, then electrophoresis, the primer for observing screening are
It is no normally to be acted in great amount of samples, judge the application feasibility of primer.Shelled Turtle Trionyx Sinensis, the full base of Mauremys mutica are utilized simultaneously
Because group DNA is expanded as negative control, determine whether primer length polymorphism only occurs in soft-shelled turtle or have expansion
Energization power.Electrophoresis result as shown in Figure 3 it is found that can be stable using the soft-shelled turtle sample of primer amplification provided by the invention appearance
Two polymorphic segments, soft-shelled turtle can not run complete band, and Mauremys mutica can only amplify a kind of segment, to prove of the invention
The specificity of primer.
PCR uses 20 μ l reaction systems, the template DNA (20 ng) of 1 μ l, the Premix TaqTM of 10 μ l
(Takara Biotechnology, Dalian, China), the upstream and downstream primer (10 mM) of 0.5 μ l, 8 μ l water mend to
20 µl.The loop parameter of pcr amplification reaction are as follows: 94 DEG C of unwinding 4min, 94 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C extend
30s, 30 circulations, 72 DEG C of 10 min of extension.
Sequence table
<110>China's Pearl River Fishery Research Institute of Aquatic Science Research Institute
<120>a kind of labeled primer and method using Mitochondrial D-loop Region specific regions identification soft-shelled turtle
<160> 4
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<223>primer
<400> 1
ATTAATTCAT GCTTGTAGGAC 21
<210> 2
<211> 17
<212> DNA
<213>artificial sequence
<223>primer
<400> 2
CGGGGTAGGG GGTTTAG 17
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<223>primer
<400> 3
GGATTTTGGG GTTTGAACGAG 21
<210> 4
<211> 19
<212> DNA
<213>artificial sequence
<223>primer
<400> 4
TTTTGGGGTT TGACGAGAG 19
Claims (5)
1. a kind of labeled primer using Mitochondrial D-loop Region specific regions identification soft-shelled turtle, characterized in that be following primer sets:
D1F:5 '-ATTAATTCATGCTTGTAGGAC-3 '
D1R:5 '-CGGGGTAGGGGGTTTAG-3 '.
2. a kind of method using Mitochondrial D-loop Region specific regions identification soft-shelled turtle, characterized in that draw using described in claim 1
Object group carries out PCR amplification to the complete genome DNA of sample to be tested, then carries out electrophoresis, and it is more can such as to obtain two soft-shelled turtle specificity
The band of state property segment judges that the species of sample to be tested are soft-shelled turtles, on the contrary then obtain adverse consequences.
3. the method as claimed in claim 2 using Mitochondrial D-loop Region specific regions identification soft-shelled turtle, characterized in that the PCR
The loop parameter of amplified reaction are as follows: 94 DEG C of unwinding 4min, 94 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 are followed
Ring, 72 DEG C of extension 10min.
4. a kind of method using Mitochondrial D-loop Region specific regions identification soft-shelled turtle, characterized in that draw using described in claim 1
Object group carries out PCR amplification to the complete genome DNA of sample to be tested, then carries out electrophoresis, gel extraction soft-shelled turtle specific polymorphic piece
Section is attached carrier sequencing, and acquired results and existing soft-shelled turtle mitochondrial DNA compare, thus can more definitely judge to test sample
Whether the species of product are soft-shelled turtles.
5. the method as claimed in claim 4 using Mitochondrial D-loop Region specific regions identification soft-shelled turtle, characterized in that the PCR
The loop parameter of amplified reaction are as follows: 94 DEG C of unwinding 4min, 94 DEG C of denaturation 30s, 59 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 are followed
Ring, 72 DEG C of extension 10min.
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