CN109468385A - A method of identification Genus Triplophysa fish - Google Patents
A method of identification Genus Triplophysa fish Download PDFInfo
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Abstract
The invention discloses a kind of methods for identifying Genus Triplophysa fish, by designing primer amplified Triplophysa COI gene order, in combination with molecular biology method, carry out Molecular Identification to Genus Triplophysa fish.Identification method of the invention solves the problems, such as that routine cannot expand Triplophysa COI gene order using fish universal primer, and has the characteristics that detection speed is fast, identification accuracy is high, high specificity.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of side for identifying Genus Triplophysa fish
Method.
Background technique
COI gene is one section of protein coding gene on mitochondrial DNA, have structure it is simple, in matrilinear inheritance, several
Recombination, the features such as evolutionary rate is fast does not occur, is research one of inter-species molecular evolution and the most useful gene of systematic growth.
At the beginning of 2003, the systematist Paul Herbert research discovery of Canadian Guelph university utilizes mitochondrial cytochrome C oxygen
Change enzyme subunit I gene, a segment length is that the DNA fragmentation of 648bp can successfully distinguish species on DNA level, and think benefit
With COI gene from the angle of molecular evolution, it will thus provide it is a kind of quickly, easy, believable classification method, and by commodity UPC bar shaped
It is bar codes technique that code, which inspires the concept for being put forward for the first time DNA bar code to develop,.In recent years, COI gene is mainly by biological section
Field is used as systematic growth research or DNA bar code research.Under normal circumstances, fish can be amplified using universal primer
COI gene order, but the fish of certain monoids are because of reasons such as its special evolution modes, using document method and universal primer simultaneously
Product cannot be amplified, it is necessary to which target fragment can be obtained and reflect for subsequent analysis by designing peculiar primer and optimizing extension band
It is fixed.
Therefore it provides a kind of molecular biology method for identifying Genus Triplophysa fish is those skilled in the art's urgent need to resolve
The problem of.
Summary of the invention
In view of this, detecting speed the present invention provides a kind of specific primer and method for identifying Genus Triplophysa fish
Fastly, identification accuracy is high.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of primer for identifying Genus Triplophysa fish, the primer sequence are as follows:
P1:5 '-CATCCTACCTGTGGCAATCAC-3 '；SEQ ID NO.1；
P2:5 '-TGGGCTCAGACAATAAATCCT-3 '；SEQ ID NO.2.
Further, a method of identification Genus Triplophysa fish, the specific steps are as follows:
(1) according to the mitochondrial COI gene sequence of Genus Triplophysa fish not of the same race, the conservative piece between different sequences is chosen
Section；
(2) conservative fragments chosen according to step (1), design and filter out the specific primer of Genus Triplophysa fish；
(3) specific primer obtained using step (2) carries out PCR amplification to the genomic DNA of Genus Triplophysa fish,
Obtain amplified production；
(4) amplified production for obtaining step (3) carries out agarose gel electrophoresis, recycles band, is sequenced；
(5) gained sequence is sequenced in step (4) to be compared, the high and its nearly source kind sequence of downloading similarity constructs system
Development tree, estimates genetic distance, primarily determines measured sequence species ownership；
(6) form of species ownership confirms again, is directed toward according to molecular data, and combining form feature confirms the final of species
Ownership.
Further, the reaction system of the PCR amplification are as follows: 15 μ L of TaqMixture, 1 μ L of template DNA, 10 μM of primers
P11.5 μ L, 10 μM of primer P21.5 μ L, ddH211 μ L of O, totally 30 μ L.
Further, the response procedures of the PCR amplification are as follows: 94 DEG C of 5min；94 DEG C of 30S, 55 DEG C of 30S, 72 DEG C of 1min, 30
Circulation；72℃10min.
It can be seen via above technical scheme that compared with prior art, the present disclosure provides a kind of identification Triplophysas
The method for belonging to fish, by designing primer amplified Triplophysa COI gene order, in combination with molecular biology method,
Molecular Identification is carried out to Genus Triplophysa fish；Triplophysa COI gene sequence cannot be expanded using fish universal primer by solving routine
The problem of column；Identification method detection speed of the invention is fast, identification accuracy is high and high specificity.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is PCR amplification result electrophoresis detection figure of the present invention；
Wherein, M marker, band length from top to bottom be respectively 2000bp, 1000bp, 750bp, 500bp,
250bp, 100bp；D1, D2, D3, D4, D5, D6, D7, D8, D9, D10, D11, D12 are up to inner lake Triplophysa (Triplophysa
dalaica)；S1, S2 are the beautiful Triplophysas (Triplophysa selleafer) of match；S3 is like Nian Triplophysa (Triplophysa
siluroides)；R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 are sturdy Triplophysa (Triplophysa robusta)；
Fig. 2 attached drawing is the present invention is based on the Genus Triplophysa fish NJ Phylogenetic Relationships of COI gene, and outgroup is Cobitidae fish
Class.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1DNA is extracted
(1) acquisition and processing of sample
Sample source is the Triplophysa sample that Henan Province's resource investigation of fish obtains.
Multiple Triplophysa samples of acquisition are removed into back scale and skin after anaesthetizing, its muscle of back is taken to be put in respectively
The centrifuge tube of 1.5mL, soaked in absolute ethyl alcohol, -20 DEG C/- 80 DEG C preservations.
(2) preparation of reagents
Pipette tips used in power dispensers, distilled water, centrifuge tube need to by 121 DEG C high pressure sterilization 30 minutes, reagent bottle, graduated cylinder,
It is rinsed after the washes clean such as beaker with distilled water.
The EDTA of 0.5mol/L: it weighs 37.22g EDTA and is dissolved in 150mL distilled water, stirred on magnetic stirring apparatus
To its dissolution, the pH value of solution is adjusted with NaOH, making the pH value of solution is about 8.0, and EDTA all dissolves at this time, takes around and adds
Enter NaOH particle 4g, water is added to be settled to 200mL, 4 DEG C of high pressure sterilization preservations.
The Tirs-HCl of 1mol/L: weighing 24.22g Tirs alkali, be dissolved in 150mL water, and enriching hydrochloric acid tune pH value makes
Its pH value 8.0 or so about needs that 0.4mL concentrated hydrochloric acid is added, and water is added to be settled to 200mL, 4 DEG C of high pressure sterilization preservations.
The SDS that mass concentration is 5%: it weighs 5g SDS and 80mL water is added to dissolve, be finally settled to 100mL, room temperature preservation.
The sodium acetate of 3mol/L: weighing 40.83g Sodium acetate trihydrate and be dissolved in appropriate distilled water and be finally settled to 100mL, and 4 DEG C
It saves.
Proteinase K: weighing Proteinase K 60mg, is dissolved in appropriate distilled water, constant volume to 3mL, 3 pipe of packing, -20 DEG C of guarantors
It deposits.
DNA extracting solution: drawing the Tris-HCl of 0.2mL 1mol/L with liquid-transfering gun respectively, 0.4mL 0.5mol/L's
The SDS that EDTA, 2mL mass concentration are 5%, adds water to be settled to 20mL, room temperature preservation.
50 times of TAE electrophoretic buffer (pH about 8.5): weighing 24.22g Tirs alkali, adds a small amount of distilled water to dissolve, with shifting
Liquid rifle is drawn glacial acetic acid 5.71mL and is added in Tirs aqueous slkali, and graduated cylinder measures the EDTA of 20mL 0.5mol/L, and water is added to be settled to
100mL, 4 DEG C of preservations, used time are diluted to 1 times.
6 times of sample-loading buffer: bromophenol blue (bromophenol blue, PBP) 1mL of 0.25% (m/v), mass concentration
For 40% aqueous sucrose solution 5mL, 6 pipe of packing, -20 DEG C of preservations.
The Ago-Gel that mass concentration is 1%: weighing the TAE buffer 40mL that 0.4g agarose is added 1 times, and the used time is existing
Match, be added EB (ethidium bromide, ethidiumbromide, gelling temp are down to 50 DEG C or so additions)；It is ready-to-use.
Chloroform: isoamyl alcohol (24:1, v/v): chloroform 24mL, isoamyl alcohol 1mL are mixed, and 4 DEG C spare, ready-to-use.
Phenol: chloroform: isoamyl alcohol (25:24:1, v/v/v): take the chloroform prepared: isoamyl alcohol (24:1) 10mL adds 10mL's
Saturated phenol, 4 DEG C spare；It is ready-to-use.
The ethyl alcohol that volume fraction is 70%: dehydrated alcohol 70mL adds distilled water to 100mL, and 4 DEG C spare.
(3) tissue DNA is extracted using phenol-chloroform method, the specific steps are as follows:
1) 400 μ l DNA extracting solutions, 4 μ l eggs are added in centrifuge tube in the sample tissue for taking appropriate step (1) to save respectively
White enzyme k (dosage can be changed according to the size of tissue)；Mixing, which is placed in 56 DEG C of water-baths, to be digested 8 hours, until tissue is complete
Until digestion；
2) 400 μ l saturation Tris balance phenol is added to the tissue fluid digested, 10min is stood after mixing, then with temperature
10000r/min high speed centrifugation 10min shifts supernatant into new centrifuge tube；
3) step 2) is repeated once, for destroying, precipitating proteins；
4) chloroform (chloroform) of 24:1 is added into pipe: about 400 μ l of isoamyl alcohol stands 10min after mixing, then with
10000r/min high speed centrifugation 10min shifts supernatant into new centrifuge tube；
5) it repeats step 4) once, is used for deproteinized, purification of protein；
6) supernatant is taken, the 40 μ l of sodium acetate that 3mol/L is added is mixed, and adds 800 μ l of -20 DEG C of dehydrated alcohols, quiet after mixing
10min is set, then with room temperature 10000r/min high speed centrifugation 5min；
7) supernatant is abandoned, precipitating is stayed；70% cold ethyl alcohol is added, twice with room temperature 2000r/min centrifugation 3min cleaning DNA；
Blocky DNA is turned down as far as possible, is made not adherent；
(8) ethyl alcohol is abandoned, is dried, it is spare that sterilizing distilled water dissolution is added.
Embodiment 2DNA concentration and quality testing
1 μ L dissolving DNA is drawn from having diluted in sufficient each pipe, detects its concentration with Nanodrop 2000；It inhales simultaneously
Take the DNA lysate of 3 μ L under conditions of voltage is 120V, electrophoresis 15min takes pictures under gel imaging system；The result shows that
The DNA of extraction can be used for PCR detection.
The Molecular Identification of 3 Genus Triplophysa fish of embodiment
(1) design of primers: in downloading Triplophysa mitochondrial genome complete sequence short-tail Triplophysa (Triplophysa on the net
Brevicauda, KT213588.1), black back Triplophysa (Triplophysa dorsalis, KT213591.1), Jiuquan Triplophysa
(Triplophysa hsutschouensis, KT213592.1), slender Triplophysa (Triplophysa leptosoma,
KT213593.1), the river Ma Erke Triplophysa (Triplophysa markehenensis, KT213594.1), Nujiang Triplophysa
(Triplophysa nujiangensa, KT213598.1), east Triplophysa (Triplophysa orientalis,
KT213599.1), Triplophysa (Triplophysa scleroptera, KT213602.1), Wuwei Triplophysa are pierced firmly
(Triplophysa wuweiensis, KT224365.1), Xichang Triplophysa (Triplophysaxichangensis,
KT224366.1), Triplophysa ulacholica (KT259194.1) extracts the COI gene in above-mentioned sequence
And be compared, upstream primer is designed in the conservative fragments area of sequence leading portion, the conserved sequence area design downstream of sequence back segment is drawn
Object, primer are synthesized by company.
Specific primer sequence:
Upstream primer P1:CATCCTACCTGTGGCAATCAC；(this position is position 5489- after comparing to SEQ ID NO.1
5509)；
Downstream primer P2:TGGGCTCAGACAATAAATCCT；(this position is position 6348- after comparing to SEQ ID NO.2
6368)。
(2) PCR amplification
PCR amplification system is as follows: 15 μ L of TaqMixture, template DNA 1 μ L, primer P1 (10 μM) 1.5 μ L, primer P2
(10 μM) 1.5 μ L, ddH211 μ L of O, totally 30 μ L.
PCR amplification condition is as follows: 94 DEG C of 5min；94 DEG C of 30S, 55 DEG C of 30S, 72 DEG C of 1min, 30 circulations；72℃10min；
4 DEG C of product preservations.
(3) purpose band detection and sample presentation sequencing
PCR product is detected with the agarose gel electrophoresis that mass concentration is 1%, reaches the sample presentation sequencing that sequencing requires, surveys
Sequence is completed by commercial company (one Hui Yuan Biotechnology Co., Ltd of Wuhan Tian), as a result as shown in Figure 1.
(4) sequencing result analysis and processing
It is committed to NCBI after all sequencing results are annotated, obtains the following sturdy Triplophysa of species accession number
Triplophysa robusta (401391:MK250403；401394:MK250404；401401:MK250406；401402:
MK250407,401403:MK250414；401412:MK250411；401384:MK25040), Da Lihu Triplophysa
Triplophysa dalaica(401396:MK250405；401404:MK250408；401383:MK250400；401386:
MK250402；401408:MK250409；401409:MK25041；403532:MK250412；401369:MK250392；
401370:MK250393；401371:MK250394；401372:MK250395；401373:MK250396；401374:
MK250397；401375:MK250398；401382:MK250399), beautiful Triplophysa Triplophysa selleafer is matched
(203571:MK250413；203576:MK250390), like Nian Triplophysa Triplophysa siluroides (203692:
MK250391)。
Each sequencing result BLAST on NCBI finds more similar sample, combines similar sample by MEGA software
This building genealogical tree and genetic distance are estimated, carry out a point kind using≤2% genetic distance as to Genus Triplophysa between different samples
Identification, as a result as shown in Figure 2；The adjacent tree (NJ) of Fig. 2 gene Genus Triplophysa fish COI gene order building is the result shows that same
The monophyletic group of the high supporting rate of species sequence homopolymerization illustrates that COI gene has stronger species identification ability, can be used for plateau
The Molecular Identification of loach species.
(5) form of species ownership confirms again, is directed toward according to molecular data, the sample of combining form feature confirmation acquisition
This, species are Genus Triplophysa fish.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Sequence table
<110>He'nan Normal University
<120>a kind of method for identifying Genus Triplophysa fish
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 1
catcctacct gtggcaatca c 21
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 2
tgggctcaga caataaatcc t 21
Claims (4)
1. a kind of primer for identifying Genus Triplophysa fish, which is characterized in that the primer sequence are as follows:
P1:5 '-CATCCTACCTGTGGCAATCAC-3 '；SEQ ID NO.1；
P2:5 '-TGGGCTCAGACAATAAATCCT-3 '；SEQ ID NO.2.
2. a kind of method for identifying Genus Triplophysa fish, which is characterized in that specific step is as follows:
(1) according to the mitochondrial COI gene sequence of Genus Triplophysa fish not of the same race, the conservative fragments between different sequences are chosen；
(2) conservative fragments chosen according to step (1), design and filter out the specific primer of Genus Triplophysa fish；
(3) specific primer obtained using step (2) is carried out PCR amplification to the genomic DNA of Genus Triplophysa fish, obtained
Amplified production；
(4) amplified production for obtaining step (3) carries out agarose gel electrophoresis, recycles band, is sequenced；
(5) gained sequence is sequenced in step (4) to be compared, the high and its nearly source kind sequence of downloading similarity constructs systematic growth
Tree estimates genetic distance, primarily determines measured sequence species ownership；
(6) species ownership form confirm again, be directed toward according to molecular data, combining form feature confirmation species most after all
Belong to.
3. a kind of method for identifying Genus Triplophysa fish according to claim 2, which is characterized in that the PCR amplification
Reaction system are as follows: 15 μ L of Taq Mixture, template DNA 1 μ L, 10 μM of primer P1 1.5 μ L, 10 μM of 1.5 μ L of primer P2,
ddH211 μ L of O, totally 30 μ L.
4. a kind of method for identifying Genus Triplophysa fish according to claim 2, which is characterized in that the PCR amplification
Response procedures are as follows: 94 DEG C of 5min；94 DEG C of 30S, 55 DEG C of 30S, 72 DEG C of 1min, 30 circulations；72℃10min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113684284A (en) * | 2021-09-07 | 2021-11-23 | 中国水产科学研究院长江水产研究所 | Primer, kit and method for identifying fish of loach family |
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