CN104419758A - Molecular genetics identification method for channel catfishes and flathead catfishes - Google Patents
Molecular genetics identification method for channel catfishes and flathead catfishes Download PDFInfo
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- CN104419758A CN104419758A CN201310398110.3A CN201310398110A CN104419758A CN 104419758 A CN104419758 A CN 104419758A CN 201310398110 A CN201310398110 A CN 201310398110A CN 104419758 A CN104419758 A CN 104419758A
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Abstract
The invention belongs to the genetics field and relates to a molecular genetics identification method for channel catfishes and flathead catfishes. The molecular genetics identification method for channel catfishes and flathead catfishes comprises the following steps: respectively extracting genome DNA (deoxyribonucleic acid) of to-be-identified channel catfishes and flathead catfishes; performing PCR (polymerase chain reaction) amplification on amicine genes of the channel catfishes and flathead catfishes; sequencing and comparing; if AGC microsatellites, starting from the 83th locus, of the MSTN (myostatin) gene are repeated for six times, identifying the catfishes as channel catfishes, and if the microsatellites are repeated for seven times, judging the catfishes as flathead catfishes. The molecular genetics identification method disclosed by the invention can be used for quickly, simply and conveniently, accurately and effectively identifying the channel catfishes and the flathead catfishes, is good in result stability and high in repetitive rate, so that the blank of identifying channel catfishes and flathead catfishes according to the existing domestic molecular biologic criteria is filled up.
Description
Technical field
This invention belongs to genetic arts, relates to the molecular genetic identification method of a kind of channel catfish and long fin Cha Wei Channel-catfish.
Background technology
Channel catfish and long fin Cha Wei Channel-catfish originate in the U.S., are the kinds that current U.S. cultural technique is the most ripe, cultured output is the highest, cultured output about 300,000 tons.Channel catfish is after 20th century, the eighties introduced China, be developed rapidly in the twenties short years, in succession breach the link technical barriers such as cultivation, breeding, feed, export processing, define from the complete industry chain (supply chain) of seed breeding, cultivation, processing, the marketing, output is also surged from initial several kilotons, reach 22.4 ten thousand tons during climax, become one of principal item of the China's export U.S..But channel catfish and long fin Cha Wei Channel-catfish germplasm origin rely on import, quality can not get ensureing, may there is the situation that channel catfish and long fin Cha Wei Channel-catfish mix.
Still there is no at present the repetition regularity of micro-satellite of bibliographical information channel catfish and long fin Cha Wei Channel-catfish MSTN gene, and using this foundation as two kinds of fish Molecular Identification.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, the molecular genetic identification method of a kind of channel catfish and long fin Cha Wei Channel-catfish is provided.
A molecular genetic identification method of channel catfish and long fin Cha Wei Channel-catfish, comprises following steps:
(1) channel catfish to be identified and long fin Cha Wei Channel-catfish genomic dna is extracted respectively;
(2) with the genomic dna extracted for template, pcr amplification channel catfish and long fin pitch the growth-inhibiting plain gene of tail Channel-catfish;
(3), after amplified production purifying, direct Sequencing, carries out sequence alignment;
(4) according to sequencing result, judge: the AGC micro-satellite of MSTN gene from the 83rd site is sextupl as channel catfish, if this micro-satellite repeat seven times for long fin Cha Wei Channel-catfish.
The described upstream primer pitching the growth-inhibiting plain gene of tail Channel-catfish for pcr amplification channel catfish and long fin is SEQ ID NO.1, and downstream primer is SEQ ID NO.2.
Described PCR reaction system is the template DNA 1 μ L of 50 μ L:50ng/ μ L, PCR damping fluid 5 μ L, dNTP mixed solution 4 μ L, each 1 μ L of upstream and downstream primer of often kind of dNTP0.1mmol/L, 10 μm of ol/L, the Taq enzyme of 2 μ L2IU; Distilled water 36 μ L; Described PCR damping fluid by 10mmol/L Tris-HCl, pH9.0,0.5mmol/L KCl, 30mmol/L MgCl2,0.01% gelatin composition; Pcr amplification reaction program is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 45s, 60 DEG C annealing 1min, 72 DEG C extend 1min, through 35 circulation after again 72 DEG C extend 10min.
For the primer pair that the molecular genetic of channel catfish and long fin Cha Wei Channel-catfish is differentiated, upstream primer is SEQ ID NO.1, and downstream primer is SEQ ID NO.2.
Primer pair of the present invention is preparing the application in channel catfish and long fin Cha Wei Channel-catfish molecular genetic identification reagent.
A kind of channel catfish and long fin Cha Wei Channel-catfish molecular genetic identification reagent, comprise primer pair of the present invention.
Described channel catfish and long fin Cha Wei Channel-catfish molecular genetic identification reagent, also preferably include PCR damping fluid and dNTP mixed solution, described PCR damping fluid is by 10mmol/L Tris-HCl, pH9.0,0.5mmol/LKCl, 30mmol/L MgCl2,0.01% gelatin composition.
Beneficial effect:
The present invention is directed to channel catfish and long fin Cha Wei Channel-catfish muscle cell growth statin (Myostatin, the difference of the multiplicity of the micro-satellite MSTN) on gene, first using this Species distinctive segment as foundation, thus qualitative identification channel catfish and long fin Cha Wei Channel-catfish.The method can quick, easy, differentiate channel catfish and long fin Cha Wei Channel-catfish accurately and efficiently, result good stability, repetition rate is high, has filled up the blank that current domestic standard molecular biological differentiates channel catfish and long fin Cha Wei Channel-catfish.
Accompanying drawing explanation
Fig. 1 is MSTN gene sequencing sequence alignment result in the present invention, and what asterisk was indicated is from the 83rd site
(MSTN-83) (AGC) that start micro-satellite repeats.
Embodiment
Illustrate the present invention further below in conjunction with embodiment, but be not that the present invention is limited in any form, only do example explanation.
Embodiment 1
1, design of primers
Design a pair specific PCR amplimer, primer sequence is: upstream primer SEQ ID NO.1:5'-ACGGTGTTC CTGTTACTGC-3', downstream primer SEQ ID NO.2:5'-CACCAGATGTTGCTATGC-3'.
2, sample collection
Get channel catfish to be identified and individual each 30 tails of long fin Cha Wei Channel-catfish.
3, extracting genome DNA
The tail fin getting every tail fish organizes about 20mg, blots surface-moisture with filter paper, loads 1.5ml centrifuge tube, adds 420 μ L STE damping fluids (30mmol/L Tris-HCl, pH8.0,200mmol/L EDTA, 50mmol/LNaCl).The Proteinase K 10 μ L that concentration is SDS80 μ L and 20mg/mL of 10% is added again, 56 DEG C of digestion 8-10h after mixing; Add equal-volume phenol: chloroform: the centrifugal 10min of primary isoamyl alcohol (25:24:1) extracting twice, 12000r, get supernatant liquor; Add equal-volume chloroform: once, the centrifugal 10min of 12000r, gets supernatant liquor in primary isoamyl alcohol (24:1) extracting; Add equal-volume precooling dehydrated alcohol, the centrifugal 10min of 12000r, abandons clear liquid; Add the washing with alcohol precipitation of 600mL70%, the centrifugal 10min of 8000r, outwells ethanol, dry; Add 200 μ L distilled waters to dissolve, ultraviolet spectrophotometer is surveyed its OD value and is diluted to 50ng/ μ L, and-20 DEG C save backup.
4, pcr amplification and detection
The DNA extracted is template, carries out pcr amplification with above-mentioned primer (SEQ ID NO.1 and SEQ ID NO.2).PCR reaction system is 50 μ L:1 μ L template DNAs (50ng/ μ L); PCR damping fluid 5 μ L (10mmol/L Tris-HCl, pH9.0,0.5mmol/L KCl, 30mmol/L MgCl2,0.01% gelatin); DNTP mixed solution 4 μ L (often kind of dNTP0.1mmol/L); Upstream and downstream primer (10 μm of ol/L) each 1 μ L, 2 μ L Taq enzyme (2IU); Distilled water 36 μ L.Amplified reaction program is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 45s, 60 DEG C annealing 1min, 72 DEG C extend 1min, through 35 circulation after again 72 DEG C extend 10min.
PCR primer detects separation in 1% agarose gel electrophoresis, under 120V voltage after electrophoresis 30min, through size and the purity of Labworks image acquisition and analysis software determination pcr amplification product.
5, order-checking comparison
Pcr amplification product after detection directly delivers to biotech firm's purifying order-checking, obtain the sequence of about 500bp, can find after comparison that (AGC) the micro-satellite multiplicity of MSTN gene from the 83rd site (MSTN-83) there are differences: 30 tail channel catfish (AGC) micro-satellite multiplicity from the 83rd site (MSTN-83) is six times, 30 tail long fin Cha Wei Channel-catfish (AGC) micro-satellite multiplicity from the 83rd site (MSTN-83) is seven times.
Embodiment 2
According to embodiment 1 method, by sample enlargement to channel catfish and individual each 100 tails of long fin Cha Wei Channel-catfish, result still display dot Cha Wei Channel-catfish (AGC) micro-satellite multiplicity from the 83rd site (MSTN-83) is six times, and long fin Cha Wei Channel-catfish (AGC) micro-satellite multiplicity from the 83rd site (MSTN-83) is seven times.
Claims (7)
1. channel catfish and long fin pitch a molecular genetic identification method of tail Channel-catfish, it is characterized in that comprising following steps:
(1) channel catfish to be identified and long fin Cha Wei Channel-catfish genomic dna is extracted respectively;
(2) with the genomic dna extracted for template, pcr amplification channel catfish and long fin pitch the growth-inhibiting plain gene of tail Channel-catfish;
(3), after amplified production purifying, direct Sequencing, carries out sequence alignment;
(4) according to sequencing result, judge: the AGC micro-satellite of MSTN gene from the 83rd site is sextupl as channel catfish, if this micro-satellite repeat seven times for long fin Cha Wei Channel-catfish.
2. the molecular genetic identification method of channel catfish according to claim 1 and long fin Cha Wei Channel-catfish, it is characterized in that the described upstream primer pitching the growth-inhibiting plain gene of tail Channel-catfish for pcr amplification channel catfish and long fin is SEQ ID NO.1, downstream primer is SEQ ID NO.2.
3. the molecular genetic identification method of channel catfish according to claim 1 and long fin Cha Wei Channel-catfish, it is characterized in that described PCR reaction system is the template DNA 1 μ L of 50 μ L:50ng/ μ L, PCR damping fluid 5 μ L, dNTP mixed solution 4 μ L, often kind of dNTP0.1mmol/L, the each 1 μ L of upstream and downstream primer of 10 μm of ol/L, the Taq enzyme of 2 μ L2IU; Distilled water 36 μ L; Described PCR damping fluid is by 10mmol/LTris-HCl, pH9.0,0.5mmol/L KCl, 30mmol/L MgCl2, and 0.01% gelatin forms; Pcr amplification reaction program is: 94 DEG C of denaturation 2min, 94 DEG C of sex change 45s, 60 DEG C annealing 1min, 72 DEG C extend 1min, through 35 circulation after again 72 DEG C extend 10min.
4. the primer pair that the molecular genetic pitching tail Channel-catfish for channel catfish and long fin is differentiated, it is characterized in that upstream primer is SEQ ID NO.1, downstream primer is SEQ ID NO.2.
5. primer pair according to claim 4 is preparing the application in channel catfish and long fin Cha Wei Channel-catfish molecular genetic identification reagent.
6. channel catfish and long fin pitch a tail Channel-catfish molecular genetic identification reagent, it is characterized in that comprising primer pair according to claim 4.
7. channel catfish according to claim 6 and long fin Cha Wei Channel-catfish molecular genetic identification reagent, characterized by further comprising PCR damping fluid and dNTP mixed solution, described PCR damping fluid is by 10mmol/LTris-HCl, pH9.0,0.5mmol/L KCl, 30mmol/L MgCl2,0.01% gelatin composition.
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| CN201310398110.3A CN104419758B (en) | 2013-09-05 | 2013-09-05 | A kind of molecular genetic identification method of channel catfish and long fin fork-tail |
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| CN201310398110.3A CN104419758B (en) | 2013-09-05 | 2013-09-05 | A kind of molecular genetic identification method of channel catfish and long fin fork-tail |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502663A (en) * | 2017-09-19 | 2017-12-22 | 江苏省淡水水产研究所 | A kind of channel catfish microsatellite Parentage determination method |
| CN111304338A (en) * | 2020-03-13 | 2020-06-19 | 江苏省淡水水产研究所 | SNP molecular marker linked with sex of channel catfish and genetic sex identification method |
| CN114836414A (en) * | 2022-04-15 | 2022-08-02 | 江苏省淡水水产研究所 | Primer and method for molecular identification of channel catfish and channel catfish |
-
2013
- 2013-09-05 CN CN201310398110.3A patent/CN104419758B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| ARIF M. KOCABAS ET AL.: "Molecular characterization and differential expression of the myostatin gene in channel catfish (Ictalurus punctatus)", 《BIOCHIMICA ET BIOPHYSICA ACTA》, 3 May 2002 (2002-05-03), pages 100 - 103 * |
| JERRY SERAPION ET AL.: "Bioinformatic Mining of Type I Microsatellites from Expressed Sequence Tags of Channel Catfish (Ictalurus punctatus)", 《MAR. BIOTECHNOL》, 31 August 2004 (2004-08-31), pages 364 - 377 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107502663A (en) * | 2017-09-19 | 2017-12-22 | 江苏省淡水水产研究所 | A kind of channel catfish microsatellite Parentage determination method |
| CN107502663B (en) * | 2017-09-19 | 2020-07-07 | 江苏省淡水水产研究所 | Channel catfish microsatellite family identification method |
| CN111304338A (en) * | 2020-03-13 | 2020-06-19 | 江苏省淡水水产研究所 | SNP molecular marker linked with sex of channel catfish and genetic sex identification method |
| CN111304338B (en) * | 2020-03-13 | 2023-03-31 | 江苏省淡水水产研究所 | SNP molecular marker linked with sex of channel catfish and genetic sex identification method |
| CN114836414A (en) * | 2022-04-15 | 2022-08-02 | 江苏省淡水水产研究所 | Primer and method for molecular identification of channel catfish and channel catfish |
| CN114836414B (en) * | 2022-04-15 | 2023-09-22 | 江苏省淡水水产研究所 | Primers and method for molecular identification of channel catfish and channel catfish |
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