CN104419758B - A kind of molecular genetic identification method of channel catfish and long fin fork-tail - Google Patents
A kind of molecular genetic identification method of channel catfish and long fin fork-tail Download PDFInfo
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- CN104419758B CN104419758B CN201310398110.3A CN201310398110A CN104419758B CN 104419758 B CN104419758 B CN 104419758B CN 201310398110 A CN201310398110 A CN 201310398110A CN 104419758 B CN104419758 B CN 104419758B
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Abstract
This invention belongs to genetic arts, relates to the molecular genetic identification method of a kind of channel catfish and long fin fork-tail.A kind of channel catfish and the molecular genetic identification method of long fin fork-tail, comprise the steps of and extract channel catfish to be identified and long fin fork-tail genomic DNA respectively；The growth inhibited plain gene of pcr amplification channel catfish and long fin fork-tail；Carry out order-checking comparison；The micro-satellite of AGC that MSTN gene starts from the 83rd site is sextupl for channel catfish, if this micro-satellite repeat seven times be long fin fork-tail.The method can quickly, easy, differentiate channel catfish and long fin fork-tail, result good stability accurately and efficiently, repetitive rate is high, has filled up current domestic standard molecular biological and has differentiated the blank of channel catfish and long fin fork-tail.
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 fork-tail.
Background technology
Channel catfish and long fin fork-tail 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 from 20th century after the eighties introduces China, it is developed rapidly in the twenties short years, in succession breach the link technical barriers such as cultivation, breeding, feedstuff, export processing, define from the complete industry chain (supply chain) of seed breeding, cultivation, processing, the marketing, yield is also surged from initial several kilotons, reach 22.4 ten thousand tons during summit, become one of principal item of the China's export U.S..But channel catfish and long fin fork-tail germplasm origin rely on import, quality can not get ensureing, it is understood that there may be the situation that channel catfish and long fin fork-tail mix.
Still do not have bibliographical information channel catfish regular with the repetition of micro-satellite of long fin fork-tail MSTN gene at present, and using this foundation as two kinds of Fish Molecular Identification.
Summary of the invention
It is an object of the invention to the above-mentioned deficiency for prior art, it is provided that the molecular genetic identification method of a kind of channel catfish and long fin fork-tail.
A kind of channel catfish and the molecular genetic identification method of long fin fork-tail, comprise the steps of
(1) channel catfish to be identified and long fin fork-tail genomic DNA are extracted respectively；
(2) with the genomic DNA of extraction for template, the growth inhibited plain gene of pcr amplification channel catfish and long fin fork-tail；
(3) after amplified production purification, direct Sequencing, carry out sequence alignment；
(4) according to sequencing result, judge: the micro-satellite of AGC that MSTN gene starts from the 83rd site is sextupl as channel catfish, if this micro-satellite repeat seven times be long fin fork-tail.
The described forward primer for pcr amplification channel catfish with the growth inhibited plain gene of long fin fork-tail is SEQIDNO.1, and downstream primer is SEQIDNO.2.
Described PCR reaction system is each 1 μ L of upstream and downstream primer of template DNA 1 μ L, PCR buffer 5 μ L, dNTP mixed liquor 4 μ L, every kind of dNTP0.1mmol/L, 10 μm of ol/L of 50 μ L:50ng/ μ L, the Taq enzyme of 2 μ L2IU；Distilled water 36 μ L；Described PCR buffer is made up of 10mmol/LTris-HCl, pH9.0,0.5mmol/LKCl, 30mmol/LMgCl2,0.01% gelatin；Pcr amplification reaction program is: 94 DEG C of denaturation 2min, 94 DEG C of degeneration 45s, 60 DEG C annealing 1min, 72 DEG C extend 1min, through 35 circulate after again 72 DEG C extend 10min.
A kind of primer pair differentiated for the molecular genetic of channel catfish with long fin fork-tail, forward primer is SEQIDNO.1, and downstream primer is SEQIDNO.2.
The primer pair of the present invention application in preparing channel catfish and long fin fork-tail molecular genetic identification reagent.
A kind of channel catfish and long fin fork-tail molecular genetic identification reagent, comprise primer pair of the present invention.
Described channel catfish and long fin fork-tail molecular genetic identification reagent, it is also preferred that include PCR buffer and dNTP mixed liquor, described PCR buffer is made up of 10mmol/LTris-HCl, pH9.0,0.5mmol/LKCl, 30mmol/LMgCl2,0.01% gelatin.
Beneficial effect:
The present invention is directed to the difference of the number of repetition of micro-satellite on channel catfish and long fin fork-tail muscle cell growth inhibin (Myostatin, MSTN) gene, first using this Species distinctive segment as foundation, thus qualitative identification channel catfish and long fin fork-tail.The method can quickly, easy, differentiate channel catfish and long fin fork-tail, result good stability accurately and efficiently, repetitive rate is high, has filled up current domestic standard molecular biological and has differentiated the blank of channel catfish and long fin fork-tail.
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) the micro-satellite started repeats.
Detailed description of the invention
It is further elucidated with the present invention below in conjunction with embodiment, but is not that the present invention is limited in any form, only illustrate.
Embodiment 1
1, design of primers
Designing a pair specific PCR amplimer, primer sequence is: forward primer SEQIDNO.1:5'-ACGGTGTTCCTGTTACTGC-3', downstream primer SEQIDNO.2:5'-CACCAGATGTTGCTATGC-3'.
2, sample collection
Take channel catfish to be identified and individual each 30 tails of long fin fork-tail.
3, extracting genome DNA
The tail fin taking every tail fish organizes about 20mg, blots surface moisture with filter paper, loads 1.5ml centrifuge tube, adds 420 μ LSTE buffer (30mmol/LTris-HCl, pH8.0,200mmol/LEDTA, 50mmol/LNaCl).The E.C. 3.4.21.64 10 μ L of SDS80 μ L and the 20mg/mL that concentration is 10% is added, 56 DEG C of digestion 8-10h after mixing；Add equal-volume phenol: chloroform: isoamyl alcohol (25:24:1) extracting twice, 12000r is centrifuged 10min, takes supernatant；Add equal-volume chloroform: once, 12000r is centrifuged 10min, takes supernatant in isoamyl alcohol (24:1) extracting；Adding equal-volume pre-cooling dehydrated alcohol, 12000r is centrifuged 10min, abandons clear liquid；Adding the washing with alcohol precipitation of 600mL70%, 8000r is centrifuged 10min, outwells ethanol, dry；Adding 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 (SEQIDNO.1 and SEQIDNO.2).PCR reaction system is 50 μ L:1 μ L template DNA (50ng/ μ L)；PCR buffer 5 μ L (10mmol/LTris-HCl, pH9.0,0.5mmol/LKCl, 30mmol/LMgCl2,0.01% gelatin)；DNTP mixed liquor 4 μ L (every kind of dNTP0.1mmol/L)；Upstream and downstream primer (10 μm of ol/L) each 1 μ L, 2 μ LTaq enzyme (2IU)；Distilled water 36 μ L.Amplified reaction program is: 94 DEG C of denaturation 2min, 94 DEG C of degeneration 45s, 60 DEG C annealing 1min, 72 DEG C extend 1min, through 35 circulate after again 72 DEG C extend 10min.
PCR primer detects separation in 1% agarose gel electrophoresis, under 120V voltage after electrophoresis 30min, determines size and the purity of pcr amplification product through Labworks image acquisition and analysis software.
5, order-checking comparison
Pcr amplification product after detection is fed directly to biotech firm's purification order-checking, obtain the sequence of about 500bp, it appeared that (AGC) micro-satellite number of repetition that MSTN gene starts from the 83rd site (MSTN-83) there are differences after comparison: (AGC) micro-satellite number of repetition that 30 tail channel catfish start from the 83rd site (MSTN-83) is six times, (AGC) micro-satellite number of repetition that 30 tail long fin fork-tail starts from the 83rd site (MSTN-83) is seven times.
Embodiment 2
According to embodiment 1 method, by individual to sample enlargement to channel catfish and long fin fork-tail each 100 tails, (AGC) micro-satellite number of repetition that result still display dot fork-tail starts from the 83rd site (MSTN-83) is six times, and (AGC) micro-satellite number of repetition that long fin fork-tail starts from the 83rd site (MSTN-83) is seven times.
Claims (3)
1. the molecular genetic identification method of a channel catfish and long fin fork-tail, it is characterised in that comprise the steps of
(1) channel catfish to be identified and long fin fork-tail genomic DNA are extracted respectively；
(2) with the genomic DNA of extraction for template, the growth inhibited plain gene of pcr amplification channel catfish and long fin fork-tail；
(3) after amplified production purification, direct Sequencing, carry out sequence alignment；
(4) according to sequencing result, judge: the micro-satellite of AGC that growth inhibited plain gene starts from the 83rd site is sextupl as channel catfish, if this micro-satellite repeat seven times be long fin fork-tail.
2. the molecular genetic identification method of channel catfish according to claim 1 and long fin fork-tail, it is characterised in that the forward primer for pcr amplification channel catfish Yu the growth inhibited plain gene of long fin fork-tail is SEQIDNO.1, and downstream primer is SEQIDNO.2.
3. the molecular genetic identification method of channel catfish according to claim 1 and long fin fork-tail, it is characterized in that the template DNA 1 μ L that described PCR reaction system is 50 μ L:50ng/ μ L, PCR buffer 5 μ L, dNTP mixed liquor 4 μ L, every 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 buffer is by 10mmol/LTris-HCl, pH9.0,0.5mmol/LKCl, 30mmol/LMgCl2, 0.01% gelatin composition；Pcr amplification reaction program is: 94 DEG C of denaturation 2min, 94 DEG C of degeneration 45s, 60 DEG C annealing 1min, 72 DEG C extend 1min, through 35 circulate after again 72 DEG C extend 10min.
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| CN107502663B (en) * | 2017-09-19 | 2020-07-07 | 江苏省淡水水产研究所 | Channel catfish microsatellite family 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 |
| 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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