CN106399500B - A kind of molecular identification method of Big Salangid and neosalanx taihuensis - Google Patents
A kind of molecular identification method of Big Salangid and neosalanx taihuensis Download PDFInfo
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- 241000969498 Neosalanx taihuensis Species 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 13
- 230000002068 genetic effect Effects 0.000 claims abstract description 12
- 238000012408 PCR amplification Methods 0.000 claims abstract description 11
- 101150001086 COB gene Proteins 0.000 claims abstract description 7
- 101150053771 MT-CYB gene Proteins 0.000 claims abstract description 7
- 101150006264 ctb-1 gene Proteins 0.000 claims abstract description 7
- 101150088166 mt:Cyt-b gene Proteins 0.000 claims abstract description 7
- 238000012163 sequencing technique Methods 0.000 claims abstract description 6
- 238000000605 extraction Methods 0.000 claims abstract description 3
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- 238000000746 purification Methods 0.000 claims abstract description 3
- 108010075028 Cytochromes b Proteins 0.000 claims description 8
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- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 4
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 239000008273 gelatin Substances 0.000 claims description 4
- 229920000159 gelatin Polymers 0.000 claims description 4
- 235000019322 gelatine Nutrition 0.000 claims description 4
- 235000011852 gelatine desserts Nutrition 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 230000004087 circulation Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000000137 annealing Methods 0.000 claims description 2
- 239000000284 extract Substances 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 230000003252 repetitive effect Effects 0.000 abstract description 2
- 241001124553 Lepismatidae Species 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- 102100025287 Cytochrome b Human genes 0.000 description 2
- 241001062511 Salangidae Species 0.000 description 2
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Abstract
The invention discloses the molecular identification methods of a kind of Big Salangid and neosalanx taihuensis.A kind of molecular genetic identification method of Big Salangid and neosalanx taihuensis comprising the steps of: (1) extract Big Salangid and neosalanx taihuensis genomic DNA to be identified respectively；(2) using the genomic DNA of extraction as template, the Ctyb gene of PCR amplification Big Salangid and neosalanx taihuensis；(3) after purification, direct Sequencing, it is Big Salangid that the 45th site base of Cytb gene is T to amplified production, which is that C is neosalanx taihuensis.This method energy is quick, simplicity, accurately and efficiently identifies Big Salangid and neosalanx taihuensis, and as a result stability is good, and repetitive rate is high, has filled up the blank of the current country's standard molecular biological identification Big Salangid and neosalanx taihuensis.
Description
Technical field
The invention belongs to genetic arts, are related to the molecular identification method of a kind of Big Salangid and neosalanx taihuensis.
Background technique
China be world silverfish area of origin and main distributed area, divide extensively in the major water system in Eastern China and lake
Cloth.Silverfish nutritive value and economic value are very high, are important economic fish, in creating huge economic benefit.Silverfish
Life cycle is short, discrete of generation, fecundity and to settle down ability strong.As typical r- Kstrategist, silverfish is sensitive to environmental change
And be swift in response, Population Dynamic is fast.However the silverfish natural resources in China but because reclaiming land from a lake, overfishing, environmental pollution and
The influence of many factors such as Habitat Fragmentation and continuous downturn, the natural resources of various silverfishes all decline to some extent, species
Distribution is reduced significantly, and individual species are gradually endangered, and carries out Icefish Resource investigation and protection work is extremely urgent.Big Salangid and Taihu Lake
New silverfish is two kinds of common silverfish types, and the two body colour and form are similar, is difficult to distinguish and identify.
Summary of the invention
The purpose of the present invention is being directed to the difficulty of differentiating forms, the molecular genetic of a kind of Big Salangid and neosalanx taihuensis is provided
Discrimination method.
The purpose of the present invention is achieved through the following technical solutions:
A kind of molecular genetic identification method of Big Salangid and neosalanx taihuensis comprising the steps of:
(1) Big Salangid and neosalanx taihuensis genomic DNA to be identified are extracted respectively；
(2) using the genomic DNA of extraction as template, the cytochrome b gene of PCR amplification Big Salangid and neosalanx taihuensis
(Ctyb)；
(3) after purification, direct Sequencing, it is Big Salangid that the 45th site base of Cytb gene is T to amplified production, if the site
It is C is neosalanx taihuensis.
Described is L14321 for the upstream primer of PCR amplification Big Salangid and the cytochrome b gene of neosalanx taihuensis:
SEQ ID NO.1, downstream primer are H15634:SEQ ID NO.2.
The PCR reaction system is 1 μ L, PCR buffer of template DNA, 5 μ L, the dNTP mixed liquor of 50 μ L:100ng/ μ L
4 μ L, every kind of dNTP 0.1mmol/L, each 1 μ L of the upstream and downstream primer of 10 μm of ol/L, the Taq enzyme of 2 μ L 2.5IU；36 μ of distilled water
L；The PCR buffer is by 10mmol/L Tris-HCl, pH9.0,0.5mmol/L KCl, 30mmol/L MgCl2,
0.01% (g/100ml) gelatin composition；Pcr amplification reaction program are as follows: 94 DEG C of initial denaturation 4min, 94 DEG C of denaturation 40s, 55 DEG C are annealed
40s, 72 DEG C of extension 90s, 72 DEG C of extension 10min again after 35 circulations.
A kind of primer pair identified for the molecular genetic of Big Salangid and neosalanx taihuensis, upstream primer L14321:SEQ
ID NO.1, downstream primer are H15634:SEQ ID NO.2.
Primer pair of the present invention identifies the application in Big Salangid and neosalanx taihuensis in molecular genetic.
Primer pair of the present invention is preparing the application in Big Salangid and neosalanx taihuensis molecular genetic identification reagent.
A kind of Big Salangid and neosalanx taihuensis molecular genetic identification reagent include primer pair of the present invention.
The Big Salangid and neosalanx taihuensis molecular genetic identification reagent further preferably includes PCR buffer and dNTP mixed
Liquid is closed, the PCR buffer is by 10mmol/L Tris-HCl, pH9.0,0.5mmol/L KCl, 30mmol/L MgCl2,
0.01% gelatin composition.
The utility model has the advantages that
The present invention is directed to Big Salangid and neosalanx taihuensis cytochrome b (Cytochrome b, Cytb) gene order difference,
For the first time using the cytochrome b gene full length sequence of Big Salangid and neosalanx taihuensis as foundation, thus Qualitive test Big Salangid and
Neosalanx taihuensis.This method can quickly, it is easy, accurately and efficiently identify Big Salangid and neosalanx taihuensis, as a result stability
Good, repetitive rate is high, has filled up the blank that current domestic standard molecular biological identifies Big Salangid and neosalanx taihuensis, has been also beneficial to
Icefish Resource protection.
Detailed description of the invention
Fig. 1 is Cytb gene sequencing sequence alignment result in the present invention, and what asterisk was indicated is from the 45th site (Cytb-45)
The base sequence difference of beginning.
Specific embodiment
Next with reference to embodiments the present invention is furture elucidated, but is not to limit in any form to the present invention, only
Only illustrate.
Embodiment 1
1, design of primers
Design a pair of of specific PCR amplimer, primer sequence are as follows: L14321:5'-CCAGTGA-
CTTGAAAAACCACCG-3'(SEQ ID NO.1), downstream primer H15634:5'-CTTAGCTTTGGGAGT-TAAGGGT-
3'(SEQ ID NO.2)。
2, sample collection
Take Big Salangid and each 30 tail of neosalanx taihuensis individual to be identified.
3, extracting genome DNA
The tail fin tissue about 30mg for taking every tail fish blots surface moisture with filter paper, is packed into 1.5ml centrifuge tube, 420 μ L are added
STE buffer (30mmol/L Tris-HCl, pH8.0,200mmol/L EDTA, 50mmol/L NaCl).It is added after mixing
Proteinase K 10 the μ L, 56 DEG C of digestion 8-10h of SDS 80 μ L and 20mg/mL that concentration is 10%；Isometric phenol: chloroform is added:
Isoamyl alcohol (volume ratio 25:24:1) extracts twice, and 12000r is centrifuged 10min, takes supernatant；Isometric chloroform: isoamyl alcohol is added
(volume ratio 24:1) extracting is primary, and 12000r is centrifuged 10min, takes supernatant；Be added isometric pre-cooling dehydrated alcohol, 12000r from
Heart 10min abandons clear liquid；The ethanol washing precipitating of 600mL 70% is added, 8000r is centrifuged 10min, outwells ethyl alcohol, dry；It is added
The dissolution of 200 μ L distilled waters, ultraviolet specrophotometer survey its OD value and are diluted to 100ng/ μ L, and -20 DEG C save backup.
4, PCR amplification and detection
Extracted DNA is template, carries out PCR amplification with above-mentioned primer (L14321 and H15634).PCR reaction system is
50 μ L:1 μ L template DNAs (100ng/ μ L)；5 μ L of PCR buffer (10mmol/L Tris-HCl, pH9.0,0.5mmol/LKCl,
30mmol/L MgCl2, 0.01% gelatin)；4 μ L of dNTP mixed liquor (every kind of dNTP 0.1mmol/L)；Upstream and downstream primer (10 μ
Mol/L) each 1 μ L, 2 μ L Taq enzymes (2.5IU)；36 μ L of distilled water.Amplified reaction program are as follows: 94 DEG C of initial denaturation 4min, 94 DEG C of changes
Property 40s, 55 DEG C of annealing 40s, 72 DEG C of extension 90s, 72 DEG C of extension 10min again after 35 circulations.
PCR product detects separation in 1% agarose gel electrophoresis, under 120V voltage after electrophoresis 30min, through gel at
As analysis system determines the size and purity of pcr amplification product.
5, sequencing compares
Pcr amplification product after detection is fed directly to biotech firm's purifying sequencing, obtains 1141bp sequence, can be with after comparison
It was found that Cytb gene has differences from the base sequence in the 45th site (Cytb-45): the 45th site (Cytb-45) of 30 tail Big Salangid
Base is T, and the 45th site (Cytb-45) of 30 tail neosalanx taihuensis is C.
Embodiment 2
According to 1 method of embodiment as a result sample enlargement to Big Salangid and each 80 tail of neosalanx taihuensis individual are still shown
Big Salangid cytochrome b gene the 45th site (Cytb-45) base is T, and neosalanx taihuensis the 45th site (Cytb-45) base is
C。
Claims (3)
1. the molecular genetic identification method of a kind of Big Salangid and neosalanx taihuensis, it is characterised in that comprise the steps of:
(1) Big Salangid and neosalanx taihuensis genomic DNA to be identified are extracted respectively；
(2) using the genomic DNA of extraction as template, the cytochrome b gene Ctyb of PCR amplification Big Salangid and neosalanx taihuensis；
It is L14321:SEQ ID NO.1 for the upstream primer of PCR amplification Big Salangid and the cytochrome b gene of neosalanx taihuensis, under
Trip primer is H15634:SEQ ID NO.2;
(3) after purification, direct Sequencing, it is Big Salangid that the 45th site base of Cytb gene is T to amplified production, if the site is C
It is neosalanx taihuensis;The Cytb gene order is as follows:
ATGGCCAACCTCCGAAAAACTCACCCTTTGCTGAAAATGACTAATCACGCTTTAGTCGACCTACCTGCCCCT
TCAAACCTTTCAGTTTGGTGAAACTTTGGCTCCCTTTTGGGAATCTGCCTAGTTCTCCAAATCCTAACAGGCCTAT
TCATGGCCATGCACTACGCCCCCGAAACCGCGAATGCATTCTCTTCTGTCGCCCACATATGTCGGGACGTCAACAA
CGGCTGACTAATACGCAACATGCATGCCAACGGAGCATCTTTCTTCTTCATCTGTGTTTACCTCCACATCGGCCGA
GGTCTTTACTACGGCTCATACCTTTACCAAGCAACATGAAATGTTGGAGTAGTCCTTCTCCTCCTGCTAATAATAA
CTGCCTTTGTAGGCTACGTCCTCCCCTGAGGACAAATGTCGTTCTGAGGGGCAACAGTAATCACCAACCTCCTCTC
TGCCGCCCCCTACGTGGGATTCGACCTAGTTTTATGACTATGAGGGGGGTTCTCTGTAGACAATGCCACCCTCACT
CGATTCTTCGCCTTCCACTTCATTCTCCCTTTCATCATTGCCGCCGCCACTGTCATTCACCTCCTTTTCCTCCACG
AAACGGGATCAAACAACCCACTTGGCCTTAGCTCAGACGTAGATAAAATCCCTTTCTTGCCATACTATATTATCAA
GGACGTAGTCGGCTTCCTAGTCTTTTTCCTCGCCTTCTTCTCAATCACCCTGTTCTTCCCCAACCTCCTCGGCGAC
CCAGATAATTTTACAGAGGCCAACCCCCTCGTCACCCCAGCCCACATTAAACCTGAGTGGTACTTTCTTTTCGCCT
ACGCTATCCTCCGGTCTATTCCCAGCAAACTGGGCGGTGTTTTAGCCCTCCTCTTCTCTATCCTGGTGCTCCTTCT
AGTGCCATTCCTTCACACCTCTAAACAGCAAGGCCTAGCTTTTCGCCCACTCACCCAACTACTCTTCTGGTCTCTC
GTGGCTGATGTTTTTATCCTTACATGAATCGGGGGAATACCTGTAGAACACCCCTACATCGTAATTGGCCAAATTG
CTTCCGTAATCTACTTCTCCATCTTCCTGATTCTTTTCCCCTTTGTAGGCTGGGCCGAAAATAAAATCCTCAAATG
AGCCT。
2. molecular genetic identification method according to claim 1, it is characterised in that PCR reaction system is 50 μ L:100ng/
1 μ L, PCR buffer of template DNA, 5 μ L, the dNTP mixed liquor 4 μ L of μ L, every kind of dNTP 0.1mmol/L, 10 μm of ol/L's
Each 1 μ L of upstream and downstream primer, the Taq enzyme of 2 μ L, 2.5 IU；36 μ L of distilled water；The PCR buffer is by 10mmol/L Tris-
HCl, pH9.0,0.5mmol/L KCl, 30mmol/L MgCl2, 0.01%(w/v) and gelatin composition.
3. molecular genetic identification method according to claim 1, it is characterised in that pcr amplification reaction program are as follows: 94 DEG C pre-
4 min, 94 DEG C of denaturation 40 s, 55 DEG C of annealing 40 s, 72 DEG C of 90 s of extension are denaturalized, 72 DEG C of extensions 10 again after 35 circulations
min。
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| CN107858437A (en) * | 2017-09-27 | 2018-03-30 | 浙江海洋大学 | A kind of molecular biology method of quick discriminating silver color schizothoracin and Yi Li schizothoracins |
| CN110699460A (en) * | 2019-09-23 | 2020-01-17 | 江苏省淡水水产研究所 | Cytb gene sequence based molecular identification method for identifying beta and beta |
| CN113913534B (en) * | 2021-11-19 | 2022-05-13 | 中国水产科学研究院黑龙江水产研究所 | Primer for identifying big whitebait and Taihu new whitebait and identification method thereof |
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| CN104152451A (en) * | 2014-08-19 | 2014-11-19 | 淮南师范学院 | Primer and method for molecular identification of new whitebait species in Taihu Lake |
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Non-Patent Citations (2)
| Title |
|---|
| molecular phylogeny of icefish salangidae based on complete mtDNA cytochrome b sequences, with comments on estuarine fish evolution;Jie Zhang等;《biological journal of the linnean society》;20071231;摘要、第331页跨栏、第332页跨栏、图3、图4 |
| 太湖大银鱼（protosalanx chinensis）细胞色素b基因序列多态性分析;李大命等;《江苏农业学报》;20151231;全文 |
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