CN108048576A - Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof - Google Patents
Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof Download PDFInfo
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Abstract
The invention discloses a kind of methods for differentiating Dai-ju stock Pseudosciaena crocea and east Guangdong Larimichthys crocea, identify that primer is：J1：F1:5’‑GGCATTTACACACATGGAC‑3’;R1:5’‑GGGATCACAACAGAGCGG‑3’；J2：F2:5’‑CATTCCCTTCCGCGCTAGT‑3’;R2:5’‑GGCTGCAAGTTCCTGAC‑3’.It has the beneficial effect that：The high frequency difference site of two kinds of geographical population Larimichthys crocea genes is made full use of, while takes into account the secondary structure of primer, using the method for introducing base mismatch, the identification primer of the present invention designed.The identification primer specificity that design obtains is strong, and PCR amplification is efficient, compared to the prior art, differentiates that the time is shorter, accuracy rate higher.
Description
Technical field
The present invention relates to field of molecular biotechnology, more particularly, to a kind of discriminating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race
Method of Larimichthys crocea and application thereof.
Background technology
Larimichthys crocea (Pseudosciaena crocea (Richardso n)) is subordinate to Perciformes (Perciformes) perch
Suborder (Percoidei) Sciaenidae (Sciaenidae) yellow croaker category (Pseudosciaena), it is near to belong to subtropical zone sociability
Ocean fish class is distributed on the south Middle of The Huanghai Sea to the China's Mainland coastal waters to the east of Qiongzhou Strait and Korea West Coast, the Lezhou Peninsula
To the west of also even be found.According to the formalness of Larimichthys crocea and ecological geography feature, China coast Larimichthys crocea is divided into 3 geography
Population.Wherein, tai-chu race is distributed in southern Yellow Sea to fish community, including Lv Siyang, Dai Quyang, cat head ocean, Dongtou ocean extremely
Near the island of Fujian Yu mountains；Fujian-East Guangdong race is mainly distributed on southern East China Sea, the Taiwan Straits and Northern Part of South China Sea (on the south the island of Yu mountains extremely
The mouth of the Zhujiang River).
Because of advantage of the Dai-ju stock Pseudosciaena crocea in terms of body colour, build, nutritive value and flavor etc., valency on the market
Lattice are higher than Fujian-East Guangdong race Larimichthys crocea;In terms of cultivation, its growth speed, survival rate, incidence, feed coefficient and cold-resistant
Ability etc. is also better than Fujian-East Guangdong race Larimichthys crocea in Fujian.And it is in the Larimichthys crocea overwhelming majority that Ningbo, Zhoushan Region cultivate
Fujian-East Guangdong race Larimichthys crocea, it is much like with Dai-ju stock Pseudosciaena crocea in appearance, using traditional character observation, subjectivity
By force, it also is difficult to effectively be distinguished.
The content of the invention
Differentiate that the time is short it is an object of the present invention to a kind of, can effectively avoid false negative result, the high mirror of accuracy rate
Other Dai-ju stock Pseudosciaena crocea and the method for Fujian-East Guangdong race Larimichthys crocea.
The second object of the present invention is the method for Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea will be differentiated for quick
The defects of differentiating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea exactly, overcoming identification by morphological characters, solves germplasm and mixes
The problem of miscellaneous, for Conservation, rationally the biodiversity research of utilization and germplasm provides technical support.
The problem of present invention in background technology for mentioning, the technical solution taken is：Differentiate Dai-ju stock Pseudosciaena crocea and Fujian-
The method of East Guangdong race Larimichthys crocea identifies that primer is：
J1：F1:5’- GGCATTTACACACATGGAC-3’;R1: 5’-GGGATCACAACAGAGCGG -3’
J2：F2:5’- CATTCCCTTCCGCGCTAGT-3’;R2: 5’- GGCTGCAAGTTCCTGAC -3’.Make full use of two
The high frequency difference site of kind of geographical population Larimichthys crocea gene, while take into account the secondary structure of primer, using introducing base mismatch
Method, the identification primer of the present invention designed.The identification primer specificity that design obtains is strong, and PCR amplification is efficient, compares
In the prior art, differentiate that the time is shorter, accuracy rate higher.
Differentiate the method for Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, concrete operation step：
1）Using standard phenol-chloroform method extraction Larimichthys crocea total DNA as template DNA, agar electrophoresis detects DNA mass, ultraviolet point
Light photometric determination DNA concentration, and design identification primer：
J1：F1:5’- GGCATTTACACACATGGAC-3’;R1: 5’-GGGATCACAACAGAGCGG -3’
J2：F2:5’- CATTCCCTTCCGCGCTAGT-3’;R2: 5’- GGCTGCAAGTTCCTGAC -3’；
2）Configure PCR amplification system, total volume be 25 μ L, each ingredient and final concentration of Buffer10mM, dNTP 0.2mM,
MgCl21.5mM, 4 kinds of primers each 0.2mM, Taq 1U, Larimichthys crocea DNA 50ng, with distilled water polishing to 25 μ L.PCR
Amplification, 95 DEG C of pre-degeneration 5min, then 95 DEG C of denaturation 20s, 60 DEG C of annealing 20s, 72 DEG C of extension 1min carry out 30 Xun Huans, most
72 DEG C of extension 5min afterwards.Above-mentioned PCR amplification condition can be such that the PCR amplification of two pairs of primers synchronously carries out.Take the product that amplification obtains
It is detected into row agarose gel electrophoresis, and using the observation of ultraviolet gel imaging system, photograph, the concentration of Ago-Gel is 1%-
1.5%.Electrophoresis result and standard diagram are compared, if only having the band of a 1250bp in collection of illustrative plates in 1000 ~ 1500bp sections,
It is then Dai-ju stock Pseudosciaena crocea；If only has the band of a 1085bp in collection of illustrative plates in 1000 ~ 1500bp sections, for Fujian-East Guangdong race
Larimichthys crocea.It can differentiate Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea only by observation gel electrophoresis figure, both save
The Morphological Identification program of very complicated, and saved cost, it is often more important that it greatly improves work efficiency.
To optimize said program, the measure taken is：PCR amplification system is configured, total volume is 25 μ L, each ingredient and end
Concentration is Buffer10mM, dNTP 0.2mM, MgCl21.5mM, 4 kinds of primers each 0.2mM, Taq 1U, Larimichthys crocea DNA
50ng adds 1.6ng 2- chloro-5-nitrobenzoic acids and 0.1ng benzohydrols, with distilled water polishing to 25 μ L.PCR expands
Increase, 95 DEG C of pre-degeneration 5min, then 95 DEG C of denaturation 20s, 60 DEG C of annealing 20s, 72 DEG C of extension 1min carry out 30 Xun Huans, finally
72 DEG C of extension 5min.2- chloro-5-nitrobenzoic acids, benzohydrol and magnesium ion have synergistic effect, can not only be greatly
The activity of Taq polymerase is activated, amplified reaction efficiency is improved, shortens the time of discriminating, and can be passivated contained by DNA profiling
PCR mortifiers, avoid the appearance of false negative result, make identification result more accurate.
Compared with prior art, the advantage of the invention is that：Make full use of the high frequency of two kinds of geographical population Larimichthys crocea genes
Difference site, while the secondary structure of primer is taken into account, using the method for introducing base mismatch, the identification of the present invention designed is drawn
Object.The identification primer specificity that design obtains is strong, and PCR amplification is efficient, compared to the prior art, differentiates that the time is shorter, accurately
Rate higher.It can differentiate Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea only by observation gel electrophoresis figure, both eliminate
The Morphological Identification program of very complicated, and saved cost, it is often more important that it greatly improves work efficiency.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
Differentiate the method for Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, concrete operation step：
1）Using standard phenol-chloroform method extraction Larimichthys crocea total DNA as template DNA, agar electrophoresis detects DNA mass, ultraviolet point
Light photometric determination DNA concentration, and design identification primer：
J1：F1:5’- GGCATTTACACACATGGAC-3’;R1: 5’-GGGATCACAACAGAGCGG -3’
J2：F2:5’- CATTCCCTTCCGCGCTAGT-3’;R2: 5’- GGCTGCAAGTTCCTGAC -3’；
2）Configure PCR amplification system, total volume be 25 μ L, each ingredient and final concentration of Buffer10mM, dNTP 0.2mM,
MgCl21.5mM, 4 kinds of primers each 0.2mM, Taq 1U, Larimichthys crocea DNA 50ng, with distilled water polishing to 25 μ L.PCR
Amplification, 95 DEG C of pre-degeneration 5min, then 95 DEG C of denaturation 20s, 60 DEG C of annealing 20s, 72 DEG C of extension 1min carry out 30 Xun Huans, most
72 DEG C of extension 5min afterwards.Above-mentioned PCR amplification condition can be such that the PCR amplification of two pairs of primers synchronously carries out.Take the product that amplification obtains
It is detected into row agarose gel electrophoresis, and using the observation of ultraviolet gel imaging system, photograph, the concentration of Ago-Gel is 1%-
1.5%.Electrophoresis result and standard diagram are compared, if only having the band of a 1250bp in collection of illustrative plates in 1000 ~ 1500bp sections,
It is then Dai-ju stock Pseudosciaena crocea；If only has the band of a 1085bp in collection of illustrative plates in 1000 ~ 1500bp sections, for Fujian-East Guangdong race
Larimichthys crocea.
Embodiment 2：
Differentiate the method for Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, concrete operation step：
1）Using standard phenol-chloroform method extraction Larimichthys crocea total DNA as template DNA, agar electrophoresis detects DNA mass, ultraviolet point
Light photometric determination DNA concentration, and design identification primer：
J1：F1:5’- GGCATTTACACACATGGAC-3’;R1: 5’-GGGATCACAACAGAGCGG -3’
J2：F2:5’- CATTCCCTTCCGCGCTAGT-3’;R2: 5’- GGCTGCAAGTTCCTGAC -3’；
2）Configure PCR amplification system, total volume be 25 μ L, each ingredient and final concentration of Buffer10mM, dNTP 0.2mM,
MgCl21.5mM, 4 kinds of primers each 0.2mM, Taq 1U, Larimichthys crocea DNA 50ng, adds the chloro- 5- nitrobenzenes of 1.6ng 2-
Formic acid and 0.1ng benzohydrols, with distilled water polishing to 25 μ L.PCR amplification, 95 DEG C of pre-degeneration 5min, then 95 DEG C of denaturation
20s, 60 DEG C of annealing 20s, 72 DEG C of extension 1min, carries out 30 Xun Huans, last 72 DEG C of extensions 5min.Above-mentioned PCR amplification condition energy
The PCR amplification of two pairs of primers is made synchronously to carry out.The product that amplification obtains is taken to be detected into row agarose gel electrophoresis, and using ultraviolet
Gel imaging system observation, photograph, the concentration of Ago-Gel is 1%-1.5%.Electrophoresis result and standard diagram are compared, if
Only have the band of a 1250bp in collection of illustrative plates in 1000 ~ 1500bp sections, be then Dai-ju stock Pseudosciaena crocea；If 1000 in collection of illustrative plates ~
Only have the band of a 1085bp in 1500bp sections, be then Fujian-East Guangdong race Larimichthys crocea.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Zhejiang Ocean university
<120>Differentiate the method and its application of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>Larimichthys crocea (Pseudosciaena crocea)
<400> 1
ggcatttaca cacatggac 19
<210> 2
<211> 18
<212> DNA
<213>Larimichthys crocea (Pseudosciaena crocea)
<400> 2
gggatcacaa cagagcgg 18
<210> 3
<211> 19
<212> DNA
<213>Larimichthys crocea (Pseudosciaena crocea)
<400> 3
cattcccttc cgcgctagt 19
<210> 4
<211> 17
<212> DNA
<213>Larimichthys crocea (Pseudosciaena crocea)
<400> 4
ggctgcaagt tcctgac 17
Claims (8)
1. differentiate the method for Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that identify that primer is：
J1：F1:5’- GGCATTTACACACATGGAC-3’;R1: 5’-GGGATCACAACAGAGCGG -3’
J2：F2:5’- CATTCCCTTCCGCGCTAGT-3’;R2: 5’- GGCTGCAAGTTCCTGAC -3’.
2. the method according to claim 1 for differentiating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that with
Lower step：
1）Larimichthys crocea total DNA is extracted as template DNA, agar electrophoresis detection DNA mass, it is dense that ultraviolet specrophotometer measures DNA
Degree, and design identification primer：
J1：F1:5’- GGCATTTACACACATGGAC-3’;R1: 5’-GGGATCACAACAGAGCGG -3’
J2：F2:5’- CATTCCCTTCCGCGCTAGT-3’;R2: 5’- GGCTGCAAGTTCCTGAC -3’；
2）PCR amplification system is configured, and carries out PCR amplification, the product that amplification obtains is taken to be detected into row agarose gel electrophoresis, and
Using the observation of ultraviolet gel imaging system, photograph, Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race are determined according to obtained agar electrophoresis illustrated handbook
Larimichthys crocea.
3. the method according to claim 1 for differentiating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that：
The PCR amplification system is 25 μ L, each ingredient and final concentration of Buffer10mM, dNTP 0.2mM, MgCl21.5mM, 4
Kind primer each 0.2mM, Taq 1U, Larimichthys crocea DNA 50ng, with distilled water polishing to 25 μ L.
4. the method according to claim 1 for differentiating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that：
The PCR response procedures are 95 DEG C of pre-degeneration 5min, then 95 DEG C of denaturation 20s, 60 DEG C of annealing 20s, 72 DEG C of extension 1min,
Carry out 30 Xun Huans, last 72 DEG C of extensions 5min.
5. the method according to claim 1 for differentiating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that：
The extraction of the Larimichthys crocea DNA uses standard phenol-chloroform method.
6. the method according to claim 1 for differentiating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that：
The concentration of the Ago-Gel is 1%-1.5%.
7. the method according to claim 1 for differentiating Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that：
The described method that Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea are determined according to agar electrophoresis illustrated handbook is by electrophoresis result and standard
Collection of illustrative plates compares, if only having the band of a 1250bp in collection of illustrative plates in 1000 ~ 1500bp sections, for Dai-ju stock Pseudosciaena crocea；If figure
Only have the band of a 1085bp in spectrum in 1000 ~ 1500bp sections, be then Fujian-East Guangdong race Larimichthys crocea.
8. differentiate the purposes of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea, it is characterised in that for quickly and accurately differentiating Mount Tai
Thoroughfare race Larimichthys crocea and the purposes of Fujian-East Guangdong race Larimichthys crocea.
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| CN201711348834.1A CN108048576A (en) | 2017-12-15 | 2017-12-15 | Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof |
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| CN201711348834.1A CN108048576A (en) | 2017-12-15 | 2017-12-15 | Differentiate method of Dai-ju stock Pseudosciaena crocea and Fujian-East Guangdong race Larimichthys crocea and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897440B (en) * | 2021-10-29 | 2023-07-04 | 浙江省农业科学院 | Molecular marker for rapidly identifying sex of large yellow croaker in daiqu and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966723A (en) * | 2006-11-28 | 2007-05-23 | 厦门大学 | Determination primer and method for yellow croaker in different population |
| CN102776284A (en) * | 2012-07-12 | 2012-11-14 | 宁波大学 | Primer and method for identifying pseudosciaena crocea of daiqu species and min-yuedong species |
-
2017
- 2017-12-15 CN CN201711348834.1A patent/CN108048576A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966723A (en) * | 2006-11-28 | 2007-05-23 | 厦门大学 | Determination primer and method for yellow croaker in different population |
| CN102776284A (en) * | 2012-07-12 | 2012-11-14 | 宁波大学 | Primer and method for identifying pseudosciaena crocea of daiqu species and min-yuedong species |
Non-Patent Citations (1)
| Title |
|---|
| 沈锡权等: "岱衢族大黄鱼(Pseudosciaena crocea)线粒体基因组全序列的扩增及其特异鉴别引物的开发 ", 《海洋与湖沼》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113897440B (en) * | 2021-10-29 | 2023-07-04 | 浙江省农业科学院 | Molecular marker for rapidly identifying sex of large yellow croaker in daiqu and application |
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