CN108531620A - Identify the primer and method of Amur Sturgeon, siberia platform and its filial generation - Google Patents
Identify the primer and method of Amur Sturgeon, siberia platform and its filial generation Download PDFInfo
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Abstract
The invention discloses the primers and method of identification Amur Sturgeon, siberia platform and its filial generation, it is made of HLJS41 Amur Sturgeon primer pair and micro-satellite primers, wherein, the forward direction primer sequence of Amur Sturgeon primer pair is as shown in SEQ ID No.1, and backward primer sequence is as shown in SEQ ID No.2；Micro-satellite primers are to the forward direction primer sequence of HLJS41 as shown in SEQ ID No.3, and backward primer sequence is as shown in SEQ ID No.4.Primer using the present invention and method can quickly and easily can distinguish siberia platform, Amur Sturgeon and its filial generation.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to identify siberia platform, Amur Sturgeon and its filial generation
Primer and method.
Background technology
Amur Sturgeon and siberia platform are 2 important breed varieties in China.Under natural conditions, Amur Sturgeon is only distributed in black
Longjiang (Amur) water system, is endemic species and the Important Economic fish in Heilungkiang, growth speed, but disease-resistant force difference,
Intolerant to transport, and more difficult domestication is excessively relied on live bait, the death rate is higher in the breeding process, increases sturgeon cultivation industry
Risk.And siberia platform is distributed mainly on the Central Asia and Eastern Europe, in China Eerqisihe River, water system has a small amount of distribution, growth speed
Degree is slow, but premunition is strong, resistance to transport.
China bred the cross combination of siberia platform and Amur Sturgeon successfully before and after 2007, because of its positive and negative friendship
Effect difference is little, so being referred to as " western miscellaneous ".It is western it is miscellaneous have an apparent production advantage compared with parents, adaptability is relatively strong, growth speed
Degree is very fast, premunition is strong, transport survival rate is high.It is western it is miscellaneous breed successfully after, rapidly in China promote cultivation, at
For northeast, North China, East China, Central China main breed variety, be current Chinese commodity sturgeon cultivation scale maximum with yield
Kind.
Carry out part in terms of western miscellaneous and its parent paternity identification to study, is concentrated mainly on isodynamic enzyme biochemical marker
In research.Wu Yi etc. is using polyacrylamide gel vertical slab electrophoresis to Amur Sturgeon, siberia platform and its positive and negative cenospecies 4
3 kinds of esterase (EST), malic dehydrogenase (MDH) and the alcohol dehydrogenase (ADH) of 5 kinds of tissues (heart, liver, flesh, eye, kidney) of group
Isodynamic enzyme is analyzed, it is found that 3 kinds of isodynamic enzymes have apparent tissue specificity in respective group；In 4 different sturgeons
Also apparent rule sex differernce is shown between group, can be used as the biochemical genetic marker for distinguishing this 4 kinds of sturgeons.Yin Hongbin etc. is same
The enzyme band that 9 kinds of Their Isozymes Expression analyses find cenospecies is carried out using the group 5 of polyacrylamide gel electrophoresis pair 4 tissue
Expression is more complicated than parent, but mostly close with the expression of maternal enzyme band, and shows Amur Sturgeon, Amur Sturgeon (♀) × west by clustering
Berli Asia sturgeon (♂) cenospecies gathers for same branch, and siberia platform, siberia platform (♀) × Amur Sturgeon (♂) cenospecies, which gather, is
Another branch.It is identified using such method, although easy to operate, is easy to learning and mastering, sample preparation is complicated, sample
Between the band difference that generates it is smaller, be highly prone to the influence of the various conditions in sample collection method and experimentation, therefore close
Report less, the sturgeon individual health assessment for being chiefly used in detecting under Artificial feeding conditions and measurement year in sturgeon differentiates.In order to
Preferably western miscellaneous and its parent's siberia platform and Amur Sturgeon can be identified, therefore be badly in need of that exploitation is a kind of quickly, stablize, can
The method leaned on provides reliable basis for subsequent research.
In the present invention it is western it is miscellaneous refer to using siberia platform be maternal Amur Sturgeon as paternal hybrid kind；It refers to Amur to apply miscellaneous
Sturgeon is that maternal siberia platform is paternal hybrid kind.
Invention content
The technical problems to be solved by the invention are：How quickly, stablize, reliably to siberia platform, Amur Sturgeon and
Their cenospecies is identified.
The technical scheme is that：Identify Amur Sturgeon, siberia platform and its primer of filial generation, it is by Amur Sturgeon
Primer pair and micro-satellite primers constitute HLJS41, wherein the forward direction primer sequence of Amur Sturgeon primer pair such as SEQ ID No.1 institutes
Show (TGTGGGGTCACGGACTTTACAG), backward primer sequence is as shown in SEQ ID No.2
(TATACACCATTATCTCTATGT)；Micro-satellite primers are to the forward direction primer sequence of HLJS41 as shown in SEQ ID No.3
(GCGCCACACACTCAACTCT), backward primer sequence is as shown in SEQ ID No.4 (GCGTCCCAATAGACCACATT).
Identify Amur Sturgeon, siberia platform and its primer of filial generation after siberia platform, Amur Sturgeon and its hybridization
Purposes in generation identification.
A kind of Amur Sturgeon, siberia platform and its identification method of filial generation, include the following steps：
(1) using sturgeon DNA to be identified as masterplate, PCR amplification is carried out as primer to HLJS41 using micro-satellite primers, if
There is the amplified production of 255bp without there is the amplified production of 202bp, is then Amur Sturgeon；If there is the amplified production of 202bp
Then it is siberia platform without there is the amplified production of 255bp；If occurring the amplified production of 255bp and 202bp simultaneously,
It is western miscellaneous or apply miscellaneous；
(2) to being accredited as western miscellaneous or applying miscellaneous sturgeon DNA and identify：Using sturgeon DNA to be identified as masterplate, with Amur
Sturgeon primer pair is that primer carries out PCR amplification, if there is the amplified production of 252bp, then miscellaneous to apply；Otherwise it is western miscellaneous.
The forward direction primer sequence of Amur Sturgeon primer pair is as shown in SEQ ID No.1, backward primer sequence such as SEQ ID No.2
It is shown；Micro-satellite primers to the forward direction primer sequence of HLJS41 as shown in SEQ ID No.3, backward primer sequence such as SEQ ID
Shown in No.4.
Compared with prior art, the invention has the advantages that：
Primer using the present invention and method, can quickly and easily can will be after siberia platform, Amur Sturgeon and its hybridization
In generation, distinguishes.
Description of the drawings
The polyacrylamide gel electrophoresis collection of illustrative plates of Fig. 1 Amur Sturgeon primer pair amplifies products；
Polyacrylamide gel electrophoresis collection of illustrative plates of Fig. 2 micro-satellite primers to HLJS41 amplified productions.
Specific implementation mode
1.1. material
1.1 experiment material：
Parental animal：34 parts of siberia platform, 10 parts of Amur Sturgeon (take part fin ray to be preserved with absolute ethyl alcohol)
Filial generation sample：Western miscellaneous 12 tail applies miscellaneous 12 tail (rounding body is preserved with absolute ethyl alcohol)
1.2 experiment equipment：
PCR instrument：Bio-Rad iCycler and MJ100；
Centrifuge：Eppendorf Centrifuge5415D；
Voltage stabilization and current stabilization electrophoresis apparatus：Bio-Rad powerPAC300；
Gel imaging system：Alphalmage Multimage Light Cabinet；
Electronic balance, constant temperature water tank, incubator, steam sterilizer, shaking table, centrifuge and some common experimental equipment.
1.3 primary drugs and reagent：
Distilled water (MilliQ), two water edetate disodium (EDTA-Na.2H2O), Proteinase K (Merck companies)
(10mg/mL), Tris alkali, concentrated hydrochloric acid, NaOH, MgCl2, Taq enzyme buffer solution, dNTPs (10mM) etc..
2. method
The extraction of 2.1 total genomic dnas
1. cutting the not more than organization material of 30mg, it is put into the centrifuge tube equipped with 200 μ lGA buffer solutions that (centrifuge tube is carried out
Label), vortex oscillation 15sec.
2. 20 μ l Proteinase K (20mg/ml) solution are added, vortex mixing, brief centrifugation is to remove cap wall
Droplet, at 56 DEG C place (digestion), until organize be completely dissolved, brief centrifugation is to remove the droplet of cap wall, then carries out
Next step.
3. 200 μ l buffer solution GB are added, fully reverse mixing, 70 DEG C of placement 10min, limpid, the brief centrifugation of solution strain
To remove the droplet of cap wall.(pay attention to：It should be put into 4 DEG C of refrigerator when waiting for)
4. 200 μ l absolute ethyl alcohols are added, fully reverse mixing, at this time it is possible that flocculent deposit, brief centrifugation is to go
Except the droplet of cap wall.
5. previous step acquired solution and flocculent deposit are all added in an adsorption column CB3 (adsorption column is put into collecting pipe,
And mark), 12000rpm (~13,400 × g) centrifuges 30sec, outwells waste liquid, adsorption column CB3 is put back in collecting pipe.
6. 500 μ l buffer solutions GD (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column CB3,
12000rpm (~13,400 × g) centrifuges 30sec, outwells waste liquid, adsorption column CB3 is put into collecting pipe.
7. 600 μ l buffer solutions PW (please first checked whether before use and absolute ethyl alcohol has been added) are added into adsorption column CB3,
12000rpm (~13,400 × g) centrifuges 30sec, outwells waste liquid, adsorption column CB3 is put into collecting pipe.
8. repeating step 7.
9. adsorption column CB3 is put back in collecting pipe, 12000rpm (~13,400 × g) centrifuges 30sec, outwells waste liquid, will
Adsorption column CB3, which is placed in, is placed at room temperature for several minutes (general 10min), thoroughly to dry rinsing liquid remaining in sorbing material.(note
Meaning：The purpose of this step is to remove rinsing liquid remaining in adsorption column, and the residual of ethyl alcohol can influence subsequent enzyme in rinsing liquid
React (digestion, PCR etc.) experiment)
10. adsorption column CB3 is transferred in a clean centrifuge tube, 50~200 are vacantly added dropwise to the intermediate position of adsorbed film
μ l elution buffers TE (this experiment ddH2O), it is placed at room temperature for 2~5min, 12000rpm (~13,400 × g) centrifuges 2min,
Solution is collected into centrifuge tube.
2.2 agarose gel electrophoresis detect DNA
1. preparing 1% Ago-Gel:It weighs in the balance and takes 0.4~0.6g agaroses, 1 times of TAE for pouring into 45mL or so is slow
Fliud flushing shakes up and is placed on micro-wave oven high temperature and is heated to being completely dissolved, and takes out after slightly cool, EB 2uL are added.
2. prepared by offset plate：Plastic plate is positioned over horizontal position, is inserted into sample comb.By agar made from previous step
Sugared coagulant liquid pours into glue board slot.Cooling half an hour gently extracts comb after gel fixation, and gel is put into electrophoresis tank.
TAE liquid, which is added, makes it submerge gel.
3. being loaded：The spotting buffer (bromophenol blue) of upper 1/6 volume is put on point template, after with rifle suction 2uL DNA solutions
Spotting buffer and mixing, record point sample sequence and point sample amount is added.
4. running electrophoresis：100V voltages, 400mA electric currents, 15~20min of electrophoresis.According on ultraviolet transmission detector after electrophoresis
The brightness of electrophoresis determines the concentration of template DNA, if concentration is high, can add appropriate MilliQ (ddH2O it) dilutes, it can if concentration is low
Increase addition in right amount in PCR amplification.DNA after detection is placed in -20 DEG C of refrigerators and saves backup.
2.3 design of primers
According to sturgeon mtDNA sequence and microsatellite sequence, two pairs of primers are devised, Amur Sturgeon primer pair is respectively designated as
With micro-satellite primers to HLJS41, primer is synthesized by Chengdu Qing Ke Zi Xi Bioisystech Co., Ltd.
The forward direction primer sequence of Amur Sturgeon primer pair is TGTGGGGTCACGGACTTTACAG (shown in SEQ ID No.1)
The backward primer sequence of Amur Sturgeon primer pair is TGTGGGGTCACGGACTTTACAG (shown in SEQ ID No.2)
Micro-satellite primers are GCGCCACACACTCAACTCT (SEQ ID No.3 institutes to the forward direction primer sequence of HLJS41
Show)
Micro-satellite primers are GCGTCCCAATAGACCACATT (SEQ ID No.4 institutes to the backward primer sequence of HLJS41
Show)
2.4PCR amplification systems and condition
Amur Sturgeon primer pair PCR amplification：PCR reaction systems add up to 25 μ L (1 μ L of template, 12.5 Mix μ L, ddH2O10.5 μ
L, 0.5 μ L of sense primer, 0.5 μ L of downstream primer).PCR reaction conditions are as follows：95 DEG C of pre-degeneration 5min；Then cycle 35 times, often
One cycle includes 94 DEG C of denaturation 40s, 55 DEG C of annealing 35s, 72 DEG C of extension 50s；Extend 10min at last 72 DEG C.Use 120v
After 1.5% agarose is to PCR product gel electrophoresis, the segment after being expanded with UV detection.
Micro-satellite primers expand HLJS41PCR：PCR reaction systems add up to 25 μ L (1 μ L of template, 12.5 Mix μ L,
DdH2O10.5 μ L, 0.5 μ L of sense primer, 0.5 μ L of downstream primer).PCR reaction conditions are as follows：95 DEG C of pre-degeneration 5min；Then
Cycle 35 times, each cycle include 94 DEG C of denaturation 40s, 55 DEG C of annealing 35s, 72 DEG C of extension 50s；Extend at last 72 DEG C
10min.PCR reaction products use 10% non-denaturing polyacrylamide gel (Polyacrylamide Gel, abbreviation PAAG) electricity
Swimming detection, dye-develop using improvement argentation after electrophoresis-scanning imagery after preserve data result.
3. result
Amur Sturgeon primer pair is being maternal filial generation DNA using Amur Sturgeon and using Amur Sturgeon as in the PCR amplification of masterplate
There are length 252bp specific bands, and using the filial generation DNA that siberia platform is female parent as in the PCR amplification of masterplate
There is not specific band (Fig. 1).
There is the band of 255bp to HLJS41 in using Amur Sturgeon DNA as the PCR amplification of masterplate in micro-satellite primers, with
Siberia platform DNA be masterplate PCR amplification in 202bp band, and using its hybridize germplasm DNA as the PCR amplification of masterplate
The middle heterozygosis band (Fig. 2) for 255bp and 202bp occur.
4 interpretations of result
According to the above results as it can be seen that using sturgeon DNA to be identified as masterplate, using micro-satellite primers to HLJS41 as primer into
Row PCR amplification, if there is 255bp amplified production without there is the amplified production of 202bp, then be Amur Sturgeon；If there is
The amplified production of 202bp is then siberia platform without there is the amplified production of 255bp；If occur simultaneously 255bp and
The amplified production of 202bp is then western miscellaneous or apply miscellaneous；To being accredited as western miscellaneous or applying miscellaneous sturgeon DNA and further identified：With
Sturgeon DNA to be identified is masterplate, and PCR amplification is carried out by primer of Amur Sturgeon primer pair, and the amplification if there is 252bp is produced
Object, then it is miscellaneous to apply；Otherwise it is western miscellaneous.
Embodiment
Using sturgeon DNA to be identified as masterplate (extracting method uses the extracting method of 2 total genomic dna of front), with micro-
Satellite primers to HLJS41 be primer carry out PCR amplification, PCR reaction systems add up to 25 μ L (1 μ L of template, Mix12.5 μ L,
DdH2O10.5 μ L, 0.5 μ L of sense primer, 0.5 μ L of downstream primer).PCR reaction conditions are as follows：95 DEG C of pre-degeneration 5min；Then
Cycle 35 times, each cycle include 94 DEG C of denaturation 40s, 55 DEG C of annealing 35s, 72 DEG C of extension 50s；Extend at last 72 DEG C
10min.PCR reaction products use 10% non-denaturing polyacrylamide gel (Polyacrylamide Gel, abbreviation PAAG) electricity
Swimming detection, dye-develop using improvement argentation after electrophoresis-scanning imagery after preserve data result.If there is
The amplified production of 255bp is then Amur Sturgeon without there is the amplified production of 202bp；If there is 202bp amplified production without
The amplified production for 255bp occur, then be siberia platform；If occurring the amplified production of 255bp and 202bp simultaneously, for west
It is miscellaneous or apply miscellaneous；
To being accredited as western miscellaneous or applying miscellaneous sturgeon DNA and further identified：Using sturgeon DNA to be identified as masterplate,
Using Amur Sturgeon primer pair as primer carry out PCR amplification, PCR reaction systems add up to 25 μ L (1 μ L of template, 12.5 Mix μ L,
DdH2O10.5 μ L, 0.5 μ L of sense primer, 0.5 μ L of downstream primer).PCR reaction conditions are as follows：95 DEG C of pre-degeneration 5min；Then
Cycle 35 times, each cycle include 94 DEG C of denaturation 40s, 55 DEG C of annealing 35s, 72 DEG C of extension 50s；Extend at last 72 DEG C
10min.With the agarose of 120v 1.5% to PCR product gel electrophoresis after, the segment after being expanded with UV detection.If gone out
The amplified production of existing 252bp, then it is miscellaneous to apply；Otherwise it is western miscellaneous.
The specific implementation mode of the application above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the application protection domain therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, under the premise of not departing from technical scheme design, various modifications and improvements can be made, these belong to this
The protection domain of application.
Claims (3)
1. identify Amur Sturgeon, siberia platform and its filial generation primer, which is characterized in that it is by Amur Sturgeon primer pair and micro-
Satellite primers constitute HLJS41, wherein the forward direction primer sequence of Amur Sturgeon primer pair draws backward as shown in SEQ ID No.1
Object sequence is as shown in SEQ ID No.2；Micro-satellite primers to the forward direction primer sequence of HLJS41 as shown in SEQ ID No.3, after
To primer sequence as shown in SEQ ID No.4.
2. identification Amur Sturgeon according to claim 1, siberia platform and its filial generation primer siberia platform,
Purposes on Amur Sturgeon and its first familiar generation identification.
3. a kind of Amur Sturgeon, siberia platform and its identification method of filial generation, which is characterized in that include the following steps：
(1) using sturgeon DNA to be identified as masterplate, PCR amplification is carried out as primer to HLJS41 using micro-satellite primers, if there is
The amplified production of 255bp is then Amur Sturgeon without there is the amplified production of 202bp；If there is 202bp amplified production without
The amplified production for 255bp occur, then be siberia platform；If occurring the amplified production of 255bp and 202bp simultaneously, for west
It is miscellaneous or apply miscellaneous；
(2) to being accredited as western miscellaneous or applying miscellaneous sturgeon DNA and identify：Using sturgeon DNA to be identified as masterplate, drawn with Amur Sturgeon
Object is if there is the amplified production of 252bp, then miscellaneous to apply to carrying out PCR amplification for primer；Otherwise it is western miscellaneous；
Wherein, the forward direction primer sequence of Amur Sturgeon primer pair is as shown in SEQ ID No.1, backward primer sequence such as SEQ ID
Shown in No.2；Micro-satellite primers to the forward direction primer sequence of HLJS41 as shown in SEQ ID No.3, backward primer sequence such as SEQ
Shown in ID No.4.
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