CN108660224A - Identify the primer and method of siberia platform, Amur Sturgeon and its filial generation - Google Patents
Identify the primer and method of siberia platform, Amur Sturgeon and its filial generation Download PDFInfo
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Abstract
The invention discloses the primers and method of a kind of identification siberia platform, Amur Sturgeon and its filial generation, it is made of As1 siberia platform primer pair and micro-satellite primers, wherein, the forward direction primer sequence of siberia platform 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 As1 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 sturgeonCenospecies gathers for same branch, siberia platform, siberia platform (♀) × Amur SturgeonCenospecies is poly-
For another branch.It is identified using such method, although easy to operate, is easy to learning and mastering, sample preparation is complicated, sample
The band difference of this generation is smaller, is highly prone to the influence of the various conditions in sample collection method and experimentation, therefore
In recent years less, the sturgeon individual health assessment for being chiefly used in detecting under Artificial feeding conditions and measurement are reported in sturgeon differentiates.For
Preferably western miscellaneous and its parent's siberia platform and Amur Sturgeon can be identified, thus be badly in need of exploitation is a kind of quickly, stablize,
Reliable method 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 offspring；It refers to apply to apply miscellaneous
Family name sturgeon is that maternal siberia platform is paternal hybrid offspring.
Invention content
The technical problems to be solved by the invention are：How quickly, stablize, reliably to siberia platform, Amur Sturgeon and
Its filial generation is identified.
The technical scheme is that：Identify siberia platform, Amur Sturgeon and its primer of filial generation, it is by western Berli
Sub- sturgeon primer pair and micro-satellite primers constitute As1, wherein the forward direction primer sequence of siberia platform primer pair such as SEQ ID
(CAGATGCCAGTAACAGGCTGA) shown in No.1, backward primer sequence is as shown in SEQ ID No.2
(TATACACCATTATCTCTATGT)；Micro-satellite primers are to the forward direction primer sequence of AS1 as shown in SEQ ID No.3
(AACAAGCGACGAACAGTGTG), backward primer sequence is as shown in SEQ ID No.4 (GAAAGGACACCAGCAGTG).
Above-mentioned identification siberia platform, Amur Sturgeon and its primer of filial generation identification siberia platform, Amur Sturgeon and
Purposes on its filial generation.
The present invention also provides a kind of identification siberia platform, Amur Sturgeon and its methods of filial generation, including walk as follows
Suddenly：
(1) using sturgeon DNA to be identified as masterplate, PCR amplification is carried out as primer to As1 using micro-satellite primers, if gone out
The amplified production of existing 200bp is then Amur Sturgeon without there is the amplified production of 105bp；If there is 105bp amplified production and
Do not occur the amplified production of 200bp, is then siberia platform；If occurring the amplified production of 105bp and 200bp simultaneously, for
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, Yi Xibai
It is then western miscellaneous that Leah sturgeon primer pair carries out PCR amplification if there is the amplified production of 215bp for primer；Otherwise miscellaneous to apply；
The forward direction primer sequence of siberia platform 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 As1 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 siberia platform primer pair amplifies products；
Polyacrylamide gel electrophoresis collection of illustrative plates of Fig. 2 micro-satellite primers to As1 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, siberia platform is respectively designated as and draws
To HLJS41, primer is synthesized by Chengdu Qing Ke Zi Xi Bioisystech Co., Ltd for object pair and micro-satellite primers.
The forward direction primer sequence of siberia platform primer pair is CAGATGCCAGTAACAGGCTGA (SEQ ID No.1 institutes
Show)
The backward primer sequence of siberia platform primer pair is TATACACCATTATCTCTATGT (SEQ ID No.2 institutes
Show)
Micro-satellite primers are AACAAGCGACGAACAGTGTG to the forward direction primer sequence of As1 (shown in SEQ ID No.3)
Micro-satellite primers are GAAAGGACACCAGCAGTG to the backward primer sequence of As1 (shown in SEQ ID No.4)
2.4PCR amplification systems and condition
Siberia platform 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, 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.
Micro-satellite primers expand As1PCR：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
3.1 mitochondrias are identified
Siberia platform primer pair is being maternal filial generation DNA using siberia platform and using siberia platform as template
PCR amplification in there are length 215bp specific bands, and be maternal filial generation DNA with Amur Sturgeon and with Amur Sturgeon
Not occur characteristic band (Fig. 1) in the PCR amplification of template.
There is the band of 200bp to As1 in using Amur Sturgeon DNA as the PCR amplification of template in micro-satellite primers, primary to the west of
Leah sturgeon DNA is hybridizing germplasm DNA as the PCR amplification of template to occur the band of 105bp in the PCR amplification of template using it
The middle heterozygosis band for 105bp and 200bp occur, the specific band amplified can effectively differentiate 3 germplasm (Fig. 2)；
4 interpretations of result
According to the above results as it can be seen that using sturgeon DNA to be identified as masterplate, As1 is carried out as primer using micro-satellite primers
PCR amplification, if there is 200bp amplified production without there is the amplified production of 105bp, then be Amur Sturgeon；If there is
The amplified production of 105bp is then siberia platform without there is the amplified production of 200bp；If occur simultaneously 105bp and
The amplified production of 200bp is then western 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, with
It is then western miscellaneous that siberia platform primer pair carries out PCR amplification if there is the amplified production of 215bp for primer；Otherwise it is to apply
It is miscellaneous.
Embodiment
(1) 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 As1 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 200bp is then Amur Sturgeon without there is the amplified production of 105bp；If there is 105bp amplified production without
The amplified production for 200bp occur, then be siberia platform；If occurring the amplified production of 105bp and 200bp simultaneously, for west
It is miscellaneous or apply miscellaneous；
(2) to being accredited as western miscellaneous or applying miscellaneous sturgeon DNA and further identified：Using sturgeon DNA to be identified as masterplate,
Using siberia platform primer pair as 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.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 215bp, then be western miscellaneous；Otherwise miscellaneous to apply.
Claims (3)
1. identifying siberia platform, Amur Sturgeon and its primer of filial generation, which is characterized in that it is by siberia platform primer pair
With micro-satellite primers to As1 constitute, wherein the forward direction primer sequence of siberia platform primer pair as shown in SEQ ID No.1, after
To primer sequence as shown in SEQ ID No.2；Micro-satellite primers to the forward direction primer sequence of As1 as shown in SEQ ID No.3, after
To primer sequence as shown in SEQ ID No.4.
2. identification siberia platform according to claim 1, Amur Sturgeon and its primer of filial generation are identifying western Berli
Purposes on sub- sturgeon, Amur Sturgeon and its filial generation.
3. a kind of identification siberia platform, Amur Sturgeon and its 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 As1 using micro-satellite primers, if there is
The amplified production of 200bp is then Amur Sturgeon without there is the amplified production of 105bp；If there is 105bp amplified production without
The amplified production for 200bp occur, then be siberia platform；If occurring the amplified production of 105bp and 200bp 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, with Siberia
It is then western miscellaneous that sturgeon primer pair carries out PCR amplification if there is the amplified production of 215bp for primer；Otherwise miscellaneous to apply；Western Berli
The forward direction primer sequence of sub- sturgeon primer pair is as shown in SEQ ID No.1, and backward primer sequence is as shown in SEQ ID No.2；It is micro- to defend
The forward direction primer sequence of star primer pair As1 is as shown in SEQ ID No.3, and backward primer sequence is as shown in SEQ ID No.4.
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