CN109182540A - Screen the Strongylocentrotus intermedius primer and method with high-temperature stability - Google Patents
Screen the Strongylocentrotus intermedius primer and method with high-temperature stability Download PDFInfo
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- CN109182540A CN109182540A CN201811177791.XA CN201811177791A CN109182540A CN 109182540 A CN109182540 A CN 109182540A CN 201811177791 A CN201811177791 A CN 201811177791A CN 109182540 A CN109182540 A CN 109182540A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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Abstract
The invention discloses the Strongylocentrotus intermedius primers that a kind of screening has high-temperature stability, it is made of 1 ~ No. 6 primer pair, respectively as shown in SEQ ID NO:1 ~ SEQ ID NO:12, screening technique successively carries out the DNA sequence dna of upstream primer F1 ~ F6 and downstream primer R1 ~ R6 in accordance with the following steps: extracting Strongylocentrotus intermedius RNA；Strongylocentrotus intermedius RNA reverse transcription is synthesized into cDNA；Using obtained cDNA as template, quantitative PCR reaction is carried out with 1 ~ No. 6 primer pair respectively；It selects the amplified production of quantitative PCR reaction while meeting the Strongylocentrotus intermedius of following Relative Expression values for the Strongylocentrotus intermedius with high-temperature stability: No. 1 ~ No. 6 primer pair amplifies product Relative Expression values 7.57 ~ 8.34,8.41 ~ 10.04,28.50 ~ 31.82,5.13 ~ 6.00,0.20 ~ 0.23 and 0.32 ~ 0.38.
Description
Technical field
The invention belongs to biotechnology more particularly to it is a kind of screening have high-temperature stability Strongylocentrotus intermedius primer and
Method.
Background technique
Strongylocentrotus intermedius (Strongylocentrotus intermedius) originate in Hokkaido, Japan and Russia is remote
The ground such as east are coastal, introduce China from Japan within 1989, form certain artificial breeding scale on Dalian, Shandong and other places, be
The coastal most important economic sea urchin type of northern China, the domestic market demand amount and export volume cumulative year after year of sexual gland.Largely
Studies have shown that ocean temperature is to influence the principal element of Strongylocentrotus intermedius growth and gonad development, Strongylocentrotus intermedius is at 15 ~ 18 DEG C
Left and right is ingested the most active, when water temperature is more than 23 DEG C, then can cause death.In recent years, the northern Yellow Sea in Summer ocean temperature of China
Often occur resulting in the massive mortality of cultivation Strongylocentrotus intermedius in 25 DEG C or more of situation for a long time, seriously affect
Between ball sea urchin industry sound development.But there is no the intermediates about Vivo Studies on Screening with high-temperature stability so far
The relevant report of sea urchin.
Summary of the invention
The present invention is to solve above-mentioned technical problem present in the prior art, and providing a kind of screen has high temperature resistance super
The Strongylocentrotus intermedius primer and method of property.
The technical solution of the invention is as follows: a kind of screening has the Strongylocentrotus intermedius primer of high-temperature stability, special
Sign is to be made of 1 ~ No. 6 primer pair, the DNA sequence dna of 1 ~ No. 6 primer pair upstream primer F1 ~ F6 and downstream primer R1 ~ R6
It is as follows respectively:
No. 1 F1:5'- cagaagaccctgtggtgaagttg-3'；
R1:5'- aagaggtagctgatgaggattgg-3'；
No. 2 F2:5'- gatggcagcgtctatctttcagt-3'；
R2:5'- ccaaccagtatttccagaacacg-3'；
No. 3 F3:5'- tggtcttgcccctccctcacttg-3'；
R3:5'- tccctaagattacttccttcggt-3'；
No. 4 F4:5'- ttctaaaacgcatcggatacgct-3'；
R4:5'- agtccagtcccacatcattcaag-3'；
No. 5 F5:5'- tgtttacatcagcatctcagcgtc-3'；
R5:5'- cccttctttcccacatccttcaa-3'；
No. 6 F6:5'- atgaagatgctgatgttgtttgc-3'；
R6:5'- aggacctccagtgttgagtgttt-3'.
A method of with above-mentioned primer screening have high-temperature stability Strongylocentrotus intermedius, successively in accordance with the following steps into
Row:
A. Strongylocentrotus intermedius RNA is extracted；
B. Strongylocentrotus intermedius RNA reverse transcription is synthesized into cDNA；
C. using obtained cDNA as template, quantitative PCR reaction is carried out with 1 ~ No. 6 primer pair respectively；
D. it selects the amplified production of quantitative PCR reaction while meeting the Strongylocentrotus intermedius of following Relative Expression values for high temperature resistant
The Strongylocentrotus intermedius of characteristic:
No. 1 primer pair amplifies product Relative Expression values 7.57 ~ 8.34；
No. 2 primer pair amplifies product Relative Expression values 8.41 ~ 10.04；
No. 3 primer pair amplifies product Relative Expression values 28.50 ~ 31.82；
No. 4 primer pair amplifies product Relative Expression values 5.13 ~ 6.00；
No. 5 primer pair amplifies product Relative Expression values 0.20 ~ 0.23；
No. 6 primer pair amplifies product Relative Expression values 0.32 ~ 0.38.
The present invention solves the problems, such as the Strongylocentrotus intermedius that Vivo Studies on Screening has high-temperature stability, and that is screened has anti-height
The Strongylocentrotus intermedius of temperature characteristics can normally survive in 25 DEG C of water temperatures, and screening operation is easy, low in cost, reproducible, quasi-
True property is higher, realizes the marker assisted selection of high temperature resistant Strongylocentrotus intermedius breeding, further improve breeding accuracy and
Breeding Efficiency.
Detailed description of the invention
Fig. 1 is the embodiment of the present invention and control group amplified production relative expression quantity schematic diagram.
Specific embodiment
The method that screening of the invention has the Strongylocentrotus intermedius of high-temperature stability, successively carries out in accordance with the following steps:
A. Strongylocentrotus intermedius RNA is extracted:
Select 100 Strongylocentrotus intermedius at random, respectively extract sea urchin coelomic fluid, be put into the centrifuge tube of 1.5mL, then with
4000r/min is centrifuged 10 minutes, is discarded supernatant, is repeated 3 times；Lysate is added, tissue is thoroughly ground with grinding pestle, is utilized
RNA kit extracts RNA.The RNA kit is purchased from TIANGEN company, model total RNA from animal tissues extracts kit
(centrifugal column type) 50preps.
B. Strongylocentrotus intermedius RNA reverse transcription is synthesized into cDNA；
Reverse transcription is carried out using reverse transcription reagent box to extracted Strongylocentrotus intermedius RNA, synthesizes cDNA.Used kit buying
From TaKaRa company, 200 Rxns of model PrimeScript RT Reagent Kit, reaction system is 10 μ L.
C. using obtained cDNA as template, quantitative fluorescent PCR reaction is carried out with 1 ~ No. 6 primer pair respectively, i.e., with each
Primer pair individually carries out first order fluorescence quantitative PCR reaction；1 ~ No. 6 primer pair upstream primer F1 ~ F6 and downstream primer R1 ~
The DNA sequence dna difference of R6 is as follows:
No. 1 F1:5'- cagaagaccctgtggtgaagttg-3'；
R1:5'- aagaggtagctgatgaggattgg-3'；
No. 2 F2:5'- gatggcagcgtctatctttcagt-3'；
R2:5'- ccaaccagtatttccagaacacg-3'；
No. 3 F3:5'- tggtcttgcccctccctcacttg-3'；
R3:5'- tccctaagattacttccttcggt-3'；
No. 4 F4:5'- ttctaaaacgcatcggatacgct-3'；
R4:5'- agtccagtcccacatcattcaag-3'；
No. 5 F5:5'- tgtttacatcagcatctcagcgtc-3'；
R5:5'- cccttctttcccacatccttcaa-3'；
No. 6 F6:5'- atgaagatgctgatgttgtttgc-3'；
R6:5'- aggacctccagtgttgagtgttt-3'.
Each quantitative fluorescent PCR response procedures are as follows:
Note: being referring to setting with 7500 quantitative fluorescent PCR instrument of ABI.
Quantitative fluorescent PCR reaction system is 20 μ L (18 μ L of primer, 2 μ L of cDNA template), and the amount of DNA template usually exists
100 ng or less, it may be necessary to dilute.
D. the relative expression quantity for calculating each fluorescent quantitative PCR product selects amplified production while meeting following phase
Strongylocentrotus intermedius to expression value is the Strongylocentrotus intermedius with high-temperature stability:
No. 1 primer pair amplifies product Relative Expression values range 7.57 ~ 8.34；
No. 2 primer pair amplifies product Relative Expression values ranges 8.41 ~ 10.04；
No. 3 primer pair amplifies product Relative Expression values ranges 28.50 ~ 31.82；
No. 4 primer pair amplifies product Relative Expression values ranges 5.13 ~ 6.00；
No. 5 primer pair amplifies product Relative Expression values ranges 0.20 ~ 0.23；
No. 6 primer pair amplifies product Relative Expression values ranges 0.32 ~ 0.38.
Finally select 58 Strongylocentrotus intermedius with high-temperature stability.
Comparative experiments:
The embodiment of the present invention is to be comparison for internal reference (β-actin) primer, using above-mentioned obtained cDNA as template and with same
Reaction system and response procedures carry out quantitative fluorescent PCR reaction, and amplified production relative expression quantity comparing result is as shown in Figure 1.
High temperature resistant experiment:
By 42 surplus intermediate seas of 58 selected by the embodiment of the present invention Strongylocentrotus intermedius and choosing with high-temperature stability
Gallbladder is respectively placed in two cylinder moulds and cultivates 1 month in same aquaculture pond.During cultivation in addition to temperature of cultivation is 25 DEG C, remaining
Cultivating condition is compared with technology.After cultivation 1 month, the selected 58 Strongylocentrotus intermedius survival rates with high-temperature stability
It is 100%, and other 42 Strongylocentrotus intermedius are then all dead.
Sequence table
<110>Dalian Ocean University
<120>screening has the Strongylocentrotus intermedius primer and method of high-temperature stability
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 1
cagaagaccc tgtggtgaag ttg 23
<210> 2
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 2
aagaggtagc tgatgaggat tgg 23
<210> 3
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 3
gatggcagcg tctatctttc agt 23
<210> 4
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 4
ccaaccagta tttccagaac acg 23
<210> 5
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 5
tggtcttgcc cctccctcac ttg 23
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 6
tccctaagat tacttccttc ggt 23
<210> 7
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 7
ttctaaaacg catcggatac gct 23
<210> 8
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 8
agtccagtcc cacatcattc aag 23
<210> 9
<211> 24
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(24)
<223>primer
<400> 9
tgtttacatc agcatctcag cgtc 24
<210> 10
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 10
cccttctttc ccacatcctt caa 23
<210> 11
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 11
atgaagatgc tgatgttgtt tgc 23
<210> 12
<211> 23
<212> DNA
<213>artificial sequence (Strongylocentrotus intermedius)
<220>
<221> misc_feature
<222> (1)..(23)
<223>primer
<400> 12
aggacctcca gtgttgagtg ttt 23
Claims (2)
1. the Strongylocentrotus intermedius primer that a kind of screening has high-temperature stability, it is characterised in that be made of 1 ~ No. 6 primer pair, institute
The DNA sequence dna difference for stating 1 ~ No. 6 primer pair upstream primer F1 ~ F6 and downstream primer R1 ~ R6 is as follows:
No. 1 F1:5'- cagaagaccctgtggtgaagttg-3'；
R1:5'- aagaggtagctgatgaggattgg-3'；
No. 2 F2:5'- gatggcagcgtctatctttcagt-3'；
R2:5'- ccaaccagtatttccagaacacg-3'；
No. 3 F3:5'- tggtcttgcccctccctcacttg-3'；
R3:5'- tccctaagattacttccttcggt-3'；
No. 4 F4:5'- ttctaaaacgcatcggatacgct-3'；
R4:5'- agtccagtcccacatcattcaag-3'；
No. 5 F5:5'- tgtttacatcagcatctcagcgtc-3'；
R5:5'- cccttctttcccacatccttcaa-3'；
No. 6 F6:5'- atgaagatgctgatgttgtttgc-3'；
R6:5'- aggacctccagtgttgagtgttt-3'.
2. a kind of method that primer screening described in claim has the Strongylocentrotus intermedius of high-temperature stability, it is characterised in that according to
It is secondary to carry out in accordance with the following steps:
A. Strongylocentrotus intermedius RNA is extracted；
B. Strongylocentrotus intermedius RNA reverse transcription is synthesized into cDNA；
C. using obtained cDNA as template, quantitative PCR reaction is carried out with 1 ~ No. 6 primer pair respectively；
D. it selects the amplified production of quantitative PCR reaction while meeting the Strongylocentrotus intermedius of following Relative Expression values for high temperature resistant
The Strongylocentrotus intermedius of characteristic:
No. 1 primer pair amplifies product Relative Expression values 7.57 ~ 8.34；
No. 2 primer pair amplifies product Relative Expression values 8.41 ~ 10.04；
No. 3 primer pair amplifies product Relative Expression values 28.50 ~ 31.82；
No. 4 primer pair amplifies product Relative Expression values 5.13 ~ 6.00；
No. 5 primer pair amplifies product Relative Expression values 0.20 ~ 0.23；
No. 6 primer pair amplifies product Relative Expression values 0.32 ~ 0.38.
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| CN201811177791.XA CN109182540B (en) | 2018-10-10 | 2018-10-10 | Primer and method for screening high-temperature-resistant strongylocentrotus intermedius |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112997933A (en) * | 2021-02-18 | 2021-06-22 | 大连海洋大学 | Method for cultivating sea urchin seedlings with high temperature resistance |
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| CN112997933A (en) * | 2021-02-18 | 2021-06-22 | 大连海洋大学 | Method for cultivating sea urchin seedlings with high temperature resistance |
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