CN108411002A - PCR primer and discrimination method for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies - Google Patents
PCR primer and discrimination method for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies Download PDFInfo
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Abstract
The present invention discloses a kind of PCR primer for differentiating Anthocidaris crassispina Strongylocentrotus intermedius cenospecies, and nucleotide sequence is as follows：Sense primer：5' ttttatctcctccctttttatytct 3'；Downstream primer：5' cctcwaaagtagttaagattgggac 3'；Discrimination method carries out in accordance with the following steps successively：Extract the sea urchin individual DNA to be identified with Anthocidaris crassispina shape；It is template that the DNA of extraction, which is diluted to 10 ng/ μ l, carries out PCR amplification with sense primer and downstream primer, the PCR reaction conditions are：94℃ 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 35 cycles；72℃ 5min；Electrophoresis is carried out to pcr amplification product, shaping band is then Anthocidaris crassispina Strongylocentrotus intermedius cenospecies.
Description
Technical field
The present invention relates to field of molecular marker more particularly to a kind of for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies
PCR primer and discrimination method.
Background technology
Strongylocentrotus intermedius（Strongylocentrotus intermedius）Also known as Strongylocentrotus intermedius originates in Japan
The north and Russian Far East part coastal area.Compared with other edible sea urchin types, Strongylocentrotus intermedius has sexual gland color and luster
Well, the features such as unsaturated fatty acid content is high, mouthfeel is sweet, deep to be liked by south east asia consumer, market demand is year by year
Increase, forms cultivation scale on Liaoning, Shandong and other places at present.The most suitable growth water temperature of studies have shown that Strongylocentrotus intermedius is 15
DEG C ~ 20 DEG C, when water temperature is increased to 23 DEG C, the mortality of intermediate sea urchin can be caused.Anthocidaris crassispina（Anthocidaris crassispina）It is NATURAL DISTRIBUTION in the sea urchin type in south China marine site, there is higher tolerance to hot environment.It is real
Verification is real, is Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies that parents generate, phase with Anthocidaris crassispina (♀) and Strongylocentrotus intermedius (♂)
Have better than maternal sexual gland quality compared with Anthocidaris crassispina, there are apparent high temperature resistant potentiality again compared to Strongylocentrotus intermedius, be one
Kind of fast growing, heat-resisting quantity be good, sexual gland quality brilliance sea urchin new varieties.However, due to Anthocidaris crassispina-Strongylocentrotus intermedius hybridization
Kind with Anthocidaris crassispina there is similar appearance to be easy occurring confounding issues in cultivation it is difficult to the naked eye distinguish.Therefore, effectively
Differentiate that purple-middle hybridization sea urchin and Anthocidaris crassispina are sea urchin cultivation and major issue urgently to be resolved hurrily in market management.
Currently, there are mainly two types of the methods of identification sea urchin type：One is the formalness feature according to sea urchin, such as shells
Band and interambulacrum breadth ratio and radian between shape, build symmetry, the position of mouth, step, pipe pedal aperture number and its arrangement mode etc. should
Method belongs to traditional classification identification method, not only needs to kill sea urchin, and determination rates are low, and are not suitable for the purple with similar appearance
The identification of sea urchin-Strongylocentrotus intermedius cenospecies and Anthocidaris crassispina；Another kind is typically to utilize gene by molecular biology method
Group sequencing, by comparing the method for genomics, the advantage of the specific molecular labeling of Large-scale Screening, this method is identification
Accuracy rate is higher, and deficiency is costly, time-consuming, has very to the professional and instrument and equipment precision of Operations Analyst personnel
High requirement.
Mitochondrial genomes are the genetic systems other than eukaryotic cells Matrix attachment region, are research molecular evolution and species
Phylogenetic important object.For complicated Matrix attachment region, the number gene contained by mitochondrial genomes is less, obtains
It is easy, operation is relatively easy.But there is no hybridize to Anthocidaris crassispina-Strongylocentrotus intermedius using mtdna sequence so far
The relevant report that kind is differentiated.
Invention content
The present invention is provided a kind of for differentiating Anthocidaris crassispina-to solve the above-mentioned technical problem present in the prior art
The PCR primer and discrimination method of Strongylocentrotus intermedius cenospecies.
Technical solution of the invention is：A kind of PCR primer for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies,
It is characterized in that nucleotide sequence is as follows：
Sense primer：5' ttttatctcctccctttttatytct 3'
Downstream primer：5' cctcwaaagtagttaagattgggac 3'.
A kind of above-mentioned discrimination method for differentiating Anthocidaris crassispina and the PCR primer of Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies,
It is characterized in that carrying out in accordance with the following steps successively：
A. to be identified sea urchin individual DNA of the extraction with Anthocidaris crassispina shape；
B. it is template the DNA of extraction to be diluted to 10 ng/ μ l, and PCR amplification is carried out with sense primer and downstream primer, described
PCR reaction conditions are： 94℃ 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 35 cycles；72℃ 5min；
C. electrophoresis is carried out to pcr amplification product, shaping band is then Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies.
Differentiate Anthocidaris crassispina-centre the present invention provides the universal primer of amplification sea urchin mtdna sequence and with the primer
The method of ball sea urchin cenospecies can carry out living body sampling at any time（Pipe foot etc.）, rapidly and accurately differentiate Anthocidaris crassispina-intermediate sea
Courage cenospecies and its female parent-Anthocidaris crassispina.
Description of the drawings
Fig. 1 be the embodiment of the present invention 1 using Strongylocentrotus intermedius DNA as template 1. ~ 5. organize primer PCR product electrophoretogram.
Fig. 2 be the embodiment of the present invention 1 using Anthocidaris crassispina DNA as template 1. ~ 5. organize primer PCR product electrophoretogram.
Fig. 3 be the embodiment of the present invention 1 using Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies DNA as template 1. ~ 5. group primer PCR production
Object electrophoretogram.
Fig. 4 is the electrophoresis detection figure that the embodiment of the present invention 2 differentiates Anthocidaris crassispina and Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies.
Specific implementation mode
Embodiment 1：
1. logging in ncbi database（https://www.ncbi.nlm.nih.gov/）, Strongylocentrotus intermedius is obtained respectively
（GenBank Accession No. KC490912.1）And horsedung sea urchin（Hemicentrotus pulcherrimus）
（GenBank Accession No. KC490911.1）Mitochondrial genomes sequence designed by sequence alignment
1. ~ 5. totally 5 groups of higher PCR primers pair of versatility, nucleotide sequence are as follows：
1. sense primer：5' taacggattaaagcacagcactgaa 3'；
Downstream primer：5' cgcatagagcttgaagggaatttaa 3'；
2. sense primer：5' tcttgttttcttgtttttgtgagtt 3'；
Downstream primer：5' ctcgtgtatcaacatccattcc 3'；
3. sense primer：5' tgccatgattgcaataggagt 3'；
Downstream primer：5' atctacaaagtgtcagtatcaggca 3'；
4. sense primer：5' ccacttctcaacccatcaccacttt 3'；
Downstream primer：5' ctattccttgggggcctatttcttc 3'；
5. sense primer：5' ttttatctcctccctttttatytct 3'；
Downstream primer：5' cctcwaaagtagttaagattgggac 3'.
2. respectively using the DNA of Strongylocentrotus intermedius, Anthocidaris crassispina and Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies as template, then distinguishing
PCR reactions are carried out with each group of PCR primer；
The extraction of 2.1 DNA
The middle hybridization sea urchin of Anthocidaris crassispina, purple-and Strongylocentrotus intermedius respectively take 3 individuals, and 30mg pipes is taken to organize enough respectively, add in mortar
Enter liquid nitrogen grinding into powder, DNA is extracted using RNA isolation kit.Kit is purchased from TianGen companies, model marine animal tissue
Genome DNA extracting reagent kit（Centrifugal column type）（Catalog number (Cat.No.)：DP324）.
The DNA of extraction is diluted to 10 ng/ μ l as template by 2.2, and PCR reactions are carried out with each group of PCR primer；
PCR reaction systems are 10 μ l systems：
ddH2O 6.2μl
TAKARA La Taq(5U/μl) 0.2μl
10×LA PCR bufferⅡ(Mg2+Plus) 1.0μl
dNTP Mixture (2.5mM each) 0.8μl
0.4 μ l of sense primer
Downstream primer 0.4ul
1.0 μ l of template DNA
Wherein,TAKARA La Taq(5U/μl)、10×LA PCR bufferⅡ(Mg2+) and dNTP Mixture Plus
(2.5mM each) is purchased from precious bioengineering（Dalian）Co., Ltd, primer is by giving birth to work bioengineering（Shanghai）The limited public affairs of share
Department's synthesis.
1. ~ 5. group primer PCR reaction condition be respectively：
① 94℃ 5min；94 DEG C of 30s, 61 DEG C of 30s, 72 DEG C of 3min30s, 35 cycles；72℃ 5min.
② 94℃ 5min；94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 4min, 35 cycles；72℃ 5min.
③ 94℃ 5min；94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 4min, 35 cycles；72℃ 5min.
④ 94℃ 5min；94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 4min, 35 cycles；72℃ 5min.
⑤ 94℃ 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 35 cycles；72℃ 5min.
3. electrophoresis is carried out to obtained PCR product respectively, it is as a result as shown in Figure 1, Figure 2, Figure 3 shows respectively.
Fig. 1 be using Strongylocentrotus intermedius DNA as template 1. ~ 5. organize primer PCR product electrophoretogram.It will be seen from figure 1 that 1. extremely
5. there is band in a number primer pair.
Fig. 2 be using Anthocidaris crassispina DNA as template 1. ~ 5. organize primer PCR product electrophoretogram.Figure it is seen that 1., 4. number
There is band in primer pair.
Fig. 3 be using Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies DNA as template 1. ~ 5. organize primer PCR product electrophoretogram.From Fig. 3
As can be seen that 1., 2., 4., 5., there is band in a number primer pair.
It can be seen that simultaneously from Fig. 2 ~ Fig. 3 in the similar Anthocidaris crassispina of shape and Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies
In only amplification Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies DNA PCR primer be the 2., 5. group, i.e., the 2., 5. group primer has spy
The opposite sex, but 5. group primer is more stronger than 2. organizing primer pair identification specificity, and proliferation time is shorter, therefore 5. group primer can be used as discriminating purple
The PCR primer of sea urchin and Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies.
5. it is as follows to organize primer pair nucleotide sequence：
Sense primer：5' ttttatctcctccctttttatytct 3'；
Downstream primer：5' cctcwaaagtagttaagattgggac 3'.
Embodiment 2：
The discrimination method for differentiating Anthocidaris crassispina and the PCR primer of Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies of the present invention, is pressed successively
It is carried out according to following steps：
A. the middle hybridization sea urchin of the purple-with Anthocidaris crassispina shape 3 and Anthocidaris crassispina individual 3 are taken, upsetting sequence at random extremely can not area
Point, extract as sea urchin to be identified and respectively each individual DNA；
B. it is template the DNA of extraction to be diluted to 10 ng/ μ l, carries out PCR amplification respectively with sense primer and downstream primer,
The nucleotide of primer is as follows：
Sense primer：5' ttttatctcctccctttttatytct 3'；
Downstream primer：5' cctcwaaagtagttaagattgggac 3'.
PCR reaction systems are 10 μ l systems：
ddH2O 6.2μl
TAKARA La Taq(5U/μl) 0.2μl
10×LA PCR bufferⅡ(Mg2+Plus) 1.0μl
dNTP Mixture (2.5mM each) 0.8μl
0.4 μ l of sense primer
Downstream primer 0.4ul
1.0 μ l of template DNA
Wherein,TAKARA La Taq(5U/μl)、10×LA PCR bufferⅡ(Mg2+) and dNTP Mixture Plus
(2.5mM each) is purchased from precious bioengineering（Dalian）Co., Ltd, primer is by giving birth to work bioengineering（Shanghai）The limited public affairs of share
Department's synthesis.
PCR reaction conditions are：94℃ 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 35 cycles；72℃
5min。
C. electrophoresis is carried out to pcr amplification product, electrophoresis result is as shown in Figure 4.
In Fig. 4, II, III, No. V sample amplification failure, is Anthocidaris crassispina；I, IV, No. VI sample expands successfully, band occurs,
For Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies.
Sequence table
<110>Dalian Ocean University
<120>PCR primer and discrimination method for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies
<160> 10
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Sense primer
<400> 1
taacggattaaagcacagcactgaa 25
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Downstream primer
<400> 2
cgcatagagcttgaagggaatttaa 25
<210> 3
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Sense primer
<400> 3
tcttgttttcttgtttttgtgagtt 25
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(22)
<223>Downstream primer
<400> 4
ctcgtgtatcaacatccattcc 22
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(21)
<223>Sense primer
<400> 5
tgccatgattgcaataggagt 21
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Downstream primer
<400> 6
atctacaaagtgtcagtatcaggca 25
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Sense primer
<400> 7
ccacttctcaacccatcaccacttt 25
<210> 8
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Downstream primer
<400> 8
ccacttctcaacccatcaccacttt 25
<210> 9
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Sense primer
<400> 9
ttttatctcctccctttttatytct 25
<210> 10
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<221>misc_feature
<222>(1)..(25)
<223>Downstream primer
<400> 10
cctcwaaagtagttaagattgggac 25
Claims (2)
1. a kind of PCR primer for differentiating Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies, it is characterised in that nucleotide sequence is as follows：
Sense primer：5' ttttatctcctccctttttatytct 3'
Downstream primer：5' cctcwaaagtagttaagattgggac 3'.
2. a kind of mirror with for differentiating Anthocidaris crassispina and the PCR primer of Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies described in claim 1
Other method, it is characterised in that carry out in accordance with the following steps successively：
A. to be identified sea urchin individual DNA of the extraction with Anthocidaris crassispina shape；
B. it is template the DNA of extraction to be diluted to 10 ng/ μ l, and PCR amplification is carried out with sense primer and downstream primer, described
PCR reaction conditions are： 94℃ 5min；94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 35 cycles；72℃ 5min；
C. electrophoresis is carried out to pcr amplification product, shaping band is then Anthocidaris crassispina-Strongylocentrotus intermedius cenospecies.
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| CN201810493591.9A CN108411002B (en) | 2018-05-22 | 2018-05-22 | PCR primer and method for identifying hybrid of echinus purpureus and echinus intermedius |
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|---|---|---|---|
| CN201810493591.9A CN108411002B (en) | 2018-05-22 | 2018-05-22 | PCR primer and method for identifying hybrid of echinus purpureus and echinus intermedius |
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| CN108411002B (en) | 2021-07-13 |
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