CN108070663A - Differentiate the molecular labeling primer and method of the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man - Google Patents
Differentiate the molecular labeling primer and method of the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man Download PDFInfo
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- CN108070663A CN108070663A CN201711273848.1A CN201711273848A CN108070663A CN 108070663 A CN108070663 A CN 108070663A CN 201711273848 A CN201711273848 A CN 201711273848A CN 108070663 A CN108070663 A CN 108070663A
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Abstract
The present invention provides the molecular labeling primer and method for differentiating the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man, wherein, the molecular labeling primer of the discriminating penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man, including introducing object one：16SF1：TTCAATCTACCTTTCAGT；Introduce object two：16SR1：TTTATCCTGGCTTATTCT.The present invention also provides the methods for the molecular labeling primer for differentiating the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man, include the following steps：1) the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man are chosen, forms the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man sample cluster, extracts the DNA of 2 sample populations respectively；2) using the DNA of genome as template, design PCR reaction systems are expanded respectively, obtain penaeus penicillatus young molecule labelled series and During Larvae Rearing of Penaeus Merguiensis De Man molecule labelled series；3) amplification purified, be sequenced, compared sequencing result, differentiate its species.
Description
Technical field
The invention belongs to field of molecular biotechnology, and in particular to a kind of discriminating penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man
Molecular labeling primer and method.
Background technology
Penaeus penicillatus and banana prawn are under the jurisdiction of Decapoda, Penaeidae, Penaeus, are widely distributed in the Indian Ocean, west too
Flat ocean, most commonly seen in China Fujian, Taiwan, Guangdong and ALONG GUANGXI COAST, two kinds of prawn meat exquisitenesses are delicious, have very high food
It is the important marine fishing in southern provinces and regions and cultivation object with value.The form of two seed shrimps is very close, often with frontal eminence length and
Frontal eminence base portion height distinguishes two kinds of prawns.But in the early life history stage, their young and young shrimp are difficult to be distinguished, and are caused
Breeding, the cultivation of two kinds of prawns, wild stocks supplement, the difficulty of forecast.Especially in Tropical Ocean Area, two kinds of prawn breeding season weights
It is folded, more it is difficult to differentiate between the young of two kinds of prawns and young shrimp.Currently used for identification penaeus penicillatus and During Larvae Rearing of Penaeus Merguiensis De Man intuitively
Method yet there are no formal report.Therefore the reliable molecular labeling of penaeus penicillatus and banana prawn is found, is reflected from the germplasm mixed
Not Qu Bie two kinds of prawns become a urgent problem to be solved.
This will be helpful to solve ocean species the problems such as mixing, and be prawn culturing, prawn protection of resources, rationally utilize and
Biodiversity research provides technical support.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of point for differentiating the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man
Sub- labeled primer and method help to solve two kinds of prawn germplasm mixed questionses, and the method for the present invention is easy to operate, is easy to grasp,
Accuracy is high.
The present invention is achieved by the following technical solution：
Differentiate the molecular labeling primer of the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man,
Including primer one and primer two：
Primer one：16SF1：TTCAATCTACCTTTCAGT；
Primer two：16SR1：TTTATCCTGGCTTATTCT.
The present invention also provides the method for the molecular labeling primer for differentiating the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man, including
Following steps：
1) the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man are chosen, forms the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man sample
Group extracts the DNA of 2 sample populations respectively；
2) using the DNA of the genome in step 1) as template, design PCR reaction systems are expanded respectively, are become mildewed
Prawn larvae molecule labelled series and During Larvae Rearing of Penaeus Merguiensis De Man molecule labelled series；
3) amplification purified, be sequenced, compared sequencing result, differentiate its species.
Preferably, the PCR reaction systems composition is：Ultra-pure water 17.5ul, 10 × buffer2.5ul, dNTPs
Each 1ul of 2ul, rTaq 0.15ul, DNA profiling 1ul, Primer.
Preferably, PCR amplification program is：Then 94 DEG C of pre-degeneration 5min are denatured 35sec, 50 DEG C of annealing for 94 DEG C
35sec, 72 DEG C of extension 35sec carry out 38 Xun Huans, last 72 DEG C of extensions 10min, 4 DEG C of preservations.
Preferably, the penaeus penicillatus young molecule labelled series are：
ATAACCGCGGCTGCTGGCACAAATTTTAGCCAGATATTTAATTAGGATCATCAATTCCAACTTACATTTATCTAATT
AGTACTAGGTACTGAGAATTTATTTCTTAACGAAACCTCTTCCTTTAATTAATTCATTAACCTTTAGTATCTTTAAC
AATGAATCTTATTGTCACAATAAATTTTACATGTACCGTTGACCTAAAAATGAA.
Preferably, the During Larvae Rearing of Penaeus Merguiensis De Man molecule labelled series are：
ATAACCGCGGCTGCTGGCACAAATTTTAGCCAGATATATAATTAGGATCATCAATTCCAACTTACATTTATCTAATT
AGTACTAGGTACTGAGAATTTATTTCTTAATGAAACCTCTCCCTTTAACTGATTCACTAACCTTTAGTATCCTTAAT
AATGAATCTTATTGTCACAATAAATTTTACATGTACCATTAACCTAAAAATGAA。
Two are can be seen that by the mtDNA12S gene 208bp sequence permutation results of the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man
Kind prawn larvae shows as 1 conversion, 9 transversion there are the difference of 10 bases.Its result is stablized, and repeatability is strong.5 groups
Contrast experiment's amplification is consistent, shows high consistency.Therefore the base difference based on 12S genes can be used as and become mildewed
The molecular labeling that prawn larvae and During Larvae Rearing of Penaeus Merguiensis De Man germplasm are distinguished.Meanwhile discrimination method is simple.
Specific embodiment
Technical scheme is further explained below by embodiment, but protection scope of the present invention from
The limitation of embodiment in any form.
Embodiment：
The penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man are selected, forms the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man sample cluster,
The DNA of 2 sample populations is extracted, the DNA then extracted carries out PCR amplification for template, amplification is purified, is surveyed
Sequence compares sequencing result；
The primer sequence is 16SF1：TTCAATCTACCTTTCAGT；16SR1：TTTATCCTGGCTTATTCT；Institute
The reaction system for the PCR amplification stated forms：
It is sequentially added in 0.2ml centrifuge tubes：
PCR amplification program is 94 DEG C of pre-degeneration 2min, and (94 DEG C of denaturation 15sec, 50 DEG C of annealing 15sec, 72 DEG C extend
15sec) × 38 Xun Huan, 72 DEG C of extensions 3min, 4 DEG C of preservation ∞.PCR product is sequenced after purification, and to 2 groups
Sequencing result is compared.
It can be seen that and become mildewed by the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man mtDNA12S gene 208bp sequence permutation results
Prawn larvae and During Larvae Rearing of Penaeus Merguiensis De Man show as 1 conversion, 9 transversion there are the difference of 10 bases.
The mtDNA12S gene 208bp sequence alignment results of the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man are：
The penaeus penicillatus young：ATAACCGCGG CTGCTGGCAC AAATTTTAGC CAGATATTTA
During Larvae Rearing of Penaeus Merguiensis De Man：*************************************A**
Ruler：.........10.........20.........30.........40
The penaeus penicillatus young：ATTAGGATCATCAATTCCAACTTACATTTATCTAATTAGT
During Larvae Rearing of Penaeus Merguiensis De Man：************************************
Ruler：.........50.........60.........70.........80
The penaeus penicillatus young：ACTAGGTACT GAGAATTTAT TTCTTAACGAAACCTCTTCC
During Larvae Rearing of Penaeus Merguiensis De Man：***********************T*********C**
Ruler：........90........100........110........120
The penaeus penicillatus young：TTTAATTAAT TCATTAACCT TTAGTATCTT TAACAATGAA
During Larvae Rearing of Penaeus Merguiensis De Man：*****C*G*****C**************C****T******
Ruler：........130........140........150........160
The penaeus penicillatus young：TCTTATTGTC ACAATAAATT TTACATGTAC CGTTGACCTA
During Larvae Rearing of Penaeus Merguiensis De Man：***************************A**A*****
Ruler：.........170.........180.........190........200
The penaeus penicillatus young：AAAATGAA
During Larvae Rearing of Penaeus Merguiensis De Man：********
Ruler：.......208
(wherein " * " represents identical base sequence site)
Sequencing result shows that 5 tail length hair prawn larvae sample amplification results are consistent, while 5 tail During Larvae Rearing of Penaeus Merguiensis De Man samples
Amplification always, is as a result stablized and repeatability is strong.
SEQUENCE LISTING
<110>Nanhai Aquatic Inst., Chinese Aquatic Scientific Research Inst
<120>Differentiate the molecular labeling primer and method of the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man
<130> 1
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 208
<212> DNA
<213>Penaeus penicillatus
<400> 1
ataaccgcgg ctgctggcac aaattttagc cagatattta attaggatca tcaattccaa 60
cttacattta tctaattagt actaggtact gagaatttat ttcttaacga aacctcttcc 120
tttaattaat tcattaacct ttagtatctt taacaatgaa tcttattgtc acaataaatt 180
ttacatgtac cgttgaccta aaaatgaa 208
<210> 2
<211> 208
<212> DNA
<213>Banana prawn
<400> 2
ataaccgcgg ctgctggcac aaattttagc cagatatata attaggatca tcaattccaa 60
cttacattta tctaattagt actaggtact gagaatttat ttcttaatga aacctctccc 120
tttaactgat tcactaacct ttagtatcct taataatgaa tcttattgtc acaataaatt 180
ttacatgtac cattaaccta aaaatgaa 208
Claims (6)
1. differentiate the molecular labeling primer of the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man, it is characterised in that including primer one and primer
Two：
Primer one：16SF1：TTCAATCTACCTTTCAGT；
Primer two：16SR1：TTTATCCTGGCTTATTCT.
2. the method for the molecular labeling primer of the discriminating penaeus penicillatus young described in claim 1 and During Larvae Rearing of Penaeus Merguiensis De Man is used for,
Include the following steps：
1) the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man are chosen, forms the penaeus penicillatus young and During Larvae Rearing of Penaeus Merguiensis De Man sample cluster, point
Indescribably take the DNA of 2 sample populations；
2) using the genomic DNA in step 1) as template, design PCR reaction systems are expanded respectively, obtain penaeus penicillatus children
Body molecule labelled series and During Larvae Rearing of Penaeus Merguiensis De Man molecule labelled series；
3) amplified production purified, be sequenced, compared sequencing result, differentiate its species.
3. according to the method described in claim 2, it is characterized in that, in step 2), the PCR reaction systems composition is：It is super
Each 1ul of pure water 17.5ul, 10 × buffer 2.5ul, dNTPs 2ul, rTaq 0.15ul, DNA profiling 1ul, Primer.
4. according to the method described in claim 2, it is characterized in that, in step 2), PCR amplification program is：94 DEG C of pre-degenerations
5min, then 94 DEG C of denaturation 35sec, 50 DEG C of annealing 35sec, 72 DEG C of extension 35sec carry out 38 Xun Huans, last 72 DEG C of extensions
10min, 4 DEG C of preservations.
5. according to the method described in claim 2, it is characterized in that,
The penaeus penicillatus young molecule labelled series are：
ATAACCGCGGCTGCTGGCACAAATTTTAGCCAGATATTTAATTAGGATCATCAATTCCAACTTACATTTATCT
AATTAGTACTAGGTACTGAGAATTTATTTCTTAACGAAACCTCTTCCTTTAATTAATTCATTAACCTTTAGTATCTT
TAACAATGAATCTTATTGTCACAATAAATTTTACATGTACCGTTGACCTAAAAATGAA。
6. according to the method described in claim 2, it is characterized in that,
The During Larvae Rearing of Penaeus Merguiensis De Man molecule labelled series are：
ATAACCGCGGCTGCTGGCACAAATTTTAGCCAGATATATAATTAGGATCATCAATTCCAACTTACATTTATCT
AATTAGTACTAGGTACTGAGAATTTATTTCTTAATGAAACCTCTCCCTTTAACTGATTCACTAACCTTTAGTATCCT
TAATAATGAATCTTATTGTCACAATAAATTTTACATGTACCATTAACCTAAAAATGAA。
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Cited By (2)
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| CN108732284A (en) * | 2018-06-04 | 2018-11-02 | 山东出入境检验检疫局检验检疫技术中心 | A method of differentiating prawn using specificity peptide fragment group |
| CN110029173A (en) * | 2019-04-18 | 2019-07-19 | 中国水产科学研究院南海水产研究所 | A kind of molecular labeling primer and its application identifying oil Yu and the Yu that sets one's teeth on edge |
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| CN108732284A (en) * | 2018-06-04 | 2018-11-02 | 山东出入境检验检疫局检验检疫技术中心 | A method of differentiating prawn using specificity peptide fragment group |
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| CN110029173A (en) * | 2019-04-18 | 2019-07-19 | 中国水产科学研究院南海水产研究所 | A kind of molecular labeling primer and its application identifying oil Yu and the Yu that sets one's teeth on edge |
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