CN104789693B - A kind of method that prawn sex identification is carried out based on high-resolution solubility curve technology - Google Patents
A kind of method that prawn sex identification is carried out based on high-resolution solubility curve technology Download PDFInfo
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Abstract
The present invention relates to molecular biotechnology, is a kind of method that litopenaeus vannamei genetic sex identification is carried out based on high-resolution solubility curve (HRM) technology.By extracting the genomic DNA of prawn individual, high-resolution solubility curve analysis is carried out to carrying out PCR amplification, amplified production using specific primer, the qualification result of male and female gender is obtained according to analysis result.The method of the present invention is a kind of easy, accurate sex appraisal method, and whole analytic process only needs 3 hours, is appropriate for a large amount of efficient sex identifications of individual.This method has broad application prospects in the discriminating of prawn gender early stage.
Description
Technical field
The present invention relates to the prawn sex identification technology in Protocols in Molecular Biology, is a kind of simple and effective specifically
The method that prawn sex identification is realized with molecular biotechnology, be a kind of to be based on high-resolution solubility curve (HRM) technology
The method for carrying out prawn sex identification.
Background technology
Prawn is the important economic cultivated animals in China, it has very important work in the culture fishery in China
With over the past decade, since litopenaeus vannamei has the characteristics that growth is fast, adaptive faculty is strong, litopenaeus vannamei has become me
State or even the prawn kind of world's cultured output maximum, account for China's prawn culturing total output more than 80% (Fishery Bureau of the Ministry of Agriculture,
2011.2011 year China Fisheries statistical yearbook Beijing:Chinese agriculture publishing house, 129).
The male and female individual of litopenaeus vannamei cultivates full feminisation group since the difference of hormone secretion, adult individuals differ greatly
Body is a kind of technology (Li, S., F.Li, Y.Xie, B.Wang, R.Wen, C.Zhang, K.Yu and for efficiently improving cultured output
J.Xiang."Screening of Genes Specifically Expressed in Males of Fenneropenaeus
chinensis and Their Potential as Sex Markers."Journal of Marine Biology,2013,
1-9.)., it is necessary to carry out Molecular Identification to the genetic sex of prawn during full feminisation colony is cultivated, in addition ground in science
In studying carefully, it is also required to carry out the sex identification of source individual for some materials for lacking phenotypic character such as musculature, appendage etc.,
It is required to use the means of Molecular Identification in these cases.
High-resolution solubility curve (HRM) is a kind of technology for carrying out fast and accurately carrying out Genotyping, which is
According to denaturation of little by little being unwind to PCR product within the scope of certain temperature, and real-time collecting detects fluorescence during being somebody's turn to do
The change of intensity.Fluorescence intensity can be depicted as a solubility curve with temperature change.Since different DNA sequence dnas can be formed
Different melting curve shapes, therefore (Wang Jiafeng long oysters gene regions SNP marker can be distinguished to different DNA sequence dnas
Scale development and its application doctor in genetic breeding research, Postgraduate School, Chinese Academy of Sciences (institute of oceanography), 2013).
The content of the invention
Prawn gender is carried out the object of the present invention is to provide one kind based on high-resolution solubility curve (HRM) technology to reflect
Fixed method, for realize prawn gender it is inexpensive, it is efficient identification technical support is provided.
In order to achieve the above object, the scheme that uses of the present invention for：
The genomic DNA of prawn is extracted, is carried out using high-resolution solubility curve (HRM) primer for carrying out sex identification
PCR amplification, amplified production carry out high-resolution solubility curve analysis, and the qualification result of male and female gender is obtained according to analysis result.
The prawn genome DNA extracting method use Tiangeng Plant Genome extracts kit in specification into
OK.
Carry out PCR amplification primer sequence be：
LvSDHF：AAATCAGTAAACCCCGAGGG
LvSDHR：GCRGTTTCGTTTGCATTTTC.
PCR amplification is carried out using the DNA of extraction and above-mentioned primer, the cumulative volume of pcr amplification reaction therein is 20 μ L, is expanded
Increasing reaction system is：0.4 μ L, 10x Taq DNA polymerase buffers liquid of deoxynucleotide mixture (10mmol/L) 2 μ L, MgCl2
(2.5 μm of ol/L) 1.6 μ L, LvSDHF and LvSDHR (10 μm of ol/L) each 0.5 μ L, genomic DNA template (50ng/ μ L) 1 μ L,
Taq archaeal dna polymerases (2.5U/ μ L) 0.5 μ L, distilled water supply 20 μ L.PCR response procedures are 95 DEG C of pre-degeneration 10min, amplification
30 circulations (95 DEG C of 20sec, 55 DEG C of 30sec, 72 DEG C of 30sec), last 72 DEG C of extensions 10min.
Amplified production carries out high-resolution solubility curve (HRM) analysis, and step is as follows：
Internal standard and each 1ul of saturable dye LC-green are added first in each reaction tube, 3000 leave the heart 30 seconds.Then
Above-mentioned product is placed on 95 DEG C of denaturation 10min in PCR instrument, taking-up afterwards is cooled to room temperature spare.
PCR product is put into LightScanner96 Genotypings instrument or other HRM parting instruments, runs high-resolution
Melting curve program, for software set to collect the data between 55 DEG C -95 DEG C, heating rate is 0.1 DEG C/S.Make after EP (end of program)
Check that software is analyzed with the analysis solubility curve carried.
The identification of male and female gender：The solubility curve that analysis software provides, if solubility curve is identical with attached drawing 1a, except master
Outside peak, there is a smooth submaximum (smooth submaximum is in before main peak), then the individual analyzed is heterozygote, is determined as female
Property individual；If solubility curve is identical with attached drawing 1b, an only main peak, then it is homozygote to analyze individual, is determined as male
Body.
The advantages of this method：
Simply：Fluorescent dye can be directly added into after PCR amplification can analyze acquisition as a result, whole process is completely one
Carried out in a pipe, can be to avoid cross contamination.
Efficiently：HRM analytic processes only need 5 minutes, 96 samples can be analyzed at the same time, determination rates are very
It is high.
Efficiently：Simplify operating procedure, reduce manually and reagent cost, expense is far fewer than sequencing approach.
The present invention is successfully constructed by the sequence of high-resolution solubility curve (HRM) technical Analysis prawn male and female difference
The method that a set of simple and effective carries out litopenaeus vannamei sex identification, using this method from prawn developmental stage, Individual Size
Etc. the limitation of factor, though minimal amount of tissue can be identified, and since the result of HRM is in the method for solubility curve
Existing, different peak shapes represents different sequence signatures, therefore can intuitively judging result, it is possible to achieve mirror rapidly and efficiently
Not.The method of the present invention is a kind of easy, accurate sex appraisal method, and whole analytic process only needs 3 hours, is adapted to
Carry out a large amount of efficient sex identifications of individual.This method has broad application prospects in the discriminating of prawn gender early stage.
Brief description of the drawings
Fig. 1：High-resolution solubility curve (HRM) analysis result；Wherein：The corresponding individual of result of 1a is female individuals,
The corresponding individuals of 1b are male.
Embodiment
Embodiment 1：The method that one kind carries out prawn sex identification based on high-resolution solubility curve (HRM) technology
Comprise the following steps that：
1. 10 tails of random each selection from local selection and breeding parent shrimp, honest close shrimp, SIS parents shrimp, Kona gulf parent shrimp
Female shrimp and 10 tail hero shrimps amount to female 40 tail of shrimp, male 40 tail of shrimp as experimental group.
2. the DNA and mark of above-mentioned sample are extracted using Tiangeng plant genome DNA extracts kit, behaviour
Make method Reference Design and specification.The DNA of extraction is measured using Nanodrop1000 (Thermol)
DNA concentration.
3. the DNA of extraction is subjected to PCR amplification using following primer：
LvSDHF；AAATCAGTAAACCCCGAGGG
LvSDHR：GCRGTTTCGTTTGCATTTTC
The cumulative volume of pcr amplification reaction therein is 20 μ L, and amplification reaction system is：Deoxynucleotide mixture
(10mmol/L) 0.4 μ L, 10xTaq DNA polymerase buffers liquid 2 μ L, MgCl2(2.5 μm of ol/L) 1.6 μ L, LvSDHF and
LvSDHR (10 μm of ol/L) each 0.5 μ L, genomic DNA template (50ng/ μ L) 1 μ L, Taq archaeal dna polymerase (2.5U/ μ L) 0.5 μ
L, distilled water supply 20 μ L.PCR response procedures are 95 DEG C of pre-degeneration 10min, and 30 amplification cycles (95 DEG C of 20sec, 55 DEG C
30sec, 72 DEG C of 30sec) it is last 72 DEG C extension 10min.
4. high-resolution solubility curve (HRM) analysis, step are carried out to amplified production using Lightscanner96
It is rapid as follows：
Internal standard and each 1ul of saturable dye LC-green are added first in each reaction tube, 3000 leave the heart 30 seconds.Then
Above-mentioned product is placed on 95 DEG C of denaturation 10min in PCR instrument, taking-up afterwards is cooled to room temperature spare.
Start operation Lightscanner96, first preheater apparatus make machine reach working status, PCR product are put into instrument
Device, run high-resolution melting curve program, software set for collect 55 DEG C -95 DEG C between data, heating rate 0.1
℃/S.The analysis software carried after EP (end of program) using lightscanner96 is analyzed, and obtains solubility curve result.
5. the solubility curve of the female shrimp of 40 tails and 40 tail hero shrimps is analyzed, the results show that the female shrimp curve of all 40 tails
It is identical with attached drawing 1a；All male shrimp solubility curves are identical with attached drawing 1b.
Embodiment 2：The method that one kind carries out prawn sex identification based on high-resolution solubility curve (HRM) technology
Comprise the following steps that：
1. 20 individuals for treating progress sex identification are sampled and make marks.
2. the DNA and mark of above-mentioned sample are extracted using Tiangeng plant genome DNA extracts kit, operating method reference
Design and specification.The DNA of extraction uses Nanodrop1000 (Thermol) measure DNA concentrations.
3. the DNA of extraction is subjected to PCR amplification using following primer：
LvSDHF；AAATCAGTAAACCCCGAGGG
LvSDHR：GCRGTTTCGTTTGCATTTTC
The cumulative volume of pcr amplification reaction therein is 20 μ L, and amplification reaction system is：Deoxynucleotide mixture
(10mmol/L) 0.4 μ L, 10xTaq DNA polymerase buffers liquid 2 μ L, MgCl2(2.5 μm of ol/L) 1.6 μ L, LvSDHF and
LvSDHR (10 μm of ol/L) each 0.5 μ L, genomic DNA template (50ng/ μ L) 1 μ L, Taq archaeal dna polymerase (2.5U/ μ L) 0.5 μ
L, distilled water supply 20 μ L.PCR response procedures are 95 DEG C of pre-degeneration 10min, and 30 circulations of amplification (95 DEG C of 20sec, 55 DEG C
30sec, 72 DEG C of 30sec) it is last 72 DEG C extension 10min.
4. carrying out high-resolution solubility curve (HRM) analysis to amplified production using Lightscanner96, step is as follows：
Internal standard and each 1ul of saturable dye LC-green are added in each reaction tube, 3000 leave the heart 30 seconds.Then will be above-mentioned
Product is placed on 95 DEG C of denaturation 10min in PCR instrument, and taking-up afterwards is cooled to room temperature spare.
Lightscanner96, first preheater apparatus make machine reach working status, and PCR product is put into instrument, operation
High-resolution melting curve program, for software set to collect the data between 55 DEG C -95 DEG C, heating rate is 0.1 DEG C/S.Program
After the analysis software that is carried using lightscanner96 analyzed, obtain solubility curve.
5. a pair solubility curve is analyzed, it is found that the solubility curve for sharing 10 tail shrimps is identical with Fig. 1 a, be accredited as female
Body, in addition 10 tail solubility curves are identical with 1b, are accredited as male.
Claims (3)
1. a kind of method that prawn sex identification is carried out based on high-resolution solubility curve technology, it is characterized in that：Institute is extracted first
The genomic DNA of the prawn individual of sex identification is needed, utilizes the primer pair LvSDHF for carrying out sex identification；
AAATCAGTAAACCCCGAGGG and LvSDHR：GCRGTTTCGTTTGCATTTTC carries out PCR amplification, and amplified production carries out high score
Resolution solubility curve (HRM) is analyzed, and is differentiated prawn gender according to the peak figure of solubility curve, is realized the sex identification of prawn.
2. in accordance with the method for claim 1, it is characterized in that：The amplified production carries out high-resolution solubility curve (HRM)
Analysis：If analyzing the solubility curve of output in addition to a main peak, there is a smooth submaximum, then the individual is denoted as
Heterozygote, is female individuals；If the result for analyzing the solubility curve of output only has a main peak, which is homozygote,
For male.
3. in accordance with the method for claim 1, it is characterized in that：The prawn is litopenaeus vannamei.
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| CN101432442A (en) * | 2006-04-25 | 2009-05-13 | 福拉姆斯大学生物技术研究所 | A sex-specific marker for shrimps and prawns |
| CN101709332A (en) * | 2009-10-30 | 2010-05-19 | 中国科学院海洋研究所 | Prawn sex probe and acquisition method thereof |
| CN102417924A (en) * | 2011-07-15 | 2012-04-18 | 天津市农业科学院中心实验室 | Method for identifying purity of cucumber hybrids based on high-resolution melting curve |
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Patent Citations (3)
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|---|---|---|---|---|
| CN101432442A (en) * | 2006-04-25 | 2009-05-13 | 福拉姆斯大学生物技术研究所 | A sex-specific marker for shrimps and prawns |
| CN101709332A (en) * | 2009-10-30 | 2010-05-19 | 中国科学院海洋研究所 | Prawn sex probe and acquisition method thereof |
| CN102417924A (en) * | 2011-07-15 | 2012-04-18 | 天津市农业科学院中心实验室 | Method for identifying purity of cucumber hybrids based on high-resolution melting curve |
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| Molecular Characterisation of Colour Formation in the Prawn Fenneropenaeus merguiensis;Nicole G. Ertl,et al;《PLOS ONE》;20130218;第8卷(第2期);e56920 * |
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