CN103805695B - For detecting the primer of the chrysanthemum Huang east speed of growth to, kit and method - Google Patents
For detecting the primer of the chrysanthemum Huang east speed of growth to, kit and method Download PDFInfo
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- CN103805695B CN103805695B CN201310654871.0A CN201310654871A CN103805695B CN 103805695 B CN103805695 B CN 103805695B CN 201310654871 A CN201310654871 A CN 201310654871A CN 103805695 B CN103805695 B CN 103805695B
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- C—CHEMISTRY; METALLURGY
- 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
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The present invention relates to for the primer detecting the chrysanthemum Huang east speed of growth, kit and method, primer is to being the upstream primer of SEQ ID NO:3 by: base sequence and the downstream primer of SEQ ID NO:4 forms.This primer is to being additionally operable to make kit, except above-mentioned primer is external, also includes the chrysanthemum Huang east mark MARKER of fast growth in kit.Detection method is as follows: extracts the DNA in sample chrysanthemum Huang east, utilizes the primer in kit to carry out PCR amplification to by the DNA in sample chrysanthemum Huang east；Amplified production is carried out PAGE process, observe stripe size, consistent with MARKER stripe size for growing fireballing individuality, the individuality that inconsistent micro-speed of growth is slow, by the primer of the present invention, kit and method are differentiated the speed of growth in chrysanthemum Huang east, having repeatable high, accuracy is strong, simple and rapid advantage.
Description
[technical field]
The present invention relates to detection primer and the method in chrysanthemum Huang east, be particularly used for detecting chrysanthemum Huang east growth speed
The primer of degree is to, kit and method.
[background technology]
Chrysanthemum Huang east (Fugu flavidus) is commonly called as ship bar, well-behaved fish, chrysanthemum magnitude yellow, all over the sky, and classification is subordinate to shape mesh
(Tetraodontif-ormes) section (Tetraodontidae) east belongs to (Fugu), is distributed mainly on China's Huanghai Sea, east
Sea and the Bohai Sea.Due to its delicious flavour, nutritious, rather well received, it is a kind of famous and precious cultivation kind.In breeding process,
Breeding environment standardization degree is more and more higher, and the individuality of fast growth can shorten culturing time, improves the utilization rate of feed,
Saving aquaculture cost, therefore cultivating screening fast growth chrysanthemum Huang east is the target that aquaculturist pursues.
Myostatin (Myostatin, MSTN) is cloned into from mice skeletal cDNA library at first, and it is main
Great expression in musculature, belongs to TGF-β super families.MSTN knock out mice is studied and shown, mutant mice
Weight ratio heterozygote and wild-type mice body weight increase about 30%, wherein myocyte's hyperplasia (hyperplasia) and muscle fibre are fertile
The muscle hypertrophy that (hypertrophy) double action is caused greatly is the main cause causing body weight to increase, and this characteristic
Unrelated with the sex of animal and age, and do not affect the fertility of animal.Therefore, MSTN is that of muscle growth is important
Negative regulation major gene resistance.The MSTN of activation and cell membrane surface receptors (activin acceptor protein II B(ACVR2B) and activin
Receptor protein IB(ACVRB)) combine, the 2/3 and p38MAPK Cellular Signaling Transduction Mediated approach of mother S in downstream can be activated, from
And regulate growing of each tissue.
" Pelteobagrus fulvidraco MSTN Gene polymorphism and the correlation analysis with growth traits thereof " (Zhu Yuanyuan etc., heredity, 2012
January 34(1): 72-78 page) in a literary composition, disclose the MSTN impact on yellow catfish growing speed, by transgenic technology and DNAi
The muscle of the MSTN gene discovery experiment fish of technology suppression zebra fish is substantially flourishing, it is taken as that this gene is animal than normal fish
Muscle growth plays important inhibitory action in growing, it is also possible to participate in generation and the regulation of fat.In fish, MSTN mDNA
Can be at muscle, eye, brain, intestines, the cheek, kidney, the heart, spleen find, and MSTN is also divided into two classes in different fingerlings, MSTN1,
MSTN2(Paralichthys olivaceus growth inhibition plain gene is cloned, Xu Jianyong, Chen Songlin, aquatic product journal the 4th phase of volume 32, in July, 2008,
497-498).
[summary of the invention]
There is gene duplication phenomenon in east in chrysanthemum Huang, these double gene and there is multiple copy, and MSTN2(is muscle
GIF) in chrysanthemum Huang east, there is multiple copy, PCR can expand some and copy unintentionally, such as base sequence SEQ
Shown in ID NO:1, SEQ ID NO:2, this fragment is positioned at MSTN2 gene 3 untranslated region territory, chrysanthemum Huang east, wherein occurs as soon as
Repeated segments GT, the existence of these copies, have impact on the analysis to result.
On the basis of above-mentioned discovery, interference is brought to ask to solve PC amplification copy in prior art for result
Topic, and make up in prior art the blank for the speed of growth research of chrysanthemum Huang east.Present invention firstly provides a kind of for
Detection chrysanthemum Huang east primer pair, primer to by: base sequence is upstream primer and the SEQ ID NO:4 of SEQ ID NO:3
Downstream primer composition.
This primer, to can be also used for making kit, except above-mentioned primer is external, also includes the speed of growth in kit
Fast chrysanthemum Huang east mark MARKER.
Method by the above-mentioned kit detection chrysanthemum Huang east speed of growth is as follows:
Extract the DNA sequence dna of the myostatin in sample chrysanthemum Huang east,
The primer in kit is utilized to carry out PCR amplification to by the DNA in sample chrysanthemum Huang east；
Amplified production is carried out PAGE process,
Observe stripe size, consistent with MARKER stripe size for growing fireballing individuality.
Further, said method also has a following prioritization scheme:
Described pcr amplification reaction system 20 μ L, including 2uL10 × PCR buffer solution, the dNTP of 0.8ul10mmol/L
Mix, the upstream primer of 0.4 μm ol/L, the downstream primer of 0.4 μm ol/L, the sample chrysanthemum Huang east DNA of 30ng.
Described pcr amplification reaction condition is preferably, sex change 3 minutes at 94 DEG C, sex change 30 seconds at 94 DEG C, renaturation at 64 DEG C
Extending 30 seconds at 30 seconds, and 72 DEG C, totally 42 circulations, last 72 DEG C extend 10min.
Described PAGE is preferably a step:
The sex change sample-loading buffer taking equivalent is separately added in PCR primer and kit in MARKER, 99 DEG C of sex change 10 points
Clock, at urea-denatured PAGE(6%) in carry out electrophoresis and run out of PAGE to bromophenol blue, carry out silver staining, fixing, dye and develop the color.
By the primer of the present invention, kit and method are differentiated the speed of growth in chrysanthemum Huang east, has repeatable
Property high, accuracy is strong, simple and rapid advantage.This primer occupies the most at high proportion in fast growth colony, and with
The compatible degree of minimum artificially breeding parent is up to more than 96%.
[accompanying drawing explanation]
Fig. 1 is the SNPs site schematic diagram in the chrysanthemum Huang east MSTN2 gene of the present invention；
Fig. 2 is that chrysanthemum Huang east grows speed PAGE band comparison diagram.
[detailed description of the invention]
The present invention will be further described to combine accompanying drawing below by specific embodiment, and following embodiment is only for clear
Ground illustrates example of the present invention, and is not the restriction to embodiments of the present invention.
Embodiment 1:
Step one, the selection in fast growth chrysanthemum Huang east
Select the chrysanthemum Huang east taking from for half age, be divided into two groups according to weight and body length, use phenol chloroform to take out according to a conventional method
Formulation (see yellow training hall etc. and translate " Molecular Cloning: A Laboratory guide " (third edition), Science Press, in September, 2002) extract chrysanthemum Huang east
Side DNA, design a pair selective amplification primer of synthesis.
Upstream primer 5 '-CAAGCTTGTCTGTTGAGGCCTGTT-3 ',
Downstream primer 5 '-CAGCACCTTCGACCTCATTTGATG-3 '；
Its DNA is carried out PCR amplification, reaction system 20 μ L, including 2ul10 × PCR buffer solution, 0.8ul10mmol/L's
DNTP mix, the upstream primer of 0.4 μm ol/L, the downstream primer of 0.4 μm ol/L, the sample chrysanthemum Huang east DNA of 30ng, reaction
Condition is as follows, 94 DEG C of sex change 3min, then 94 DEG C of sex change 30sec, 55 DEG C of renaturation 30 seconds, and 72 DEG C extend 30 seconds, 42 circulations,
Last 72 DEG C extend 10min.
Product, through the sex change PAGE electrophoresis of 6%, through dyeing colour developing, observed result, operates as follows: take equivalent sex change loading
Buffer solution adds PCR primer (formula is shown in molecular cloning handbook), and 99 DEG C of sex change 10 minutes, at urea-denatured PAGE(6%) in carry out
Electrophoresis runs out of PAGE to bromophenol blue.Carry out silver staining, fixing, dye and develop the color.Observe stripe size, containing 187-189bp size
The individuality of band, is the individuality of fast growth.
Then using this individuality DNA sequence dna as fast growth individuality MARKER.
Step 2, the detection chrysanthemum Huang east speed of growth
Above-mentioned primer is made as kit to, MARKER, when needs detect, extracts the muscle in sample chrysanthemum Huang east
The DNA sequence dna of GIF, and with the primer in above-mentioned kit to carrying out PCR amplification.
Reaction system 20 μ L, including 2uL10 × PCR buffer solution, the dNTP mix of 0.8ul10mmol/L, 0.4 μm ol/L
Upstream primer, the downstream primer of 0.4 μm ol/L, the sample chrysanthemum Huang east DNA. reaction condition of 30ng is, sex change 3 points at 94 DEG C
Clock, sex change 30 seconds at 94 DEG C, extend 30 seconds at renaturation 30 seconds, and 72 DEG C at 64 DEG C, totally 42 circulations, last 72 DEG C of extensions
10min。
PCR primer with compare the MARKER sex change PAGE electrophoresis through 6%, through dyeing colour developing, observed result, operate as follows:
Taking equivalent sex change sample-loading buffer and add PCR primer (formula is shown in molecular cloning handbook), 99 DEG C of sex change 10 minutes, urea-denatured
PAGE(6%) carry out electrophoresis in and run out of PAGE to bromophenol blue.Carry out silver staining, fixing, dye and develop the color.Observe stripe size, with
The individuality that MARKER stripe size is consistent, is the individuality of fast growth.Its comparison diagram sees Fig. 2, and wherein L represents fast of growth
Body, it is slow individual that S represents growth, and M is kit MARKER.
Claims (2)
1. one kind for detecting the primer pair of the chrysanthemum Huang east speed of growth, it is characterised in that primer to by: base sequence is SEQ
The upstream primer of ID NO:3 and the downstream primer composition of SEQ ID NO:4.
2. the method detecting the chrysanthemum Huang east speed of growth, it is characterised in that comprise the steps of:
Extract the DNA sequence dna of the myostatin in sample chrysanthemum Huang east,
The primer in kit is utilized to carry out PCR amplification, described pcr amplification reaction body to by the DNA in sample chrysanthemum Huang east
It is 20 μ L, including 2uL 10 × PCR buffer solution, the dNTP mix of 0.8ul 10mmol/L, the upstream primer of 0.4 μm ol/L, 0.4
The downstream primer of μm ol/L, base sequence is upstream primer and the downstream primer of SEQ ID NO:4, the 30ng of SEQ ID NO:3
Sample chrysanthemum Huang east DNA；Described pcr amplification reaction condition is, sex change 3 minutes at 94 DEG C, sex change 30 seconds at 94 DEG C, 64
Extending 30 seconds at renaturation 30 seconds, and 72 DEG C at DEG C, totally 42 circulations, last 72 DEG C extend 10min；
Amplified production carries out PAGE, dyeing, and the step of described PAGE is: the sex change sample-loading buffer taking equivalent is separately added into
In PCR primer and kit in MARKER, 99 DEG C of sex change 10 minutes, urea-denatured PAGE 6% carries out electrophoresis to bromophenol blue
Run out of PAGE, carry out silver staining, fixing, dye and develop the color；Observe stripe size, consistent with the MARKER stripe size in kit
For grow fireballing individuality.
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| CN201310654871.0A CN103805695B (en) | 2013-12-05 | 2013-12-05 | For detecting the primer of the chrysanthemum Huang east speed of growth to, kit and method |
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| CN201310654871.0A CN103805695B (en) | 2013-12-05 | 2013-12-05 | For detecting the primer of the chrysanthemum Huang east speed of growth to, kit and method |
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| CN103805695B true CN103805695B (en) | 2016-08-31 |
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| CN110241225A (en) * | 2019-04-29 | 2019-09-17 | 中国水产科学研究院 | The SNP marker of species identification for river Puffer combines |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102893927A (en) * | 2011-07-26 | 2013-01-30 | 上海市水产研究所 | Feeding method of controllable bait for fries of Takifugu flavidus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102893927A (en) * | 2011-07-26 | 2013-01-30 | 上海市水产研究所 | Feeding method of controllable bait for fries of Takifugu flavidus |
Non-Patent Citations (2)
| Title |
|---|
| ACCESSION NO: KE120244, Takifugu flavidus unplaced genomic scaffold SCAFFOLD_0003, whole genome shotgun sequence;Yang,G.;《GenBank database》;20130129;全文 * |
| New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis;Kuroyanagi et al.;《BMC Genomics》;20131113;第14卷(第786期);摘要，第12页 * |
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Address after: 200433 No. 265, Jiamusi Road, Shanghai, Yangpu District Patentee after: SHANGHAI FISHERIES Research Institute SHANGHAI FISHERIES TECHNICAL EXTENSION STATION Address before: 200433 No. 265, Jiamusi Road, Shanghai, Yangpu District Patentee before: SHANGHAI FISHERIES Research Institute |
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