CN109652562A - A method of detection ox muscle FASN gene expression amount - Google Patents
A method of detection ox muscle FASN gene expression amount Download PDFInfo
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Abstract
The invention discloses a kind of methods for detecting ox muscle FASN gene expression amount, the present invention passes through design FASN gene-specific primer pair, the qPCR of internal control primer GAPDH is expanded, and the relative quantitative assay to qPCR result, accurately and rapidly analyze expression of the FASN gene in ox muscle.The qPCR detection method of detection FASN gene expression dose established by the present invention, can be used for accurately detecting the expression of different breeds of cattle muscle FASN gene.The invention the method can be used for detecting the expression of different breeds of cattle muscle FASN gene, provide reference to further investigation ox FASN gene function, method is easy, easy to operate.
Description
Technical field
The present invention relates to genetic engineering fields, refer in particular to a kind of method for detecting ox muscle FASN gene expression amount.
Background technique
The multienzyme complex system that fatty acid synthetase (fatty acid synthase, FASN) is made of 6 kinds of enzymes,
FASN is Fatty synthesis rate-limiting enzyme, and activity height directly controls the synthesis of body fat.In the mammalian body, dimer
FASN existing for form just has the activity of catalysis fatty acid synthesis, and monomeric form FASN does not have the work of catalysis fatty acid synthesis
Property, but the partial function domain of FASN still possesses catalytic activity in monomer, however acyl is transferred to by substrate in advance for needs
The base load body protein functional areas (Acyl carrier protein, ACP) and the functional domain activity to interact with ACP reduction.
Using the method for in situ hybridization and somatic hybridization, by the FASN assignment of genes gene mapping of ox on No. 19 chromosomes, DNA sequence
Column overall length 19.7Kb, cDNA sequence overall length 7542bp encode 2513 amino acid.Relative to its hetero-organization FASN gene in liver
The tissue expressions activity such as dirty, lactation period mammary gland and fat is higher, while FASN activity of gene expression is also swashed by related in animal body
Its expression effect can be improved in the regulation of element, such as sterol, prolactin, and insulin, progesterone can lower its expression effect.In addition,
Daily ration medium high carbon hydrate and fat can influence the synthesis of liver F ASN.In the collaboration of insulin and Adrenal Glucocorticoid
Under effect, Hi CHO daily ration can increase the synthesis of liver F ASN；And fat can inhibit the table of liver F ASN gene
It reaches.It is less about report of the gene on ox muscle at present.Therefore ox muscle FASN can accurately be detected by needing to develop one kind
The method of gene expression amount, in order to its application in meat research.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of detection ox muscle FASN gene expression amounts
Method.
The present invention is achieved by the following technical solutions:
A method of detection ox muscle FASN gene expression amount, specifically includes the following steps:
1) total serum IgE is extracted from beef tissue to be detected, then cDNA sample is prepared by reverse transcription reaction；
2) the cDNA sample of preparation is carried out the real-time PCR of fluorescent quantitation to detect, the qPCR amplified reaction of reference gene GAPDH
System are as follows:Rremix Ex TaqTM II (Perfect ReaL Time) 10.0 μ L, ddH28 0.5 μ of μ L, G-F of O
0.5 μ L of L, G-R, the 1.0 μ L of musculature cDNA of ox；
The qPCR amplification reaction system of ox FASN gene are as follows:10.0 μ L of Rremix Ex TaqTM II,
ddH2O8 μ L, F-F 0.5 μ L, F-R 0.5 μ L, 1.0 μ L of ox musculature cDNA；
3) it being detected on quantitative PCR apparatus, fluorescence signal detects at the end of each circulation, 3 repetitions of each sample,
Calculate the expression quantity of ox FASN gene.
In the above-mentioned technical solutions, the sequence of the primer pair F-F and F-R are as follows:
F-F:5 '-ATCGAGTGCATCAGGCAAGT-3 ' is shown in sequence table SEQ NO.1.
F-R:5 '-TGTGAGCACATCTCGAAAGCCA-3 ' is shown in sequence table SEQ NO.2.
In the above-mentioned technical solutions, the sequence of the primer pair G-F and G-R are as follows:
G-F:5 '-CGTGTCTGTTGTGGATCTGACCTG-3 ' is shown in sequence table SEQ NO.3.
G-R:5 '-CAACCTGGTCCTCAGTGTAGCCT-3 ' is shown in sequence table SEQ NO.4.
In the above-mentioned technical solutions, the reference gene GAPDH is identical with the qPCR amplified reaction program of ox FASN gene,
Are as follows: 95 DEG C of initial denaturation 30sec, 95 DEG C of denaturation 30sec, 58 DEG C of annealing 1min, 72 DEG C of extension 20sec, 40 recycle.
In the above-mentioned technical solutions, the concentration of described G-F, G-R, F-F and F-R are 20 μm of ol/L.
The invention has the following advantages:
The present invention is expanded by design FASN gene-specific primer pair, the qPCR of internal control primer GAPDH, and to qPCR
As a result relative quantitative assay accurately and rapidly analyzes expression of the FASN gene in ox muscle.The present invention is established
Detection FASN gene expression dose qPCR detection method, can be used for accurately detect different breeds of cattle muscle FASN gene table
Up to level.The invention the method can be used for detecting the expression of different breeds of cattle muscle FASN gene, to further investigation ox
FASN gene function provides reference, and method is easy, easy to operate.
Specific embodiment
In order to enable those skilled in the art to better understand the solution of the present invention, combined with specific embodiments below furtherly
Bright technical solution of the present invention.
A method of detection ox muscle FASN gene expression amount, the specific steps are as follows:
1) specific primer of FASN gene order is designed to F-F and F-R and reference gene primer pair G-F and G-R:
The FASN gene order (Genbank Accession No.NM-001012669) included with reference to Genbank, internal reference
Gene GAPDH (Genbank Accession No.0NM-001034034), utilizes 3.0 software design of Primer Premier
Primer, and pass through Blast function in NCBI, the specificity of detection primer.
F-F:5 '-GTGAAGCCCATTGAGGACAT-3 ' is shown in sequence table SEQ NO.1.
F-R:5 '-AGCTGCACGTGTTCTGTCAC-3 ' is shown in sequence table SEQ NO.2.
The sequence of the primer pair G-F and G-R are as follows:
G-F:5 '-CGTGTCTGTTGTGGATCTGACCTG-3 ' is shown in sequence table SEQ NO.3.
G-R:5 '-CAACCTGGTCCTCAGTGTAGCCT-3 ' is shown in sequence table SEQ NO.4.
2) the musculature total serum IgE and synthetic tissue cDNA of ox to be measured are extracted；
Sample acquisition: 3 years old Mongolia Cattle and Simmental each 6, acquired after butchering on the left of each test ox trunk the 12nd to
Longissimus dorsi muscle at 13rd rib cage is put in -80 DEG C of refrigerators after liquid nitrogen frozen and saves backup.
The extraction of ox musculature total serum IgE uses the Total RNAs extraction box of Thermo Fisher Scientific company
(TRIzolTMReagent it) extracts, extracting method is referring to kit specification.The integrality of total serum IgE passes through agarose gel electrophoresis
Detection determines；Total rna concentration passes through the micro ultraviolet specrophotometer measurement of NanoDrop2000.Total serum IgE is reversed using Takara
It records kit (PrimeScriptTM RT Master Mix) and carries out reverse transcription into tissue cDNA.
3) using the musculature cDNA of ox to be measured as template, using the primer pair G-F and G-R described in 1) as primer, qPCR expands
Increase reference gene GAPDH；Using the musculature cDNA of ox to be measured as template, using the primer pair F-F and F-R described in 1) as primer,
QPCR expands ox FASN gene；
Wherein, the qPCR amplification reaction system of reference gene GAPDH are as follows:Rremix Ex TaqTM II
(Perfect ReaL Time) 10.0 μ L, ddH2O 8 μ L, G-F 0.5 μ L, G-R 0.5 μ L, the musculature cDNA1.0 μ of ox
L；
Ox FASN gene qPCR amplification reaction system are as follows:Rremix Ex TaqTM II 10.0 μ L, ddH2O8μ
L, F-F 0.5 μ L, F-R 0.5 μ L, 1.0 μ L of ox musculature cDNA；
Wherein, the concentration of G-F, G-R, F-F and F-R are 20 μm of ol/L；
Reference gene GAPDH is identical with the qPCR amplified reaction program of ox FASN gene, are as follows: 95 DEG C of initial denaturation 30sec,
95 DEG C of denaturation 30sec, 58 DEG C of annealing 1min, 72 DEG C of extension 20sec, 40 recycle.
Wherein, in order to ensure the accuracy of reference gene, G-F, G-R, F-F and F-R in above-mentioned two amplification reaction system
Concentration be 10 μm of ol/L；In above-mentioned two amplification reaction system,Rremix Ex TaqTM II(Perfect
ReaL Time) it is also identical, it is purchased from precious bioengineering (Dalian) Co., Ltd.
Reference gene GAPDH is identical with the qPCR amplified reaction program of ox FASN gene, are as follows: 95 DEG C of initial denaturation 30sec,
95 DEG C of denaturation 30sec, 58 DEG C of annealing 1min, 72 DEG C of extension 20sec, 40 recycle.
Wherein, according to the production of Takara companyRremix Ex TaqTM II(Perfect ReaL Time)
Fluorescence quantitative kit illustrates preparation reaction solution, the sterile no enzyme PCR pipe of 0.2mL is placed on ice, according to each reaction
The number of the reaction of PCR needed for the quantity of various components and 1 secondary response needed for system, calculates the total amount of various reactive components,
It is added separately in the sterile no enzyme PCR pipe of above-mentioned 0.2mL by each component plus according to the total amount of calculating, after the completion of configuration, sufficiently
It mixes, bubble in managing is removed, with centrifuge wink from primary, upper machine.Whole operation process is protected from light as far as possible.
4) qPCR's as a result, calculation expression amount in detection 3).
The mRNA relative expression quantity of target gene is calculated using 2- Δ Δ Ct value.
Δ Ct=Ct (ox FASN gene recurring number)-Ct (reference gene GAPDH recurring number)
Ct value: fluorescence signal reaches recurring number experienced when given threshold in reaction system.It can be calculated by measured value
The template quantity of gene starting out.Using house-keeping gene as reference gene, target gene is standardized by reference gene, it can
Determine the relative quantity of target gene transcriptional expression.Two kind muscle samples are carried out using 20.0 software of SPSS version
Between significance test of difference, using one-way analysis of variance, P≤0.05 indicates significant difference, and all results are with average value
It is indicated with standard deviation.The results are shown in Table 1.
1 N of muscle FASN gene mRNA relative expression quantity of table
The Q-PCR detection method of established FASN gene expression dose is confirmed through the foregoing embodiment, can be used for essence
The really expression of detection different breeds of cattle muscle FASN gene.The invention the method can be used for detecting the expression of FASN gene
Level provides reference to further investigation ox FASN gene function, and method is easy, easy to operate.
Illustrative description has been done to the present invention above, it should explanation, the case where not departing from core of the invention
Under, any simple deformation, modification or other skilled in the art can not spend the equivalent replacement of creative work equal
Fall into protection scope of the present invention.
Claims (5)
1. a kind of method for detecting ox muscle FASN gene expression amount, it is characterised in that: described method includes following steps:
1) total serum IgE is extracted from beef tissue to be detected, then cDNA sample is prepared by reverse transcription reaction；
2) the cDNA sample of preparation is carried out the real-time PCR of fluorescent quantitation to detect, the qPCR amplification reaction system of reference gene GAPDH
Are as follows:Rremix Ex TaqTM II 10.0 μ L, ddH28 0.5 0.5 μ L of μ L, G-R of μ L, G-F of O, the muscle groups of ox
Knit 1.0 μ L of cDNA；
The qPCR amplification reaction system of ox FASN gene are as follows:Rremix Ex TaqTM II 10.0 μ L, ddH28 μ L of O,
F-F 0.5 μ L, F-R 0.5 μ L, 1.0 μ L of ox musculature cDNA；
3) it is detected on quantitative PCR apparatus, fluorescence signal detects at the end of each circulation, 3 repetitions of each sample, calculates
The expression quantity of ox FASN gene.
2. a kind of method for detecting ox muscle FASN gene expression amount according to claim 1, it is characterised in that: described to draw
Sequence of the object to G-F and G-R are as follows:
G-F:5 '-CGTGTCTGTTGTGGATCTGACCTG-3 ',
G-R:5 '-CAACCTGGTCCTCAGTGTAGCCT-3 '.
3. a kind of method for detecting ox muscle FASN gene expression amount according to claim 1, it is characterised in that: described to draw
Sequence of the object to P-F and P-R are as follows:
F-F:5 '-ATCGAGTGCATCAGGCAAGT-3 ',
F-R:5 '-TGTGAGCACATCTCGAAAGCCA-3 '.
4. a kind of method for detecting ox muscle FASN gene expression amount according to claim 1, it is characterised in that: in described
It is identical with the qPCR amplified reaction program of ox FASN gene to join gene GAPDH, are as follows: 95 DEG C of initial denaturation 30sec, 95 DEG C of denaturation
30sec, 58 DEG C of annealing 1min, 72 DEG C of extension 20sec, 40 recycle.
5. a kind of method for detecting ox muscle FASN gene expression amount according to claim 1, it is characterised in that: described to draw
The concentration of object G-F, G-R, F-F and F-R are 20 μm of ol/L.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8124344B2 (en) * | 2006-09-07 | 2012-02-28 | National Livestock Breeding Center (Nlbc) Incorporated Administrative Agency | Method of determining an amount of fatty acid contents in bovine intramuscular fat on the basis of genotype of fatty acid synthase gene and method of determining goodness of eating quality of beef on the basis of the results thereof |
| KR20130102845A (en) * | 2012-03-08 | 2013-09-23 | 대한민국(농촌진흥청장) | Diagnosis method of meat quality using dna marker associated with marbling score in hanwoo |
| CN104232632A (en) * | 2014-09-16 | 2014-12-24 | 中国农业大学 | Application of molecular marker in detection of milk production character of milk cow |
| CN104232648A (en) * | 2014-09-19 | 2014-12-24 | 中国农业科学院北京畜牧兽医研究所 | Regulation factor of targeted FAS (fatty acid synthetase) and application thereof |
| CN105861721A (en) * | 2016-01-29 | 2016-08-17 | 广西大学 | Method for detecting triglyceride content in pig muscle based on gene FASN |
-
2019
- 2019-01-18 CN CN201910046863.5A patent/CN109652562A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8124344B2 (en) * | 2006-09-07 | 2012-02-28 | National Livestock Breeding Center (Nlbc) Incorporated Administrative Agency | Method of determining an amount of fatty acid contents in bovine intramuscular fat on the basis of genotype of fatty acid synthase gene and method of determining goodness of eating quality of beef on the basis of the results thereof |
| KR20130102845A (en) * | 2012-03-08 | 2013-09-23 | 대한민국(농촌진흥청장) | Diagnosis method of meat quality using dna marker associated with marbling score in hanwoo |
| CN104232632A (en) * | 2014-09-16 | 2014-12-24 | 中国农业大学 | Application of molecular marker in detection of milk production character of milk cow |
| CN104232648A (en) * | 2014-09-19 | 2014-12-24 | 中国农业科学院北京畜牧兽医研究所 | Regulation factor of targeted FAS (fatty acid synthetase) and application thereof |
| CN105861721A (en) * | 2016-01-29 | 2016-08-17 | 广西大学 | Method for detecting triglyceride content in pig muscle based on gene FASN |
Non-Patent Citations (1)
| Title |
|---|
| 赵称赫: ""蒙古牛肉品质及背最长肌脂肪代谢相关基因mRNA表达量的研究"", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Application publication date: 20190419 |