CN110468213A - A kind of black chicken inosinicacid of gold thatch and the relevant molecular labeling of intramuscular fat content and application - Google Patents

A kind of black chicken inosinicacid of gold thatch and the relevant molecular labeling of intramuscular fat content and application Download PDF

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CN110468213A
CN110468213A CN201910699521.3A CN201910699521A CN110468213A CN 110468213 A CN110468213 A CN 110468213A CN 201910699521 A CN201910699521 A CN 201910699521A CN 110468213 A CN110468213 A CN 110468213A
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chicken
inosinicacid
fat content
thatch
molecular labeling
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CN110468213B (en
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张涛
陈兰
王金玉
谢凯舟
张跟喜
戴国俊
巩用双
张闪闪
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Yangzhou University
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Abstract

The present invention relates to field of poultry breeding, it is related to a kind of black chicken inosinicacid of golden thatch and the relevant molecular labeling of intramuscular fat content and application.The molecular labeling is located at chicken GAS6 gene 27463bp, is denoted as g.C27463T.The invention also discloses application of the above-mentioned molecular labeling in the black chicken breeding of golden thatch, and method is simple and fast, and free from the influence of the external environment, provide reference for the black chicken inosinicacid of golden thatch and the relevant molecular breeding of intramuscular fat content.

Description

A kind of black chicken inosinicacid of gold thatch and the relevant molecular labeling of intramuscular fat content and application
Technical field
The present invention relates to field of poultry breeding, and in particular to one kind is based on GAS6 (i.e. growth retardation specific gene 6) gene Molecular labeling relevant to the black chicken inosinicacid of golden thatch and intramuscular fat content and application.
Background technique
Currently, chicken has become the second largest consumption of meat product that China is only second to pork.Broiler chicken is after genetic improvement, body Weight gain, feed conversion rate, the speed of growth and chest muscle dramatically increase again, this selection course has produced modern commerce chicken strain, Has the characteristics that the high broiler chicken strain of Seedling height speed, breast meat yield.However, the excessive pursuit to the speed of growth, leads to meat There is abdominal fat over-deposit and the lipopenic phenomenon of intramuscular fat in chicken.Studies have shown that inosinicacid and intramuscular fat content with The flavor of chicken, tenderness are closely related, influence chicken meat quality.Currently, increase inosinicacid and intramuscular fat content have become meat One of main target of chicken breeding.
Meat Quality is complicated quantitative character, and by all polygenic control, traditional breeding way is difficult to take Meat Quality Obtain biggish genetic progress.SNPs is as third generation molecular labeling, and application prospect is extensively sent out in animal breeding, find and identify with Chicken inosinicacid and the relevant SNPs of intramuscular fat content, and assisted Selection is marked using it, can speed up chicken inosinicacid and The genetic improvement speed of intramuscular fat content character.
Growth retardation specific gene products 6 (growth arrest-specific gene 6, GAS6), at first by Nagata etc. has found and clones in the NIH3T3 cell of serum free medium culture, and the albumen of gene coding is by 678 The framework albumen of Amino acid profile is the ligand of TAM receptor in receptor tyrosine kinase family, has extensive biology effect It answers.Studies have shown that GAS6 gene differential expression in chicken Preadipocyte atomization, closely related with chicken fat deposition.
However still lacks improve the black chicken inosinicacid of golden thatch and intramuscular fat content using the molecular labeling of GAS6 gene at present Technology.
Summary of the invention
In order to overcome drawbacks described above, the technical problem to be solved in the present invention is to provide the black chicken inosinicacids of a kind of golden thatch and intramuscular The relevant molecular labeling of fat content and application improve the golden black chicken inosinicacid of thatch and intramuscular rouge using GAS6 gene molecule marker Fat content.
To solve the above problems, the invention discloses a kind of based on GAS6 gene with the black chicken inosinicacid of golden thatch and intramuscular rouge The relevant molecular labeling of fat content, the molecular labeling are located at chicken GAS6 gene 27463bp, are denoted as g.C27463T.Chicken Genotype at GAS6 gene 27463bp is TT or CC or CT.
It is above-mentioned to the black chicken inosinicacid of golden thatch and relevant point of intramuscular fat content for detecting that the invention also discloses one kind The primer sets of son label, the primer sets include one group of GAS6-P1 primer pair,
The nucleic acid sequence of GAS6-P1 primer pair are as follows:
Upstream primer F:5 '-GACACCTGTGCGGTCAGACT-3 ', as shown in SEQ ID NO.1；
Downstream primer R:5 '-TCACTGTCTGCCACATGCCA-3 ', as shown in SEQ ID NO.2.
The invention also discloses a kind of above-mentioned molecule marks relevant to the black chicken inosinicacid of golden thatch and intramuscular fat content of detection The method of note, which comprises using chicken genomic DNA as template, PCR amplification is carried out using above-mentioned specific primer group, Obtain PCR product, PCR product parting after sscp analysis.
PCR amplification system is by chicken DNA profiling, upstream primer F, downstream primer R, ddH2O, Taq polymerase, dNTPs, MgCl2, reaction buffer composition.Optionally, PCR amplification system are as follows: 2 × Taq Master Mix for Page 10 μ L, DNA 1 μ L of template, 0.8 μ L of upstream primer, downstream primer 0.8 μ L, ddH2O 7.4μL；Taq Master Mix for Page includes Taq polymerase, dNTPs, MgCl2, reaction buffer.
Optionally, PCR amplification program is 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 61 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.
SSCP response procedures are as follows: take PCR product and sample-loading buffer to mix, be first denaturalized, then rapid ice bath, be in DNA Single-chain state；The PCR product being denaturalized is taken to carry out native polyacrylamide gel electrophoresis, then silver staining colour developing.More specifically, SSCP response procedures are as follows: take 2.5 μ LPCR products and 7.5 μ L sample-loading buffers (98% deionized formamide, 0.025% bromine phenol Blue, 0.025% dimethylbenzene FF, 10mmol/LEDTA (pH8.0), 2% glycerol) mixing, first 98 DEG C of denaturation 10min, then rapid ice 10min is bathed, DNA is made to be in single-chain state.The 8 μ L of PCR product being denaturalized is taken to carry out non-denaturing polyacrylamide gel (PAGE30%, Acr: Bis=29: 1) electrophoresis, after 220V voltage prerunning 10min, 120V electrophoresis 10-12h, silver staining is aobvious Color.
The invention also discloses it is a kind of it is above-mentioned with the molecular labeling of the black chicken inosinicacid of golden thatch and intramuscular fat content in chicken Application in breeding or the black chicken inosinicacid of golden thatch and intramuscular fat content improvement.
Compared with prior art, the present invention can obtain following technical effect:
GAS6 gene at 27463bp there are one at mutational site, mutational site and chicken inosinicacid and intramuscular rouge at this The significant correlation of fat content, and it is easy detection.Therefore, the present invention provides one kind based on GAS6 gene and chicken inosinicacid and intramuscular rouge The relevant molecular labeling of fat content, and provide the detection method of this molecular labeling and its contain in chicken inosinicacid and intramuscular fat Application in amount improvement provides a kind of new method for the molecular mark of chicken Meat Quality.
Detailed description of the invention
Fig. 1 is mutation polyacrylamide gel electrophoresis figure, i.e. GAS6 gene P1 at chicken GAS6 gene 27463bp of the present invention The PCR-SSCP electrophoretogram of product (i.e. GAS6-P1 primer sets expand PCR product)；
Fig. 2 is mutation sequencer map, the i.e. sequence of GAS6 gene C C, TT and CT at chicken GAS6 gene 27463bp of the present invention Compare；
Fig. 3 is the nucleotide fragments that GAS6-P1 primer sets of the present invention expand, and underscore is primer sequence, at box It is mutational site.
Specific embodiment
Carry out the specific embodiment that the present invention will be described in detail below by way of specific example.
Jiangsu Suntory animal husbandry Development Co., Ltd is collected with a batch of 120 112 black chickens of age in days gold thatch, acquires blood Sample extracts genomic DNA, is dissolved in TE, -20 DEG C of preservations are standby after NANODROP1000 nucleic acid concentration analyzer measures concentration and purity With.
According to the GAS6 gene order (accession number are as follows: NC_006088.5) delivered in GenBank, using Primer Premier 5.0 designs 1 pair of primer.Amplified production size is 211bp.Primer sequence is as follows:
GAS6-P1: upstream primer: 5 '-GACACCTGTGCGGTCAGACT-3 ' (SEQ ID NO:1)
Downstream primer: 5 '-TCACTGTCTGCCACATGCCA-3 ' (SEQ ID NO:2)
Using above-mentioned primer sets, PCR amplification, PCR reaction system are as follows: 2 × Master are carried out by template of chicken genomic DNA 10 μ L of Mix for Page, 0.8 μ L of upstream primer, downstream primer 0.8 μ L, DNA profiling μ L, ddH2O 7.4μL.PCR reacts item Part: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 61 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 recycle；72 DEG C of extension 7min, 4 DEG C save.
Take 2.5 μ L PCR products and 7.5 μ L sample-loading buffers (98% deionized formamide, 0.025% bromophenol blue, 0.025% dimethylbenzene FF, 10mmol/LEDTA (pH8.0), 2% glycerol) mixing, first 98 DEG C of denaturation 10min, then rapid ice bath 10min makes DNA be in single-chain state.The 8 μ L of PCR product being denaturalized is taken to carry out non-denaturing polyacrylamide gel (PAGE30%, Acr: Bis=29: 1) electrophoresis, after 220V voltage prerunning 10min, 120V electrophoresis 10-12h, silver staining is aobvious Color, discovery of taking pictures are respectively designated as CC, TT and CT genotype (Fig. 1) there are three kinds of different banding patterns.
Every kind of genotype takes 3 samples that Shanghai Sangon Biological Engineering Technology And Service Co., Ltd is transferred to be sequenced.It surveys Sequence result and GAS6 gene original sequence (GenBankID:NC_006088.5), which compare, to be found, at 27463bp there are one at C → T mutation, is denoted as g.C27463T (Fig. 2).
Genetic Variation Analysis is carried out to 3 kinds of genotype of GAS6 gene, calculates separately out each gene frequency and genotype Frequency, the results show that C and T gene frequency is respectively 0.39 and 0.61, CC, CT and TT genotype frequency are respectively 0.26,0.26 It is 0.543 with the mutational site 0.48, g.C27463T heterozygosity, polymorphism information content (Polymorphism Information Content, PIC) value be 0.352, effective number of allele 1.84.
To establish the related of the mutational site gene g.C27463T GAS6 and the black chicken inosinicacid of Jin Mao and intramuscular fat content Property, 120 112 black chickens of age in days gold thatch, slaughter determining inosinicacid and thick rouge are collected by Yu Jiangsu Suntory animal husbandry Development Co., Ltd Fat content analyzes data, will using the general linear model (general linear model, GLM) in 22.0 software of SPSS The mutational site gene g.C27463T GAS6 and inosinicacid and intramuscular fat content are associated analysis, as a result with average value ± Standard deviation indicates.
As shown in Table 1, CT genotype individuals inosine acid content is higher than TT genotype individuals, is significantly higher than CC genotype individuals (P < 0.05), CT genotype individuals crude fat content be higher than TT genotype individuals, it is extremely significant be higher than CC genotype individuals (P < 0.01).In conclusion CT genotype is preponderant genotype of the present invention, in the black chicken Breeding Process of golden thatch, by selecting and remain CT gene Type individual improves the black chicken inosinicacid of golden thatch and intramuscular fat content, can improving pork quality character.
The association analysis of 1 GAS6 gene g.C27463T of table mutation and the black chicken Meat Quality of golden thatch
Note: colleague's shoulder mark difference lowercase indicates significant difference (P < 0.05), and not marking-up is female or same letter indicates poor Different not significant (P > 0.05)
The above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited merely to above-described embodiment, All technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It should be pointed out that for the art For those of ordinary skill, several improvements and modifications without departing from the principles of the present invention should be regarded as protection of the invention Range.
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Claims (8)

1. a kind of molecular labeling relevant to the black chicken inosinicacid of golden thatch and intramuscular fat content based on chicken GAS6 gene, feature It is, the molecular labeling is located at chicken GAS6 gene 27463bp, is denoted as g.C27463T.
2. according to claim 1 a kind of based on chicken GAS6 gene with the black chicken inosinicacid of golden thatch and intramuscular fat content phase The molecular labeling of pass, which is characterized in that the genotype at chicken GAS6 gene 27463bp is TT or CC or CT.
3. molecular labeling relevant to the black chicken inosinicacid of golden thatch and intramuscular fat content described in being used to detect as claimed in claim 1 or 22 Primer sets, which is characterized in that the primer sets include one group of GAS6-P1 primer pair,
The nucleic acid sequence of GAS6-P1 primer pair are as follows:
Upstream primer F:5 '-GACACCTGTGCGGTCAGACT-3 ', as shown in SEQ ID NO.1；
Downstream primer R:5 '-TCACTGTCTGCCACATGCCA-3 ', as shown in SEQ ID NO.2.
4. the kit containing primer sets described in claim 3.
5. it is a kind of detect as claimed in claim 1 or 22 described in molecular labeling relevant to the black chicken inosinicacid of golden thatch and intramuscular fat content Method, which is characterized in that the described method includes: utilizing primer sets as claimed in claim 3 using chicken genomic DNA as template PCR amplification is carried out, PCR product, PCR product parting after sscp analysis are obtained.
6. according to the method described in claim 5, it is characterized in that, PCR amplification system by chicken DNA profiling, upstream primer F, under Swim primer R, ddH2O, Taq polymerase, dNTPs, MgCl2, reaction buffer composition.
7. according to the method described in claim 5, it is characterized in that, PCR amplification program are as follows: 95 DEG C of 5 min of initial denaturation；95℃ It is denaturalized 30s, 61 DEG C of annealing 30s, 72 DEG C of extension 30s, 30 recycle；72 DEG C of 10 min of extension, 4 DEG C of preservations.
8. molecular labeling relevant to the black chicken inosinicacid of golden thatch and intramuscular fat content of any of claims 1 or 2 is in chicken breeding Or the application in chicken inosinicacid and intramuscular fat content improvement.
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Inventor after: Zhang Tao

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Inventor after: Gong Yongshuang

Inventor after: Zhang Shanshan

Inventor before: Zhang Tao

Inventor before: Chen Lan

Inventor before: Wang Jinyu

Inventor before: Xie Kaizhou

Inventor before: Zhang Genxi

Inventor before: Dai Guojun

Inventor before: Gong Yongshuang

Inventor before: Zhang Shanshan