CN113862380A - Molecular marker related to pH of slaughtered meat of yak Wnt3a gene and application - Google Patents
Molecular marker related to pH of slaughtered meat of yak Wnt3a gene and application Download PDFInfo
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Abstract
The invention discloses a molecular marker level application of the pH correlation of slaughtered beef of a yak Wnt3a gene, wherein a yak consumption site marker is positioned in an NC-037334.1: 3057982, namely the 3057982 th position of 7 th chromosome of ARS-UCD1.2 cattle genome, the pH value of meat 45min after the AA genotype individual is slaughtered is higher than that of the AG genotype individual, the molecular marker can be used as a related molecular marker of meat quality characters of Sichuan yaks, provides basis for the molecular marker and auxiliary selection of the beef cattle, and is used for early breeding of high-quality cattle.
Description
Technical Field
The invention relates to the technical field of yak molecular breeding, in particular to a molecular marker related to the pH of slaughtered meat of a yak Wnt3a gene and application thereof.
Background
The yak lives in a high and cold area with an altitude of more than 3000m and has the excellent characteristics of extremely high adaptability, coarse feeding resistance, cold and fatigue resistance and the like in the high altitude area. The yak meat is green, safe and pollution-free, has rich nutritive value, and is rich in amino acids and polyunsaturated fatty acids necessary for human body. The number of the existing yaks in the world is about 1700 thousands, China is a main country of yaks, more than 1600 thousands of yaks exist in China, account for more than 90 percent of the world, and are mainly distributed in Tibet, Sichuan, Qinghai and other areas.
The yak meat is rich in protein, amino acid, carotene, calcium, phosphorus and other trace elements, has low fat content and high calorie, and has obvious effects of enhancing disease resistance, cell activity and organ functions of human bodies. The yak meat has extremely high nutritional value which is incomparable with other beef.
Currently, tenderness, flavor, flesh color, juiciness and the like are main evaluation indexes of people on meat quality and sensory performance. The meat color is the most intuitive evaluation standard for consumers, and the color, the freshness and the like of the meat are observed. For the measurement of the meat color in the meat quality property, it is generally measured by a colorimeter, and the meat color is represented by L × (brightness value), a × (red value), b × (yellow value). The flesh color depends on the content of myoglobin (Mb) in muscle, which is dark red, Mb and O after muscle dissection2Contact formation MbO2Bright red, Mbo over time2Further oxidation to denatured myoglobin, light brown. Therefore, the smaller the values of L and b, the larger the value of a, the better the flesh color. The pH of meat is mainly determined by glycolysis speed after slaughtering of animals, and the glycolysis speed is high, so that the lactic acid content is increased, and the pH value of muscle is reduced. The pH value of the meat quality can directly influence the tenderness, the meat color, the cooking loss and other properties, and is an important index for measuring the meat acidification. The pH value of the pre-slaughter beef is neutral, and the pH value is reduced along with the increase of the post-slaughter time, so that the color of the beef is poor, and the water holding capacity is reduced. Therefore, the change of pH value after slaughtering is also important for measuring meat quality traits.
The Wnt protein family is a secreted glycoprotein, and can regulate a signal transduction process mediated by a cell surface receptor, thereby regulating a series of cell behaviors such as cell differentiation, proliferation, migration and the like. The Wnt signaling pathway plays an important role in various biological processes such as growth and development, and can be divided into a canonical Wnt pathway and a non-canonical Wnt pathway, wherein the canonical Wnt pathway, i.e., a β -catenin dependent pathway, is a research hotspot in recent years.
Wnt proteins can be classified into Wnt1 class and Wnt5a class, and Wnt3a is a member of Wnt1 class proteins in the Wnt family, acting through a canonical Wnt signaling pathway, and can promote cell proliferation and differentiation. The research on Wnt3a gene mainly focuses on the aspects of normal cell development and tumor formation. The MTT method is used for observing the influence of Wnt3a on the proliferation capacity of the mesenchymal stem cells, and the result shows that Wnt3a can promote the proliferation of the mesenchymal stem cells and inhibit chondrogenic differentiation of the mesenchymal stem cells; the transgenic mouse is taken as an animal model, and the Wnt3a is proved to be capable of promoting the differentiation of melanocytes and the synthesis of melanin from the aspects of in vitro and in vivo; wnt3a can inhibit the apoptosis of porcine pancreatic stem cells by activating beta-catenin, which suggests that Wnt3a may be closely related to the self-renewal and anti-apoptosis of cells; the Wnt3a protein has obvious difference in positive expression rate between bladder cancer and normal bladder tissue, and the result shows that the Wnt3a protein may be related to the invasiveness of the bladder cancer.
The prior art discloses a method for detecting single nucleotide polymorphism of Qinchuan cattle Wnt7a gene and application thereof, which can be used as a molecular marker for improving weight, body length, chest width and cross height of Qinchuan cattle; and discloses a single nucleotide polymorphism site of cattle WNT10B gene and a detection method thereof, wherein the single nucleotide polymorphism site can be used as a molecular marker for cattle growth trait selection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a molecular marker related to the pH value of slaughtered meat of a yak Wnt3a gene and application thereof.
The first purpose of the invention is to provide a detection reagent for yak molecular marker genotype.
The second purpose of the invention is to provide the application of the detection reagent in detecting and/or evaluating the pH value character of meat of cattle 45min after slaughtering.
The third purpose of the invention is to provide the application of the detection reagent in the preparation of a kit for detecting and/or evaluating the pH value character of meat of cattle 45min after slaughtering.
The fourth purpose of the invention is to provide a method for detecting, and/or evaluating the pH value character of meat of 45min after slaughtering of a yak.
The fifth purpose of the invention is to provide a kit for detecting, and/or evaluating the pH value character of meat of cattle 45min after slaughtering.
The sixth purpose of the invention is to provide the detection reagent, the method and/or the application of the kit in yak molecule breeding.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention adopts DNA sequencing technology to research polymorphism of Sichuan yak Wnt3a gene, and uses SPSS 23.0 software to analyze the correlation between the polymorphism and meat quality character, and Wnt3a gene has mutation site I2-25716A in Sichuan yak>G, and pH of AA genotype individuals45minIs significantly higher than that of AG genotype individuals (P)<0.05). I2-25716A indicating Wnt3a gene>The G locus can be used as a related molecular marker of meat quality traits of the Sichuan yaks, provides a theoretical basis for early breeding of high-quality beef cattle, and lays a foundation for constructing a core cattle group with high-quality meat quality traits.
Therefore, the invention claims a detection reagent for yak molecular marker genotype, wherein the yak molecular marker is located in NC-037334.1: 3057982, position 3057982 of chromosome 7 of cattle genome of ARS-UCD version 1.2, beef pH value 45min after slaughtering of individual AA genotype with different genotypes is higher than that of AG genotype individual.
And the application of the detection reagent in detecting and/or evaluating the pH value character of 45min beef after slaughtering of a yak.
And the application of the detection reagent in preparing a kit for detecting and/or evaluating the pH value character of 45min beef after slaughtering of a yak.
The invention also claims a method for detecting and/or evaluating the pH value character of 45min beef after slaughtering yaks, which detects the genotype of yak molecular markers, wherein the yak molecular markers are located at NC-037334.1: 3057982, position 3057982 of chromosome 7 of cattle genome of ARS-UCD version 1.2, beef pH value 45min after slaughtering of individual AA genotype with different genotypes is higher than that of AG genotype individual.
Preferably, the genotype of the yak molecular marker is detected by using a primer with a nucleotide sequence shown as SEQ ID NO. 1-2.
More preferably, the PCR amplification system (total volume 20. mu.L): taq PCR Mix 10 uL, primer 0.4 uL with nucleotide sequence shown in SEQ ID NO 1-2, DNA template 2 uL, ddH2O 7.2μL。
More preferably, the PCR amplification procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 40s, annealing at 57 ℃ for 40s, and extension at 72 ℃ for 1min for 34 cycles; further extension was carried out at 72 ℃ for 10 min.
The invention also claims a kit for detecting and/or evaluating the pH value character of 45min beef after slaughtering of a yak, which contains the detection reagent.
Preferably, the detection reagent is a primer with a nucleotide sequence shown as SEQ ID NO 1-2.
F：5′-TTGGTCTAACGGGAAAGGG-3′(SEQ ID NO:1)；
R：5′-TGGAGGTCAAGAGTGGGTGT-3′(SEQ ID NO:2)。
Preferably, PCR amplification reagents are also contained.
More preferably, the PCR amplification reagent is Taq PCR Mix.
More preferably, the PCR amplification system (total volume 20. mu.L): taq PCR Mix 10 uL, primer 0.4 uL with nucleotide sequence shown in SEQ ID NO 1-2, DNA template 2 uL, ddH2O 7.2μL。
More preferably, the PCR amplification procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 40s, annealing at 57 ℃ for 40s, and extension at 72 ℃ for 1min for 34 cycles; further extension was carried out at 72 ℃ for 10 min.
The detection reagent, the method and/or the kit are/is applied to yak molecular breeding, wherein the yak molecular breeding refers to breeding of beef pH value characters 45min after yak slaughtering.
Preferably, the yak is yak consumed in Sichuan.
Compared with the prior art, the invention has the following beneficial effects:
wnt3a gene has mutation site I2-25716A in Sichuan yaks>G, located at NC _ 037334.1: 3057982, position 3057982 of chromosome 7 of the cattle genome version 1.2 of ARS-UCD, which has different genotypes, pH of AA genotype individuals45minIs significantly higher than that of AG genotype individuals (P)<0.05). I2-25716A indicating Wnt3a gene>The G locus can be used as a related molecular marker of meat quality traits of the Sichuan yaks, provides a basis for molecular marker and auxiliary selection of the beef cattle, and is used for early breeding of high-quality cattle.
Drawings
FIG. 1 shows the PCR amplification result of Sichuan yak Wnt3a gene; m: DL-2000 Marker; 1-6: PCR amplification product of Wnt3a gene.
FIG. 2 is the sequencing map of Wnt3a gene I2-25693C > T site 3 genotypes.
FIG. 3 is the sequencing map of Wnt3a gene I2-25709T > C site 3 genotypes.
FIG. 4 is a sequencing map of Wnt3a gene I2-25716G > A site 2 genotypes
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 amplification of polymorphism of Wnt3a Gene
First, experimental material
The selected 102 Sichuan yaks are raised in a certain cattle farm in Alba, Sichuan under the same raising conditions, jugular venous blood at the same part of each cattle is collected, treated by an anticoagulant and stored in a refrigerator at the temperature of-80 ℃.
Second, Experimental methods
1. DNA extraction
Extracting DNA of a blood sample of the Sichuan yak by using the blood genome DNA extraction kit according to the instruction, detecting the concentration and purity of the DNA by using a micro spectrophotometer, and detecting the quality of the DNA by agarose gel electrophoresis.
2. Primer design
Based on the bovine Wnt3a gene sequence (ENSBTAT00000078373.1) published in Ensembl, 1 pair of primers was designed and synthesized by Biotechnology engineering (Shanghai) GmbH. The primer sequence is as follows:
F：5′-TTGGTCTAACGGGAAAGGG-3′(SEQ ID NO:1)；
R：5′-TGGAGGTCAAGAGTGGGTGT-3′(SEQ ID NO:2)，
3. PCR amplification and product sequencing
Carrying out PCR amplification by using DNA of the blood of the Sichuan yaks as a template. PCR amplification system (total volume 20. mu.L): taq PCR Mix 10. mu.L, upstream and downstream primers 0.4. mu.L each, DNA template 2. mu.L, ddH2O7.2. mu.L. PCR amplification procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 40s, annealing at 57 ℃ for 40s, and extension at 72 ℃ for 1min for 34 cycles; further extension was carried out at 72 ℃ for 10 min. After the PCR amplification product is detected by 1.5% agarose gel electrophoresis and the target band is confirmed to be correct, the PCR amplification product is sent to the company of Biotechnology engineering (Shanghai) Ltd for sequencing.
Third, experimental results
As shown in FIG. 1, 3 SNPs were found in intron 2 of Wnt3a gene, I2-25693C > T (SNP site 1), I2-25709T > C (SNP site 2), and I2-25716G > A (SNP site 3). The different genotype maps are shown in FIGS. 2-4.
Example 2 analysis of population genetic characteristics of 3 SNPs of Wnt3a Gene
First, experiment method
Calculating the gene frequency, genotype frequency, genetic heterozygosity (H), effective allele factor (Ne) of the allele using Popgene 32 software; polymorphic Information Content (PIC) was calculated using PIC 0.6 software
Second, experimental results
The population genetic characteristics of 3 SNPs sites of the Wnt3a gene are shown in Table 1, and the results show that: I2-25693C>T (SNP site 1), I2-25709T>C (SNP site 2), I2-25716G>The dominant alleles at the A (SNP site 3) site are C, T, A respectively, and the dominant alleles are CT, CT and AA respectively. I2-25693C>T (SNP site 1), I2-25709T>The gene heterozygosity (H) and the Polymorphic Information Content (PIC) of the C (SNP site 2) site are both between 0.25 and 0.50, which shows that 2 SNPs sites are in moderate polymorphism in the Wnt3a gene, I2-25716A>The H and PIC at the G site were less than 0.5, indicating that the site is of low polymorphism. Chi shape2The test shows that 3 SNPs sites are in Hardy-Weinberg equilibrium state (P) in the Sichuan yak population>0.05)。
TABLE 1 analysis of the population genetic characteristics of 3 SNPs of Wnt3a Gene
Note: chi shape2Values representing no significance (P)>0.05)，χ2 0.05(df＝2)＝5.99，χ2 0.01(df＝2)＝9.21。
Example 3 correlation analysis of different genotypes of 3 SNPs sites of Wnt3a gene and quality traits of Sichuan yak meat
First, experiment method
The data of the quality character of the Sichuan yak meat in example 1 were collected, and the following methods were used to determine the quality character of the meat:
(1) determination of flesh color: after 102 Sichuan yaks are slaughtered, the longissimus dorsi samples are placed in a refrigerator at 4 ℃ for storage, and after 24 hours, the chromaticity instrument is used for measuring three parameters of the longissimus dorsi, such as brightness (L), redness (a), yellowness (b) and the like. Each sample was measured three times and averaged for recording.
(2) Determination of pH: the eye muscles of 102 Sichuan yaks after slaughter are stored in a refrigerator at 4 ℃, and the pH value of the eye muscles is measured by a pH meter at 45min and 24h after slaughter respectively. Three measurements were made for each sample, averaged and recorded, and the data was collated.
And (3) carrying out difference significance test on the relation between the quality characters and the genotypes of the 102 Sichuan yak meat by using multiple comparisons in the single-factor variance of SPSS 23.0 software, and taking P <0.05 as a judgment standard of difference significance.
Second, experimental results
The correlation analysis of 3 SNPs sites of the Sichuan yak Wnt3a gene and the meat quality traits is shown in Table 2. As can be seen from the table: I2-25716A>pH of G site AA genotype individuals45minThe value is significantly higher than that of AG genotype individuals (P)<0.05)。I2-25693 C>T、I2-25709 T>The difference between the individuals with different genotypes at the C locus and the meat quality traits such as pH, meat color and the like is not obvious (P)>0.05)。
TABLE 2 correlation analysis results of different genotypes of 3 SNPs sites of Wnt3a gene and quality traits of Sichuan yak meat
Example 4 detection and/or evaluation method of pH value trait of 45min beef of Szechwan yak after slaughter
1. Extraction of DNA
And extracting sample DNA.
2. PCR amplification and product sequencing
And carrying out PCR amplification by using the sample DNA as a template.
PCR amplification system (total volume 20. mu.L): taq PCR Mix 10. mu.L, upstream and downstream primers with nucleotide sequences shown in SEQ ID NO 1-2 0.4. mu.L each, DNA template 2. mu.L, ddH2O 7.2μL。
PCR amplification procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 40s, annealing at 57 ℃ for 40s, and extension at 72 ℃ for 1min for 34 cycles; further extension was carried out at 72 ℃ for 10 min.
After the PCR amplification product is detected by 1.5% agarose gel electrophoresis and the target band is confirmed to be correct, the PCR amplification product is sent to the company of Biotechnology engineering (Shanghai) Ltd for sequencing.
Third, interpretation of results
The sequencing results were aligned to the ARS-UCD version 1.2 bovine genome for NC _ 037334.1: 3057982, the 3057982 th position of the 7 th chromosome of the cattle genome of ARS-UCD1.2, wherein in different genotypes, the pH value of beef is higher than that of AG genotype individuals 45min after the AA genotype individuals are slaughtered.
Example 5A kit for detecting, and/or evaluating the pH value trait of 45min post-slaughter beef of a Szechwan yak
A, make up
Upstream and downstream primers with nucleotide sequences shown as SEQ ID NO 1-2, Taq PCR Mix and ddH2O。
Second, use method
The same as in example 4.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Sequence listing
<110> Guangdong ocean university
Molecular marker related to pH of slaughtered meat of <120> yak Wnt3a gene and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttggtctaac gggaaaggg 19
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tggaggtcaa gagtgggtgt 20
Claims (10)
1. A detection reagent for the genotype of a yak molecular marker, which is characterized in that the yak molecular marker is located in NC-037334.1: 3057982, position 3057982 of chromosome 7 of the cattle genome of ARS-UCD version 1.2, for individuals with different genotypes, the beef pH value 45min after slaughter of an AA genotype individual is higher than that of an AG genotype individual.
2. The use of the detection reagent of claim 1 for detecting, and/or assessing the pH trait of 45min beef after slaughter of a yak.
3. Use of the detection reagent of claim 1 in the preparation of a kit for detecting and/or evaluating the pH value of beef from 45min after slaughtering of a yak.
4. A method for detecting and/or evaluating pH value characters of beef of 45min after slaughtering of yaks is characterized by detecting genotypes of yak molecular markers, wherein the yak molecular markers are located in NC-037334.1: 3057982, position 3057982 of chromosome 7 of the cattle genome of ARS-UCD version 1.2, for individuals with different genotypes, the beef pH value 45min after slaughter of an AA genotype individual is higher than that of an AG genotype individual.
5. The method according to claim 4, characterized in that the genotype of the yak molecular marker is detected by using a primer with a nucleotide sequence shown as SEQ ID NO. 1-2.
6. A kit for detecting and/or evaluating the pH value character of beef 45min after slaughtering of yaks, which is characterized by comprising the detection reagent according to claim 1.
7. The kit according to claim 6, wherein the detection reagent is a primer having a nucleotide sequence shown in SEQ ID NO. 1-2.
8. The kit of claim 6, further comprising PCR amplification reagents.
9. The kit of claim 8, wherein the PCR amplification reagent is Taq PCR Mix.
10. The use of the detection reagent of claim 1, the method of claim 4, and/or the kit of claim 6 in the breeding of yak molecules, wherein the breeding of yak molecules is the breeding of the pH trait of beef 45min after slaughtering of yaks.
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