CN118166118A - ANKRD50 gene as molecular marker of Tibetan sheep immune trait and application thereof - Google Patents

ANKRD50 gene as molecular marker of Tibetan sheep immune trait and application thereof Download PDF

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CN118166118A
CN118166118A CN202410434627.1A CN202410434627A CN118166118A CN 118166118 A CN118166118 A CN 118166118A CN 202410434627 A CN202410434627 A CN 202410434627A CN 118166118 A CN118166118 A CN 118166118A
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tibetan sheep
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ankrd50
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袁超
卢曾奎
刘建斌
郭婷婷
安雪姣
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Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
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Abstract

The invention relates to the technical field of molecular markers, in particular to a molecular marker of ANKRD50 gene as Tibetan sheep immune trait and application thereof. The ANKRD50 gene is used as a molecular marker of Tibetan sheep immune traits, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and y at 148 th position of the sequence represents T or C, so that the T/C polymorphism of the Tibetan sheep ANKRD50 gene at the position is caused. The invention can judge the immunoglobulin IgA, igG, igM content of Tibetan sheep individuals by detecting the base of 32776109 nucleotide locus on Tibetan sheep chromosome 17, and provides a new SNP molecular marker resource for the auxiliary selection of Tibetan sheep immune character markers for non-diagnostic purposes.

Description

ANKRD50 gene as molecular marker of Tibetan sheep immune trait and application thereof
Technical Field
The invention relates to the technical field of molecular markers, in particular to a molecular marker of ANKRD50 gene as Tibetan sheep immune trait and application thereof.
Background
In the sheep industry, the disease severely threatens the health of sheep and causes great economic loss. Although diseases can be prevented by enhancing the measures of raising management, using drug vaccines and the like, the epidemic of infectious diseases cannot be effectively controlled, and meanwhile, a large amount of drugs can cause drug resistance to animal organisms, so that hidden danger is caused to the safety of animal products.
In the long term, from the genetic basic research of disease resistance, the resistance genes are screened, the disease resistance breeding is carried out from the molecular level, the resistance of sheep to pathogens is improved from the genetic aspect, and the immunity is enhanced, so that the method is an important way for fundamentally solving the problem. The potential huge economic benefit of disease-resistant breeding and the attractive prospect of researching animal models of human diseases can be achieved, and the development of the work is being promoted vigorously. With the development of molecular biology, molecular genetics and genetic engineering techniques, disease-resistant breeding has begun to play an important role in sheep breeding.
Tibetan sheep live in Qinghai-Tibet plateau for a long time, have severe environments and need higher disease resistance to adapt to the environments. Therefore, the disease resistance of Tibetan sheep is improved, and the Tibetan sheep has stronger adaptability to severe natural environments such as high altitude, low air pressure, strong ultraviolet rays, hypoxia, cold season nutritional stress and the like.
The immune index may reflect disease resistance of the animal. The disease resistance of animals is largely dependent on the health and ability of their immune system. The primary function of the immune system is to recognize and eliminate pathogens, such as bacteria, viruses, etc., that invade the body. When a pathogen invades, the immune system reacts rapidly, initiating a series of immune response processes to fight and clear the pathogen.
Immune indicators generally include antibody levels, immune cell numbers, function, and the like. Antibodies are protein molecules produced by B cells that specifically bind to and neutralize pathogens, enhancing the effects of immune responses. The number and function of immune cells are also important indicators reflecting the state of the immune system. When the immune system is challenged, immune cells are activated and begin to proliferate to cope with pathogen invasion. These cells will differentiate further into more effector cells, such as macrophages and cytotoxic T cells, etc., to destroy the pathogen.
In addition, the disease resistance of animals is also affected by genetic factors, environmental factors, nutritional status, age, and the like. For example, genetic factors can affect the structure and function of the animal's immune system, and environmental conditions and nutritional status can affect the health and responsiveness of the immune system.
Therefore, the immune index can be used as an important reference to evaluate the disease resistance and health condition of animals. By monitoring the change of immune indexes, the immune problem of animals can be found in time, and corresponding measures are taken to improve the disease resistance of the animals and prevent and treat diseases.
Therefore, how to find the mutation site in the gene from the gene level in Tibetan sheep breeding by means of genetics, genetic engineering technology and the like, and discover the relation between the gene and the character through the association analysis between the mutation site and the character for early selection, thereby improving the seed selection efficiency and accuracy and having wide application value.
Disclosure of Invention
The invention aims at providing an ANKRD50 gene as a molecular marker of Tibetan sheep immune traits and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an ANKRD50 gene as a molecular marker of Tibetan sheep immune traits, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and y at 148 th position of the sequence represents T or C, so that T/C polymorphism of the Tibetan sheep ANKRD50 gene at the position is caused.
The invention also provides a primer pair for detecting the molecular marker, and the nucleotide sequences of the primer pair are shown in SEQ ID NO.2 and SEQ ID NO. 3.
The invention also provides a method for detecting the molecular marker of the immune trait of Tibetan sheep, which comprises the following steps:
(1) Extracting Tibetan sheep genome DNA;
(2) Amplifying the genomic DNA of step (1) using the primer pair;
(3) And (3) carrying out typing identification on the polymorphic site of the amplification product obtained in the step (2).
Preferably, the amplification system of step (2) is: 22. Mu.L of PCR premix, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer and 1. Mu.L of template.
Preferably, the PCR premix is gold medal Mix (green).
Preferably, the amplification in step (2) is performed by the following steps: 98 ℃ for 2min;98 ℃ for 10s,57.5 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
The invention also provides application of the molecular marker or the primer pair or the method in-vitro detection of the Tibetan sheep immune trait for non-diagnosis purpose.
The invention also provides application of the molecular marker or the primer pair or the method in Tibetan sheep auxiliary breeding.
Preferably, the application is to screen Tibetan sheep varieties with high immunity.
Compared with the prior art, the invention has the following beneficial effects:
The invention can judge the immunoglobulin IgA, igG, igM content of Tibetan sheep individuals by detecting the base of 32776109 nucleotide locus on Tibetan sheep chromosome 17, and provides a new SNP molecular marker resource for the auxiliary selection of Tibetan sheep immune character markers for non-diagnostic purposes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows PCR amplification products; wherein M represents Marker; 1. 2, 3 represent 3 sets of replicates.
FIG. 2 shows the peak pattern and sequence obtained after purification and sequencing of PCR products.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1 Sample collection
The sample is from Tibetan sheep group under natural grazing condition, wherein Gansu Gannan Tibetan autonomous state 40 parts, qinghai Ganya Jade Tree 45 parts, tibet autonomous region day click market 87 parts, collect 172 empty Tibetan sheep blood sample 5mL in clean procoagulant vacuum blood collection tube, stand for 30min, then 3500r/min centrifuge for 15min, suck supernatant in clean PE tube, seal and store in-20deg.C low temperature refrigerator; and collecting 5mL of blood sample in a blood collection tube added with EDTA-K2 anticoagulant, quickly and uniformly mixing the blood sample after the blood sample is collected, temporarily storing the blood sample in a sampling box containing an ice bag, and carrying the blood sample back to a laboratory for freezing and preserving the blood sample in a refrigerator at the temperature of minus 20 ℃ for DNA extraction.
2 Main reagents and instruments
EDTA-K2 vacuum blood collection tubes were purchased from Jiangsu Yuli medical instruments Co., ltd; blood genome extraction kit was purchased from tiangen biochemical technology (beijing) limited; nanoDrop 2000 spectrophotometer Thermo FISHER SCIENTIFIC, inc; DL2000 Marker, agarose, nucleic acid dye were all purchased from Beijing Soy Bao technology Co., ltd; gold medal Mix (green) was purchased from beijing engine biotechnology limited; the electrophoresis apparatus is purchased from Beijing Liuyi instrument factory; PCR instrument was purchased from BioRad corporation. IgA (E027-1-1), igG (E026-1-1), igM (E025-1-1) detection kits were purchased from Nanjing's institute of biological engineering.
3 Method
3.1 Immunoglobulin IgA, igG, igM detection
The detection kit is used for measurement by a spectrophotometry method according to IgA, igG, igM of Nanjing institute of biological engineering. Firstly, establishing a standard curve by using a standard substance; secondly, adding 7 mu L of distilled water, standard solution and sample to be detected into a blank tube, a standard tube and a measuring tube respectively, supplementing R1 solution to 900 mu L, incubating for 5min at 37 ℃, and recording the reading number as A1 at the wavelength of 340 nm; then 180. Mu.L of R2 solution is added into each tube, and the tubes are incubated for 5min at 37 ℃, and the reading number at the wavelength of 340nm is recorded as A2; finally, Δa=a2-A1 was calculated, and Δa was substituted into the standard curve equation to calculate the concentration of sample IgA, igG, igM (the specification of IgA, igG, igM detection kit describes a corresponding standard curve ;http://www.njjcbio.com/products.aspid＝841;http://www.njjcbio.com/products.aspid＝840;http://www.njjcbio.com/products.aspid＝839).
3.2 Extraction of genomic DNA from blood
Extracting genome DNA from blood sample by adopting a blood genome extraction kit of Tiangen biochemical technology (Beijing) limited company, and placing the extracted DNA under an ultraviolet spectrophotometer to detect the concentration and purity, wherein the concentration is more than 20 ng/mu L, OD 260/OD280 and is between 1.7 and 1.9, so that the experiment requirement is met, and the DNA is stored at-20 ℃ for standby.
3.3 Primer design
Referring to the international sheep genome oar_v4.0 version 17 chromosome gene sequence (GenBank accession number: nc_ 019474.2), a pair of specific primers was designed using PRIMER PREMIER 5.0.0 software, containing g32776109T > C SNP sites.
Primer sequence:
F：5'-TCAGCTTCCTATATTTACACTGG-3'(SEQ ID NO.2)；
R：5'-AAACATTCTGGAAATGGCAAA-3'(SEQ ID NO.3)。
the amplified fragment length was 499bp, and the primers were synthesized by Beijing engine biotechnology Co.
3.4 PCR amplification and sequencing
PCR amplification System 25. Mu.L: gold medal Mix (green) 22. Mu.L, 1. Mu.L each for the upstream and downstream primers, and 1. Mu.L for the template.
PCR amplification procedure: 98 ℃ for 2min;98 ℃ for 10s,57.5 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
And detecting the PCR product by using 1.5% agarose gel electrophoresis, and after the PCR product is qualified by using the agarose gel electrophoresis detection, sequencing by using a direct sequencing method, and completing sequencing by Beijing qingke biotechnology Co. The amplified nucleotide sequence is shown as SEQ ID NO. 4. The SNP marker is positioned at 148 positions of the nucleotide sequence shown in SEQ ID NO.4, and the position can be T/C (the nucleotide sequence of the SNP marker is shown as SEQ ID NO. 1).
SEQ ID NO.4：
TCAGCTTCCTATATTTACACTGGTCTGTTTTTAACTTAATGTGTCATTACGGTGTTCTGCAAATACCATACTATTTTTTTTTTAACTTGTAAACTTCATTTTTTAGAGCATTTTTAGGTTCACTGCTTTCTTCTTAAGTGGAAAGTGTAGAGAGTTTGTTTATCCCCATACCTTCACACACACAGCCTCCTCCACTATAAACATCCTGCATCACAATGGTGTGTTTGTTATAACTGATCAATCTGCATTGACATATCATTATCATCCAAAGTCCATAGTTGACATTAGGGTTCACTCTTGGTATTGTATATCCAGTTGGTTTTAAGAAATGTGTAATGATATGTATATACCATTACTATATAGTTTTGTATGGTAAAACTACACAATATTTTACTGCTCTAAAAATCCTCTGTCCTCTGCCTACTCATCACTGCCTCCCTATTCTCTGGCAGCCTGTGAGCTTTCTGCTTTAACCAGTTTTGCCATTTCCAGAATGTTT.
And comparing the sequencing results of the PCR products by using biological analysis software MEGA6.0, and analyzing a sequencing peak diagram to finish typing.
4 Statistical analysis
And counting the number of individuals with different genotypes at each site according to the genotyping result. The frequency of g32776109T > C gene, genotype frequency, effective allele (Ne), site heterozygosity (He) and Hardy-Weinberg equilibrium test are calculated by Popgen software, and the polymorphism information content is calculated by PIC (polymorphism information content, PIC for short) calculation software. The correlation of Tibetan sheep different genotypes with immunoglobulin IgA, igG, igM was analyzed using a general linear model in IBM SPSS STATISTICS software, and the results are expressed as "mean ± standard error".
5 Results
5.1 PCR amplification and sequencing results
The amplified product of the SNP locus of the Tibetan sheep chromosome 17 g32776109T > C (see FIG. 1;123 for 3 repeats) is detected by using 1.5% agarose gel, the band is clear and has no impurity band, the specificity is good, the fragment size of the PCR product is 499bp, the expected size is met, and the next experiment can be carried out.
The peak pattern and sequence obtained after the PCR product is purified and sequenced are shown in FIG. 2. As can be seen from FIG. 2, the SNP site of g32776109T > C has T-C mutation, and three genotypes of TT, CT and CC exist.
5.2 Statistical analysis results
Genotype and allele frequency of the Tibetan sheep chromosome 17 g32776109T > C SNP site were analyzed from a population genetics perspective. As can be seen from Table 1, the CC genotype was most frequently found at g32776109T > C SNP site, and the C allele frequency was 94.8% for the dominant genotype, which was expressed as the dominant allele. The SNP sites were significantly deviated from Hardy-Weinberg equilibrium (P < 0.05) as demonstrated by χ 2 fitness test (Table 1). The expected heterozygosity of the locus is 0.099, PIC is 0.094, PIC is less than 0.25, and the locus belongs to low-level polymorphism.
TABLE 1 Tibetan sheep chromosome 17 g32776109T > C SNP site polymorphism
5.3 Correlation analysis of different genotypes and immunoglobulins IgA, igG, igM
The correlation of Tibetan sheep different genotypes and immunoglobulin IgA, igG, igM content is analyzed by using a general linear model in IBM SPSS STATISTICS software, and the result shows that the immunoglobulin IgA, igG, igM of Tibetan sheep individuals with TT genotype is obviously higher than that of Tibetan sheep individuals with CC and TC genotypes (p < 0.05), and the immunoglobulin IgA, igG, igM between the individuals with CC and TC genotypes does not show obvious difference (p > 0.05), which indicates that the base of the Tibetan sheep chromosome 17 g32776109T > C SNP locus is obviously related to Tibetan sheep IgA, igG, igM and is the SNP marker related to Tibetan sheep IgA, igG, igM. The results are shown in Table 2.
TABLE 2 correlation analysis between different genotypes and immunoglobulins IgA, igG, igM
Note that: the same row of data is marked with different lower case letters to indicate that the difference is significant (P < 0.05).
In summary, the SNP molecular marker is positioned at 32776109 th base on chromosome 17 of the international sheep reference genome oar_v4.0 version; the mutation type is T/C, named g32776109T > C, three genotypes exist, and when 32776109 th base on the 17 th chromosome is T, the genotypes are TT or TC; when 32776109 bases on the 17 th chromosome are C, the genotype is CC; through correlation analysis of different genotypes and immunoglobulin IgA, igG, igM content, the immunoglobulin IgA, igG, igM of Tibetan sheep individuals with the TT genotype is found to be significantly higher than that of individuals with CC and TC genotypes (p < 0.05), and the immunoglobulin IgA, igG, igM among individuals with the CC and TC genotypes does not show significant difference (p > 0.05). By detecting the base of 32776109 nucleotide locus on the Tibetan sheep chromosome 17, the immunoglobulin IgA, igG, igM content of Tibetan sheep individuals can be judged, and the invention provides a new SNP molecular marker resource for auxiliary selection of Tibetan sheep immune trait markers for non-diagnostic purposes.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

  1. The ANKRD50 gene is used as a molecular marker of Tibetan sheep immune traits, and is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and the y at the 148 th position of the sequence represents T or C, so that the T/C polymorphism of the Tibetan sheep ANKRD50 gene at the position is caused.
  2. 2. A primer pair for detecting the molecular marker of claim 1, wherein the nucleotide sequences of the primer pair are shown in SEQ ID nos. 2 and 3.
  3. 3. A method for detecting a molecular marker associated with an immune trait in Tibetan sheep, comprising the steps of:
    (1) Extracting Tibetan sheep genome DNA;
    (2) Amplifying the genomic DNA of step (1) using the primer pair of claim 2;
    (3) And (3) carrying out typing identification on the polymorphic site of the amplification product obtained in the step (2).
  4. 4. The method of claim 3, wherein the amplification system of step (2) is: 22. Mu.L of PCR premix, 1. Mu.L of upstream primer, 1. Mu.L of downstream primer and 1. Mu.L of template.
  5. 5. The method of claim 4, wherein the PCR primer Mix is gold medal Mix.
  6. 6. The method of claim 3, wherein the amplification procedure of step (2) is: 98 ℃ for 2min;98 ℃ for 10s,57.5 ℃ for 10s and 72 ℃ for 10s, 40 cycles in total; extending at 72℃for 2min.
  7. 7. Use of the molecular marker of claim 1 or the primer pair of claim 2 or the method of any one of claims 3 to 6 for in vitro detection of an immune trait of Tibetan sheep of non-diagnostic interest.
  8. 8. Use of the molecular marker of claim 1 or the primer pair of claim 2 or the method of any one of claims 3 to 6 in Tibetan sheep assisted breeding.
  9. 9. The use according to claim 8, wherein the use is for screening of high immunity Tibetan sheep breeds.
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CN116287299A (en) * 2023-02-20 2023-06-23 民勤同泽农业有限公司 Molecular marker related to sheep immune traits and application thereof
CN117683909A (en) * 2024-01-25 2024-03-12 中国农业科学院兰州畜牧与兽药研究所 Molecular marker related to Tibetan sheep immune traits and application thereof
CN117721221A (en) * 2024-01-25 2024-03-19 中国农业科学院兰州畜牧与兽药研究所 Cloning and application of SNP molecular marker related to Tibetan sheep immune traits

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Publication number Priority date Publication date Assignee Title
CN116287299A (en) * 2023-02-20 2023-06-23 民勤同泽农业有限公司 Molecular marker related to sheep immune traits and application thereof
CN117683909A (en) * 2024-01-25 2024-03-12 中国农业科学院兰州畜牧与兽药研究所 Molecular marker related to Tibetan sheep immune traits and application thereof
CN117721221A (en) * 2024-01-25 2024-03-19 中国农业科学院兰州畜牧与兽药研究所 Cloning and application of SNP molecular marker related to Tibetan sheep immune traits

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