A bovine ATG5 gene 5apos; application of SNP molecular marker related to terminal disease resistance

Abstract

The invention discloses application of a cattle ATG5 gene 5'end disease resistance related SNP molecular marker, and belongs to the technical field of molecular marker assisted breeding. One SNP site g.T-144C related to mastitis resistance is found on the ATG5 gene of the Chinese Holstein dairy cow, and the SNP site g.T-144C is located at the 5'end of the ATG5 gene of the No.9 chromosome of the Chinese Holstein dairy cow. Through a dual-luciferase report experiment and correlation analysis of the SNP molecular marker and the staphylococcus aureus mastitis resistance of dairy cattle, direct correlation between the ATG5 gene g.T-144C site and the staphylococcus aureus mastitis resistance is disclosed and verified, and a reliable molecular target is provided for breeding for disease resistance. Therefore, the invention provides an effective molecular marker for molecular-assisted selection and disease-resistant breeding of cow mastitis resistance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular marker assisted breeding, and particularly relates to application of a bovine ATG5 gene 5' end disease resistance related SNP molecular marker. BACKGROUND

[0002] Mastitis is a common disease in dairy farms. After the mammary gland is infected by pathogenic microorganisms or stimulated by physical and chemical factors, inflammation occurs in the interstitial or parenchymal tissue of the mammary gland, which seriously affects the milk yield of dairy cows and the quality of dairy products. Mastitis can be divided into clinical mastitis and subclinical mastitis (also known as hidden mastitis) according to the degree and duration of the disease. It is mainly caused by pathogenic microorganisms, and more than 80% of the mammary gland infections are caused by bacteria. Common pathogenic bacteria include Staphylococcus aureus, Streptococcus uberis and Escherichia coli. In addition, there are also reports of mastitis induced by viruses, mycoplasma, fungi and other infections. Traditional treatment methods include antibiotic therapy, physical therapy and traditional Chinese medicine treatment, but none of them can solve the problem from the root, and antibiotic therapy may also affect the quality of dairy products and the health of dairy cows. In addition, the accuracy of the breeding method based on somatic cell count is insufficient, and the genetic progress is slow. Therefore, using the research results of molecular genetics for mastitis disease resistance breeding has become an important way to prevent and treat mastitis in dairy cows.

[0003] Due to the fact that Staphylococcus aureus exhibits multiple antibiotic resistance, and also has multiple immune escape mechanisms, such as preventing the formation of autophagy lysosomes, hindering the recruitment of neutrophils, phagocytosis and inhibiting the immune function of macrophages, etc., it makes the treatment and control of mastitis more difficult. Therefore, in the balanced breeding of dairy cows, it is particularly important to explore the breeding approach of using molecular genetic markers to improve the mastitis resistance of dairy cows. SUMMARY

[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide an application of a bovine ATG5 gene 5' end disease resistance related SNP molecular marker, so as to provide a new SNP molecular marker for breeding dairy cows with mastitis resistance.

[0005] The technical solution of the present application to solve the above technical problems is as follows: an application of a bovine ATG5 gene 5' end disease resistance related SNP molecular marker in the preparation of a product for detecting or assisting in detecting the mastitis resistance of dairy cows is provided. The SNP molecular marker is from the bovine ATG5 gene, and the nucleotide sequence is shown as SEQ ID NO. 1. The polymorphic site is located at position 857 of the sequence shown as SEQ ID NO. 1, and the polymorphism is C or T.

[0006] The present application provides a kit for detecting or assisting in detecting the mastitis resistance of dairy cows, which comprises a primer pair for detecting the above-mentioned SNP molecular marker.

[0007] Further, the upstream and downstream primer sequences of the primer pair are as follows: Forward primer F: 5'-CAGTGCCAACAGCTTTTAAGAGTAA-3'; Reverse primer R: 5'-CAACCCTGCACCTAGGCTG-3'.

[0008] The application provides application of the kit in screening or identifying a dairy cow mastitis resistance dominant strain.

[0009] The application provides a method for assisting in screening or identifying a dairy cow mastitis resistance dominant strain, comprising the following steps: (1) extracting genomic DNA of a dairy cow to be tested; (2) using the genomic DNA of the dairy cow to be tested as a template, using the kit, and amplifying the dairy cow ATG5 gene through a PCR reaction; (3) detecting the PCR amplification product, and if the base at the 305th position in the sequence of the amplification product is C, then the dairy cow to be tested belongs to a high-mastitis-resistance dairy cow dominant strain.

[0010] The application provides application of a bovine ATG5 gene 5' end disease resistance related SNP molecular marker in assisted breeding of mastitis resistant dairy cows, the SNP molecular marker is from a bovine ATG5 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1; the polymorphic site is located at the 857th position in the sequence shown in SEQ ID NO. 1, and the polymorphism is C or T.

[0011] The application provides a method for assisting in breeding of mastitis resistant dairy cows, comprising the following steps: using a high-mastitis-resistance dairy cow dominant strain obtained through the method as a parent for breeding.

[0012] The application has the following beneficial effects: the application finds an SNP site g.T-144C related to mastitis resistance in a Chinese Holstein cow ATG5 gene, which is located at the 5' end of the ATG5 gene of the chromosome 9 of the Chinese Holstein cow. Through molecular experiments and correlation analysis of the SNP molecular marker and mastitis resistance of the dairy cow, it is firstly revealed and verified that the ATG5 gene g.T-144C site is directly correlated with the Staphylococcus aureus mastitis resistance, and a reliable molecular target is provided for disease resistance breeding. Therefore, the application provides a theoretical basis for molecular assisted selection and breeding of dairy cow mastitis resistance. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 Peak value of population genetic differentiation index of the ATG5 gene region of the chromosome 9 between the infection-resistant and infection-susceptible groups; Figure 2 Figure for sequencing result of g.-144 T>C site of ATG5 gene; Figure 3 Figure for PGL3-Basic-ATG5 vector for detecting dual-luciferase activity; Figure 4 Figure for detecting dual-luciferase activity of g.-144 T>C site of ATG5 gene. DETAILED DESCRIPTION

[0014] The following examples are intended to illustrate but not limit the present application. Unless otherwise indicated, the conditions in the examples were carried out under conventional conditions or as recommended by the manufacturer. Unless otherwise indicated, the reagents or instruments used were conventional products available commercially.

[0015] Example 1: Discovery and genotyping of SNP site 1. Discovery of SNP site Through genomics analysis, the present application obtains a genomic candidate interval related to mastitis resistance of Chinese Holstein cow. Through gene annotation of the genomic candidate interval and analysis of biological function information of the gene, the ATG5 gene is successfully located (NCBI Gene ID: 282686). Figure 1 On this basis, through analysis of the variation site in the ATG5 gene, it is found that there are two SNPs, g.C-711T and g.T-144C, in the 5' end of the gene, and the regulatory gene expression activity of the two SNP sites is verified. It is found that the C allele of ATG5 gene g.T-144C has a significant up-regulation effect on gene expression. Figure 2

[0016] 2. Extraction of bovine frozen semen DNA ​Genomic DNA was extracted using phenol-chloroform extraction method. The specific experimental steps are as follows: take 200 μL of frozen semen sample, add 400 μL of normal saline, invert and mix well, then centrifuge at 12000 rpm for 10 min, discard the supernatant, and repeat the washing step; then add 600 μL of frozen semen DNA extraction buffer and 10 μL of proteinase K to the precipitate, vortex well, and incubate at 56°C overnight to complete the lysis. Add 500 μL of mixed solution of phenol, chloroform and isoamyl alcohol (volume ratio 25:24:1) to the lysis solution, invert and mix well, centrifuge at 12000 rpm for 10 min to separate the phases; then transfer the upper aqueous phase to a new tube, add an equal volume of chloroform and isoamyl alcohol (v / v 24:1) for back extraction, mix well, then centrifuge at 12000 rpm for 10 min, collect the supernatant, and add 800 μL of pre-cooled anhydrous ethanol to precipitate the DNA, mix gently until the floccules are precipitated, centrifuge at 12000 rpm for 2 min at 4°C to collect the precipitate; then add 800 μL of 70wt% ethanol to the precipitate, mix well, then centrifuge at 12000 rpm for 2 min at 4°C, repeat once. Collect the precipitate, dry at room temperature, then dissolve the DNA in 50 μL of ultrapure water, and store for use.

[0017] 3. Extraction of blood genomic DNA Blood genomic DNA was extracted using the Tiangen blood genomic DNA extraction kit (DP348). The extraction steps were performed according to the kit instructions.

[0018] 4. Primer design Primer3Plus and NCBI Primer-BLAST were used to design primers in the 5' flanking region of the ATG5 gene, and the amplified fragment contained the ATG5 gene g.T-144C site. The following principles were followed during primer design: The length of the PCR amplified fragment was about 400; 2) the G / C content of the primer was between 40-60%; 3) the length of the primer was no more than 25 bp, and the annealing temperature (Tm value) was between 58-62°C; 4) the 3' end of the primer should not end with A or T as much as possible, and the 3' end should avoid the presence of consecutive 3 identical bases to avoid the formation of hairpin structures and primer dimers; 5) try to avoid the presence of hairpin structures or primer dimers in the upstream and downstream primers or the primer itself; 6) predict the specificity of the primer to ensure the uniqueness of the amplified band. The sequences of the designed primer pairs are shown below: Forward primer F: 5'-CAGTGCCAACAGCTTTTAAGAGTAA-3' (SEQ ID NO. 2); Reverse primer R: 5'-CAACCCTGCACCTAGGCTG-3' (SEQ ID NO. 3).

[0019] PCR amplification was performed with the above primer pair, and the reaction system (20 μL) was as follows: 1 μL of template DNA, 0.4 μL of each of the upper and lower primers corresponding to the 10 mM primer pair, 10 μL of (2x) Taq PCR Mix, and double-distilled water to 20 μL. The reaction conditions were as follows: ① pre-denaturation at 94℃ for 3 min; ② amplification reaction: denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s, 30 cycles; ③ final extension at 72℃ for 5 min; and ④ cooling at 4℃. The PCR product was subjected to routine sequencing.

[0020] Example 2: Functional verification of the SNP site A genomic fragment containing different alleles (T or C) of the 5'UTR of the ATG5 gene was cloned into a pGL3-Basic luciferase reporter vector to construct recombinant plasmids, respectively. The pGL3-basic plasmid was purchased from Promega Company. The constructed vector PGL3-Basic-ATG5 is shown in Figure 3 An equal amount of the recombinant plasmid and a reference plasmid were used to edit a bovine mammary epithelial cell line by a liposome transfection method, the cells were lysed after a period of culture, and the fluorescence value was determined using a dual luciferase detection system. The specific operation steps were performed according to the kit instructions.

[0021] Example 3: Association analysis of the SNP site and the resistance phenotype According to the bovine herd management records of the breeding center, the bovine herd was divided into a mastitis susceptible group and a resistant group, and the genotype frequency distribution of the g.-144 T>C site was compared between the two groups.

[0022] Result analysis: (1) Effect of 5'UTR mutation on gene expression activity The present application successfully identified the g.-144 T>C polymorphic site in the 5'UTR region of the ATG5 gene and verified the effect of the SNP in the 5'UTR region of the ATG5 gene on gene expression activity. It can be seen from Figure 2 that the luciferase activity of the CC genotype at the g.T-144C site in the 5'UTR region of the ATG5 gene was significantly enhanced (P<0.01) compared with the wild type, proving that the site has an effect on resistance to mastitis (see Figure 4 ).

[0023] (2) Detection of molecular marker technology and application The application provides a method for detecting a SNP molecular marker in a promoter region of an ATG5 gene related to resistance of dairy cow mastitis by a conventional primer amplification technology. 62 healthy dairy cows and 62 dairy cows with mastitis are genotyped (detection results are shown in Tables 1-2), and the C allele frequency of the healthy cow group is 56.5% and the C allele frequency of the dairy cow with mastitis group is 41.2%; statistical test shows that the difference between the two is significant (P<0.05), which indicates that the site is significantly related to the resistance of dairy cow mastitis. Therefore, the SNP molecular marker of the application can be used for molecular assisted selection and disease-resistant breeding of dairy cow mastitis.

[0024] Table 1 Genotype of the g.-144 T>C site of the ATG5 gene in the resistant infection group

[0025] Table 2 Genotype of the g.-144 T>C site of the ATG5 gene in the susceptible infection group

[0026] The nucleotide sequence (containing the 5' end sequence of the gene) of the ATG5 gene in the application is as follows: (1) The nucleotide sequence of the ATG5 gene is as follows: The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a bovine ATG5 gene 5' end disease-associated SNP molecular marker in the preparation of a product for detecting or aiding in the detection of resistance to mastitis in dairy cattle, characterized in that, The SNP molecular marker is from a bovine ATG5 gene, the nucleotide sequence of which is shown as SEQ ID NO. 1; the polymorphic site is located at position 857 of the sequence shown as SEQ ID NO. 1, and the polymorphism is C or T.

2. A kit for detecting or aiding in the detection of resistance to mastitis in a dairy cow, characterized in that, The kit comprises a primer pair for detecting the SNP molecular marker of claim 1.

3. The kit of claim 2, wherein The sequences of the upstream and downstream primers of the primer pair are as follows: Forward primer F: 5'-CAGTGCCAACAGCTTTTAAGAGTAA-3'; Reverse primer R: 5'-CAACCCTGCACCTAGGCTG-3'.

4. Use of the kit of claim 2 or 3 in screening or identifying a dairy cow mastitis-resistant superior strain.

5. A method of assisting in the selection or identification of a dairy cow mastitis resistant superior line, characterized in that, The method comprises the following steps: (1) extracting the genomic DNA of the dairy cow to be tested; (2) using the genomic DNA of the dairy cow to be tested as a template, the kit of claim 2 or 3 is used to amplify the bovine ATG5 gene through a PCR reaction; (3) detecting the PCR amplification product, if the base at position 305 of the sequence of the amplification product is C, then the dairy cow to be tested belongs to a mastitis-resistant superior strain of dairy cow.

6. The application of bovine ATG5 gene 5' end disease resistance related SNP molecular marker in the assisted breeding of mastitis resistant cows, characterized in that, The SNP molecular marker is from a bovine ATG5 gene, the nucleotide sequence of which is shown as SEQ ID NO. 1; the polymorphic site is located at position 857 of the sequence shown as SEQ ID NO. 1, and the polymorphism is C or T.

7. A method of assisted breeding of a cow against mastitis, characterized in that, The method comprises the following steps: The mastitis-resistant superior strain of dairy cow obtained by the method of claim 5 is used as a parent for breeding.

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