A method for detecting POLB gene deletion markers in cattle

Abstract

The present invention belongs to the technical field of livestock molecular genetics breeding, and specifically relates to a method for detecting a deletion marker in the POLB gene of cattle. The method uses cattle genomic DNA as a template and a specific primer pair (F: 5'-ACTCCAGGTTCCTTTTACTGGC-3', R: 5'-GTCGGTGATGCCCCCGT-3') as primers to amplify a partial gene fragment upstream of the CDS region of an individual cattle by PCR, and detects the PCR amplification product by electrophoresis. The PCR product is then directly digested with the restriction endonuclease MaeI, and the digested product is subjected to agarose gel electrophoresis. The genotype of the individual cattle is determined based on the electrophoresis detection result.

Description

Technical Field

[0001] The invention belongs to the technical field of livestock molecular genetics breeding, and particularly relates to a method for detecting a deletion marker of a cattle POLB gene. Background Art

[0002] As living standards in my country improve, beef consumption has increased year by year. However, my country's overall beef supply is less than its consumption, and some beef needs to be imported to fill the gap. To further improve the growth performance of beef cattle in my country and enhance breeding efficiency, integrating molecular breeding with conventional breeding techniques has become an important approach. Molecular marker-assisted selection (MAS) is a key technology in molecular breeding. It utilizes molecular markers associated with growth traits to rapidly and accurately select for dominant alleles at the molecular level.

[0003] POLB is a DNA polymerase first isolated from bovine thymus by Weissbach et al. in 1971. This gene belongs to the DNA polymerase X family. Members of this family of DNA polymerases play important roles in DNA replication, DNA damage repair, and cell cycle regulation. In 1986, Zmudzka et al. cloned the DNA sequences of the POLB gene from mice and humans, respectively. The POLB protein is the smallest DNA polymerase in eukaryotic cells, encoding 335 amino acids with a molecular weight of only 39 ku.

[0004] The primary function of POLB is to repair DNA damage by participating in the base excision repair (BER) system. Failure to effectively repair DNA damage in cells can lead to cellular senescence, apoptosis, and carcinogenesis. Studies have found that blocking the protein kinase A (PKA) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways and reducing POLB gene expression in human esophageal cancer cells can alter the biological characteristics of these cells, inhibiting cell proliferation and promoting apoptosis. Furthermore, silencing or reducing POLB gene expression in mouse esophageal cancer cells and human oral squamous cell carcinoma cells can regulate the cell cycle, leading to abnormal mitosis and promoting cell proliferation. Using RNA interference to inhibit POLB gene expression in breast cancer cells (MCF-7) also significantly inhibited cell proliferation. Shen Liang's research has shown that POLB may regulate the proliferation and migration of MCF-7 cells through the PI3K / Akt signaling pathway. Zhu Yanyan used N-nitrosomethylbenzylamine (NMBA) to induce expression in esophageal epithelial tissue of POLB knockout mice. mRNA transcriptome microarray analysis and GO function enrichment analysis of differentially expressed genes revealed 33 differentially expressed genes associated with cell proliferation regulation and 27 associated with cell cycle regulation, including signaling pathways such as PI3K / Akt and MAPK-ERK. These reports indicate that changes in POLB gene expression are closely related to cell proliferation, apoptosis, and cell cycle regulation.

[0005] However, there are few studies on the POLB gene in cattle growth traits, and no reports on gene polymorphism have been found. Summary of the Invention

[0006] In view of the defects and problems existing in the prior art, the present invention provides a method for detecting a deletion marker of the POLB gene in cattle.

[0007] The solution adopted by the present invention to solve the technical problem is: a method for detecting the deletion marker of the POLB gene in yellow cattle, using yellow cattle genomic DNA as a template and a specific primer pair as primers, amplifying a partial gene fragment upstream of the CDS region of an individual yellow cattle by PCR, and detecting the PCR amplification product by electrophoresis; then directly digesting the PCR product with the restriction endonuclease MaeI, and detecting the digested product by agarose gel electrophoresis, and determining the genotype of the individual yellow cattle based on the electrophoresis detection result; the primer pair is:

[0008] F: 5'-ACTCCAGGTTCCTTTACTGGC-3';

[0009] R: 5'-GTCGGTGATGCCCCCGT-3'.

[0010] The PCR amplification reaction system for the above-mentioned method for detecting the POLB gene deletion marker in cattle is as follows: 2× TaqPCR Master Mix 5.00 μL, ddH2O 3.00 μL, PF 0.5 μL, PR 0.5 μL, and DNA template 1.0 μL.

[0011] The PCR amplification reaction procedure for the above-mentioned method for detecting the deletion marker of the cattle POLB gene is as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 15 seconds, annealing at 63°C for 15 seconds, extension at 72°C for 15 seconds, 38 cycles; extension at 72°C for 10 minutes; and storage at 4°C.

[0012] The above-mentioned method for detecting the deletion marker of the cattle POLB gene, the enzyme digestion reaction system is: Bfal 0.4μL, Buffer 2μL, PCR product 2μL, ddH2O 15.6μL, reaction temperature 37°C, reaction time 8h.

[0013] In the above method for detecting cattle POLB gene deletion markers, the agarose gel electrophoresis uses agarose gel with a mass concentration of 3%.

[0014] In the above method for detecting deletion markers of cattle POLB gene, the wild genotype showed two bands of 93 bp and 267 bp; the deletion genotype showed a 360 bp band; and the heterozygous genotype showed three bands of 93 bp, 267 bp and 360 bp.

[0015] A kit for detecting deletion polymorphism of cattle POLB gene, characterized in that the kit comprises a primer pair for PCR amplification of a 12 bp deletion mutation site at position c.-221_-210del upstream of the CDS region of NC_037354.1 of cattle POLB gene, wherein the primer pair is:

[0016] F: 5'-ACTCCAGGTTCCTTTACTGGC-3'

[0017] R: 5'-GTCGGTGATGCCCCCGT-3'.

[0018] The invention relates to an application of the method for detecting a cattle POLB gene deletion marker in cattle molecular marker-assisted selection breeding.

[0019] In the above application, at the deletion marker site amplified by the primer pair, the growth advantage genotype corresponding to the individual is the wild type.

[0020] The present invention has the following beneficial effects: Using bovine whole-genomic DNA as a template, the method amplifies a portion of the bovine POLB gene by PCR, and uses agarose gel electrophoresis to identify the genotype of an individual with a 12bp deletion mutation site upstream of the CDS region of the POLB gene. Trait association analysis shows that the wild-type genotype of this 12bp deletion mutation site in the POLB gene is significantly correlated with oblique body length in cattle. This can be used for marker-assisted selection in cattle breeding, enhancing the selection of wild-type individuals and improving the growth performance of cattle. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is the enzyme electrophoresis pattern of the present invention.

[0022] Figure 2 This is the wild-type sequencing map.

[0023] Figure 3 This is a deletion-type sequencing map.

[0024] Figure 4 This is the heterozygous sequencing map. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and examples.

[0026] Example 1: This example provides a method for detecting deletion mutation markers of the cattle POLB gene, which mainly includes the following contents.

[0027] 1. Primer design

[0028] The present invention uses Oligo7 and NCBI to design primers for the c.-221_-210del deletion site upstream of the POLB gene CDS based on the bovine POLB gene published in GenBank (NCBI GenBank accession number NC_037354.1). The primer sequences are:

[0029] F (upstream primer) 5′-ACTCCAGGTTCCTTTTACTGGC-3′;

[0030] R (downstream primer) 5′―GTCGGTGATGCCCCCGT―3′.

[0031] 2. Extraction of cattle whole genome DNA

[0032] In this example, whole genomic DNA was extracted from a blood sample of Jiaxian Red Cattle using a genomic DNA extraction kit. The specific steps are as follows:

[0033] (1) Take 200 μL of Jiaxian Red Bull blood sample and add it to a 1.5 mL centrifuge tube;

[0034] (2) Add 20 μL Proteinase K and mix well;

[0035] (3) Add 200 μL GB buffer, mix well, and let stand at 70°C for 10 min;

[0036] (4) Add 200 μL of anhydrous ethanol and shake on a shaker for 15 seconds to mix;

[0037] (5) Add the mixed solution from the previous step to the adsorption column CB3 and centrifuge at 12,000 rpm for 30 seconds;

[0038] (6) Discard the solution, add 500 μL of GD buffer to the adsorption column CB3, and centrifuge at 12,000 rpm for 30 s;

[0039] (7) Discard the solution, add 600 μL of PW rinse solution to the adsorption column CB3, and centrifuge at 12,000 rpm for 30 s;

[0040] (8) Repeat step (7);

[0041] (9) Discard the liquid and centrifuge at 12,000 rpm for 30 seconds;

[0042] (10) Discard the liquid and let it dry;

[0043] (11) Transfer the adsorption column CB3 to a new sterilized 1.5 mL centrifuge tube, add 30 μL of sterile water, let it stand at room temperature for 5 min, and centrifuge at 12,000 rpm for 2 min;

[0044] (12) Repeat step (11), then collect the solution and store it at -20°C.

[0045] 3. DNA purity and concentration testing

[0046] (1) Turn on the spectrophotometer and select it for DNA detection. Then, aspirate 1 μL of sterile water to rinse the sample spot on the instrument and wipe it dry with filter paper.

[0047] (2) Pipette 1 μL of sterile water as a blank control, then take 1 μL of sample and spot it, and record the concentration and purity readings of each sample;

[0048] (3) The purity of the measured sample must ensure OD 260 / OD 280 The value is between 1.8-2.0.

[0049] 4. PCR reaction

[0050] The designed primer sequences were used to extract DNA from Jiaxian Red Cattle:

[0051] F (upstream primer) 5′-ACTCCAGGTTCCTTTTACTGGC-3′;

[0052] R (downstream primer) 5′―GTCGGTGATGCCCCCGT―3′, (primers synthesized by Sangon Biotech (Shanghai) Co., Ltd.), PCR amplification was performed. The PCR reaction system is shown in Table 1 below; the PCR amplification program is shown in Table 2 below.

[0053] Table 1 PCR reaction system

[0054]

[0055] Table 2 PCR amplification procedure

[0056]

[0057]

[0058] After PCR amplification, the band size of the PCR amplified product was detected by agarose gel electrophoresis to determine whether the amplification was correct. The PCR product should be 360 ​​bp.

[0059] 5. Enzyme digestion and electrophoresis

[0060] The c.-221_-210del deletion site was analyzed using SnapGene Viewer software, and a Bfal (or Mael) (C / TAG) restriction enzyme site was found at the site. The PCR product was directly digested using the restriction endonuclease MaeI. The enzyme digestion system is shown in Table 3 below.

[0061] Table 3 Enzyme digestion system

[0062]

[0063] Mix well and digest at 37°C for 8 hours.

[0064] The enzyme digestion products were then subjected to typing using 3% agarose gel electrophoresis. The specific steps were as follows:

[0065] (1) Weigh 0.75 g of agarose using an electronic balance and add it to a 250 mL conical flask;

[0066] (2) Use a graduated cylinder to measure 25 mL of 1× TAE solution, add it to the conical flask, shake gently to mix, and then place it in a microwave oven and heat on medium heat for 1.5 min until the agarose is completely dissolved.

[0067] (3) After the solution cools to about 40°C, add 2 μL of nucleic acid dye using a pipette, shake gently to mix, and pour into the gel plate with the comb inserted. After solidification for about 30 minutes, remove the comb from the gel and place it in the gel electrophoresis apparatus. Add 1× TAE solution until the liquid surface covers the gel and expel the air in the gel pores.

[0068] (4) Spot 15 μL of the digested product into the gel wells and run the electrophoresis at 100 V and 100 A for 1 h.

[0069] (5) Take out the gel after electrophoresis from the electrophoresis tank, turn on the UV gel imaging system, put the gel in, close the door, turn on the UV light to observe the electrophoresis bands, and determine the individual genotype based on the band size. The enzyme digestion pattern is shown in the figure. Figure 1 , the sequencing results of different genotypes are shown in Figure 2-4 Specifically:

[0070] Wild type: 93 bp 267 bp

[0071] Deletion type: 360bp

[0072] Heterozygous type: 93bp 267bp 360bp.

[0073] Example 2: This example provides a kit for detecting deletion polymorphism of cattle POLB gene, which includes a primer pair for PCR amplification of the 12 bp deletion mutation site of c.-221_-210del upstream of the CDS region of the cattle POLB gene NC_037354.1, wherein the primer pair is:

[0074] F: 5'-ACTCCAGGTTCCTTTACTGGC-3';

[0075] R: 5'-GTCGGTGATGCCCCCGT-3';

[0076] It also includes 2×Taq MasterMix 5.00 μL, ddH2O 3.00 μL, restriction endonuclease MaeI and Buffer 2 μL.

[0077] Example 3: In this example, blood samples from 71 Jiaxian Red Cattle were used as experimental materials, DNA was extracted, and genotype identification was performed using Jiaxian Red Cattle DNA as a template.

[0078] A general linear model was used to analyze the association between the c.-221_-210del polymorphism and the body plagiogrowth trait of Jiaxian Red Cattle. The results were shown in Table 4.

[0079] Table 4 Results of association analysis between the c.-221_-210del polymorphism and the plagiocephalic trait in Jiaxian Red Cattle

[0080]

[0081] As shown in Table 4, wild-type cattle outperform the missing genotype for the trait. The wild type is the dominant genotype, and selection of wild-type individuals should be strengthened to improve the growth performance of Jiaxian Red cattle. Body trait is an important indicator of cattle growth performance and is highly genetically correlated with body weight. For example, the genetic correlation coefficient between finishing body length and carcass weight in Hereford cattle is 0.86. In production practice, body trait length and chest circumference data can be used to estimate cattle weight. Selecting for body trait length through molecular marker-assisted selection is crucial for improving cattle growth performance and meat production.

Claims

1. A method for detecting a deletion marker of the cattle POLB gene for use in cattle molecular marker-assisted selection breeding, characterized in that: The method for detecting the POLB gene deletion marker in cattle is as follows: using cattle genomic DNA as a template and a specific primer pair as primers, amplifying a partial gene fragment upstream of the CDS region of an individual cattle by PCR, and detecting the PCR amplification product by electrophoresis; then directly digesting the PCR product with the restriction endonuclease MaeI, performing agarose gel electrophoresis on the digested product, and determining the genotype of the individual cattle according to the electrophoresis detection result; The primer pair is used for PCR amplification of the 12 bp deletion mutation site at c.-221-210del upstream of the CDS region of the bovine POLB gene NC_037354.

1. The primer pair is: F: 5'-ACTCCAGGTTCCTTTACTGGC-3'; R: 5'-GTCGGTGATGCCCCCGT-3'.

2. The use according to claim 1, characterized in that The reaction system for PCR amplification was: 2× Taq PCR Master Mix 5.00 μL, ddH2O 3.00 μL, primer F 0.5 μL, primer R 0.5 μL, and DNA template 1.0 μl.

3. The use according to claim 1, characterized in that The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 15 s, annealing at 63°C for 15 s, extension at 72°C for 15 s, 38 cycles; extension at 72°C for 10 min; and storage at 4°C.

4. The use according to claim 1, characterized in that The enzyme digestion reaction system was: Bfal 0.4 μL, Buffer 2 μL, PCR product 2 μL, ddH2O 15.6 μL, reaction temperature 37°C, reaction time 8 h.

5. The use according to claim 1, characterized in that The agarose gel electrophoresis uses agarose gel with a mass concentration of 3%.

6. The use according to claim 1, characterized in that The wild genotype showed two bands of 93bp and 267bp; the deletion genotype showed a 360bp band; and the heterozygous genotype showed three bands of 93bp, 267bp and 360bp.

7. The use according to any one of claims 1 to 6, characterized in that At the deletion marker site amplified by the primer pair, the growth advantage genotype corresponding to the individual is the wild type.

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