A primer pair for detecting the DNA methylation level of the promoter of sheep KAP8 gene and its application and methylation detection method

Abstract

The present invention relates to a primer pair for detecting the DNA methylation level of the promoter of the sheep KAP8 gene, and an application and a methylation detection method thereof, and belongs to the field of gene detection technology. The present invention relates to a primer pair for detecting the DNA methylation level of the promoter of the sheep KAP8 gene, and the sequence of the primer pair is shown in SEQ ID NO: 1-2. The primer pair can be used to specifically detect the DNA methylation level of the promoter region of the KAP8 gene of different breeds of sheep. The DNA methylation level of the region can be used to determine the thickness of sheep wool fibers, providing a scientific basis for later sheep breeding and selection.

Description

Technical Field

[0001] The present invention relates to the technical field of gene detection, and in particular to a primer pair for detecting the DNA methylation level of a sheep KAP8 gene promoter, an application thereof, and a methylation detection method. Background Art

[0002] DNA, as a carrier for storing biological information, contains four bases: A, G, C, and T. However, these bases often undergo some chemical modifications in regulating gene function as organisms grow and develop. Methylation is the most common form of natural DNA modification. DNA methylation plays an important role in epigenetic regulation and is a common way of regulating gene expression in animal growth and development. This change usually occurs in densely populated areas of cytosine (C) and guanine (G), which is CpG islands. CpG islands are mostly located in the promoter region of genes and are also key areas for gene regulation. Methylation modification of CpG islands is an important way to affect gene transcription expression. Studies have confirmed that DNA methylation regulates gene expression by changing the binding of upstream promoter sequences of genes to transcription factors, thereby achieving genetic control of gene function over phenotype, and even DNA methylation modification can determine the sex of species.

[0003] Wool growth and development is a complex process regulated by multiple genes. The main component of wool is keratin-associated protein (KAP), and its content and structural composition have an important influence on the physical, chemical and phenotypic properties of wool. In particular, the gene expression level of KAP8 is highly correlated with wool fineness. However, existing studies have not clarified how the KAP8 gene regulates sheep wool development, nor have they disclosed the mechanism of how to regulate sheep wool development through DNA methylation modification and its corresponding detection method.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a primer pair for detecting the DNA methylation level of sheep KAP8 gene promoter and its application and methylation detection method.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a primer pair for detecting the DNA methylation level of a sheep KAP8 gene promoter. The sequence of the primer pair is shown in SEQ ID NOs: 1-2.

[0008] The present invention also provides a method for detecting the DNA methylation level of the promoter of sheep KAP8 gene using the primer pair, comprising the following steps:

[0009] (1) extracting DNA from in vitro sheep skin tissue, and performing methylation treatment on the DNA to obtain methylated genomic DNA to be tested;

[0010] (2) Using the methylated genomic DNA to be tested as a template, using the primer pair to perform PCR amplification, detecting the PCR amplification product, screening the positive product for sequencing, comparing the sequencing results, and determining the DNA methylation level of the sheep KAP8 gene promoter according to the comparison results.

[0011] Preferably, the ex vivo sheep skin tissue is sheep ear skin tissue and / or shoulder blade skin tissue.

[0012] Preferably, the methylation treatment is bisulfite methylation treatment.

[0013] Preferably, the conditions for the PCR amplification are:

[0014] Pre-denaturation at 94°C for 4.5-5.5 min; denaturation at 94°C for 29-35 s, annealing at 58-65°C for 38-50 s, extension at 72°C for 43-60 s, 34-40 cycles; then maintain at 72°C for 6.5-7.5 min.

[0015] Preferably, the size of the PCR amplification product is 193 bp.

[0016] Preferably, the detection in step (2) is agarose gel electrophoresis detection;

[0017] The initial concentration of agarose during the agarose gel electrophoresis detection is 1.5-2.5 wt %.

[0018] Preferably, the sequencing is bidirectional sequencing or sequencing by connecting the positive product to an empty vector;

[0019] The empty vector is pMD18-T;

[0020] The alignment was performed using Megalign software.

[0021] The present invention also provides the application of the primer pair or the method in judging sheep breeds, detecting wool traits or selecting sheep breeds.

[0022] Preferably, when judging sheep breeds, detecting wool traits or selecting sheep breeds, the method is performed based on the DNA methylation level of the sheep KAP8 gene promoter;

[0023] When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene was >2, it was determined to be a fine-wool sheep breed;

[0024] When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene was ≤1, it was determined to be a coarse-wool sheep breed;

[0025] When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene = 2, it is determined to be a breed between fine-wool sheep and coarse-wool sheep.

[0026] The present invention provides a primer pair for detecting the DNA methylation level of the promoter of sheep KAP8 gene and its application and methylation detection method. The method has the following advantages:

[0027] The present invention is based on PCR and second-generation sequencing technology platforms, has low requirements on laboratory equipment and technicians, and is suitable for most testing and research units. Compared with existing phenotypic breeding technology, it greatly improves the detection efficiency and accuracy, especially for wool fineness trait breeding that can only be carried out after adulthood or offspring testing, it greatly shortens the breeding cycle and speeds up the breeding process, which is of great significance for fine wool sheep breeding and variety identification.

[0028] On the basis of the previous experiments, the inventors carried out the cloning of the promoter region of KAP8 gene and the full-length cloning of mRNA, discovered the gene polymorphism and breed specificity of the promoter region, predicted the methylated CpG island, discovered the difference between the DNA of Chinese Merino fine-wool sheep and Suffolk sheep and the hybrid offspring of Chinese Merino fine-wool sheep, and carried out methylation detection. The test results show that 3 sites of the KAP8 gene promoter of fine-wool sheep have undergone methylation modification, while only 1 site of the KAP8 gene promoter in the DNA of coarse-wool sheep has undergone methylation modification or no methylation, and 2 sites of the KAP8 gene promoter of the hybrid offspring of fine-wool sheep have undergone methylation modification. This result can be used for the identification of fine-wool sheep and the early determination of wool fineness, and can be used for molecular marker-assisted breeding to accelerate the breeding process. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram for designing primers for upstream segment amplification of KAP8.

[0030] Figure 2 This is a diagram showing the CpG enrichment in the promoter region of the KAP8 gene of Chinese Merino sheep.

[0031] Figure 3 It is a potential methylation site in the promoter of KAP8 gene (the upstream sequence is the original sequence, and the downstream sequence is the methylated sequence).

[0032] Figure 4 This is a diagram of the methylation-specific PCR amplification results (M represents the Marker solution, 1-2 represents Suffolk rough-wool sheep, and 3-4 represents Chinese Merino fine-wool sheep).

[0033] Figure 5 This is the sequencing peak diagram of the promoter region of the KAP8 gene (A represents Chinese Merino fine-wool sheep, and B represents Suffolk coarse-wool sheep).

[0034] Figure 6 is the lollipop model of the methylation level of the KAP8 gene promoter (rows 1 to 8 represent the 8 methylation sites predicted in the promoter region of the KAP8 gene, columns 1 to 3 of columns 1 to 6 represent the positive clones of Chinese Merino fine-wool sheep, and columns 4 to 6 represent the positive clones of Suffolk sheep. In the figure, white dots represent no methylation, and black dots represent GC sites with methylation modification).

[0035] Figure 7 This is the prediction result of transcription factor at the third methylation site. DETAILED DESCRIPTION

[0036] The invention provides a primer pair for detecting the DNA methylation level of a sheep KAP8 gene promoter. The sequence of the primer pair is shown in SEQ ID NOs: 1-2.

[0037] The present invention also provides a method for detecting the DNA methylation level of the promoter of sheep KAP8 gene using the primer pair, comprising the following steps:

[0038] (1) extracting DNA from in vitro sheep skin tissue, and performing methylation treatment on the DNA to obtain methylated genomic DNA to be tested;

[0039] (2) Using the methylated genomic DNA to be tested as a template, using the primer pair to perform PCR amplification, detecting the PCR amplification product, screening the positive product for sequencing, comparing the sequencing results, and determining the DNA methylation level of the sheep KAP8 gene promoter according to the comparison results.

[0040] In the present invention, the ex vivo sheep skin tissue is sheep ear skin tissue and / or shoulder blade skin tissue. The ex vivo sheep skin tissue is collected from the lamb after birth to before sexual maturity. In the present invention, the method for collecting sheep skin tissue is: shearing the wool and disinfecting and wiping the skin tissue, soaking the cut skin tissue in 75% alcohol for 15 minutes and then taking it out for use. In the present invention, the method for extracting DNA from ex vivo sheep skin tissue refers to a commercial DNA extraction kit or refers to the "Molecular Cloning Experiment Guide" for DNA extraction.

[0041] In the present invention, the methylation treatment is bisulfite methylation treatment.

[0042] In the present invention, the conditions for the PCR amplification are:

[0043] Pre-denaturation at 94°C for 4.5-5.5 min; denaturation at 94°C for 29-35 s, annealing at 58-65°C for 38-50 s, extension at 72°C for 43-60 s, 34-40 cycles; then keep at 72°C for 6.5-7.5 min. Preferably, pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 62.5°C for 40 s, extension at 72°C for 45 s, 35 cycles; then keep at 72°C for 7 min.

[0044] In the present invention, the size of the PCR amplification product is 193 bp.

[0045] In the present invention, the detection in step (2) is agarose gel electrophoresis detection;

[0046] The initial concentration of agarose during the agarose gel electrophoresis detection is 1.5-2.5wt%, preferably 2wt%. In the present invention, the agarose gel electrophoresis detection system also includes Gelred nucleic acid dye. In the present invention, the concentration of Gelred nucleic acid dye is 0.02-0.5‰, preferably 0.1‰.

[0047] In the present invention, the sequencing is direct bidirectional sequencing or sequencing by connecting the positive product to an empty vector; the empty vector is pMD18-T; and the alignment is performed using Megalign software.

[0048] The present invention also provides the application of the primer pair or the method in judging sheep breeds, detecting wool traits or selecting sheep breeds.

[0049] In the present invention, when judging sheep breeds or detecting wool traits or breeding sheep breeds, the DNA methylation level of the sheep KAP8 gene promoter is used;

[0050] When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene was >2, it was determined to be a fine-wool sheep breed;

[0051] When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene was ≤1, it was determined to be a coarse-wool sheep breed;

[0052] When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene = 2, it is determined to be a breed between fine-wool sheep and coarse-wool sheep.

[0053] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1 Analysis of differential expression of KAP8 gene in fine wool sheep and coarse wool sheep

[0055] Based on the difference in the content of keratin-associated protein KAP8 in fine and coarse wool and its high correlation with wool fineness, the study of the difference in KAP8 gene expression level is an important basis for proving its role in wool production and regulation. Using the fluorescence real-time quantitative PCR method, the full-length cDNA sequence of the KAP8 gene was cloned from the dermis of the left anterior scapula (the tissue layer where the hair follicles are located) of three Chinese Merino fine-wool sheep and Suffolk sheep, and primers were designed according to the obtained cDNA sequence (the accession number of the KAP8 gene sequence in NCBI is X05639.1) and the mRNA level of the KAP8 gene in the skin hair follicles of fine-wool sheep and coarse-wool sheep was detected by fluorescence real-time quantitative PCR. The results showed that the mRNA level of the KAP8 gene in the coarse-wool sheep Suffolk was significantly higher than that in the Chinese Merino fine-wool sheep (the relative reference gene expression levels were 14.80±2.91 and 2.52±0.36, respectively), reaching 5.87 times, indicating that wool fineness is positively correlated with the expression of the KAP8 gene. As an important element in regulating gene expression, the regulatory role of promoter in the gene expression process deserves in-depth study.

[0056] The primer sequences of KAP8 are shown in SEQ ID NOs: 3-4; the primer sequences of internal reference GAPDH are shown in SEQ ID NOs: 5-6.

[0057] Table 1 Primers for KAP8 gene fluorescence quantitative PCR

[0058]

[0059] Example 2 Cloning and analysis of sheep KAP8 promoter sequence

[0060] According to the KAP8 gene sequence (X05639.1), specific primers were designed, and TAIL-PCR (thermal asymmetric interlaced PCR) was used to obtain the upstream 5′ sequence of the KAP8 gene. After sequencing and splicing, a full-length sequence of 3297 bp (submitted to NCNI, accession number MT090064) was obtained, of which the upstream length of the coding sequence was 1826 bp. Based on the obtained sequence, primers for amplifying the upstream of the transcription start point were designed (as shown in Table 2 and SEQ ID NO: 7-14) (the schematic diagram of the design of the upstream segment amplification primers of the KAP8 gene is shown in Figure 1 PCR amplification was performed using 30 DNA samples from Chinese Merino fine-wool sheep and 30 DNA samples from Suffolk sheep. Positive PCR products were directly sequenced and spliced.

[0061] According to the sequencing results, representative sequences of fine-wool sheep and coarse-wool sheep were selected for further analysis according to the analysis of two sheep populations. The upstream sequences of KAP8 of 60 samples obtained by PCR cloning were manually compared and spliced, and four haplotype representative sequences of Chinese Merino fine-wool sheep and two haplotype representative sequences of Suffolk coarse-wool sheep were obtained according to genotype classification.

[0062] Table 2 Primer sequences for upstream segment amplification of KAP8 gene

[0063]

[0064] Example 3 Prediction of CpG island in KAP8 gene promoter sequence

[0065] The four haplotype representative sequences of Chinese Merino fine-wool sheep and the two haplotype representative sequences of Suffolk coarse-wool sheep were entered into the online website one by one and analyzed, and the core promoter sequence of the KAP8 gene of the two sheep and the CpG island region that may be methylated were predicted. The possible existence of CpG islands was predicted using methprimer (http: / / www.urogene.org / methprimer / ) and SMS (http: / / www.bioinformatics.org / sms2 / cpg islands.html). The results of bioinformatics analysis showed that a 108bp long CpG island was predicted in the promoter region of the KAP8 gene of Chinese Merino fine-wool sheep, with a specific location of -518 to -410 ( Figure 2 ), including up to 8 potential methylation sites in this region ( Figure 3 ), while Suffolk sheep had no or only one methylation site under the same circumstances.

[0066] Figure 2 The results showed that there was a CpG island in the promoter region of KAP8 gene.

[0067] Figure 3 It was shown that there were 8 potential methylation sites in the promoter region of the KAP8 gene of fine-wool sheep.

[0068] Example 4 KAP8 gene promoter DNA methylation analysis

[0069] Three DNA samples were taken from each haplotype of Chinese Merino fine wool sheep and Suffolk rough wool sheep, and the methylation detection and differential analysis of the KAP8 gene promoter region were performed using the bisulfite sequencing technology BSP method, and specific primers for predicting methylation sites in the KAP8 gene promoter region were designed based on the prediction results. The specific primer sequences are shown in SEQ ID NO: 1-2.

[0070] Upstream primer K8CpGL: 5′-TTTTATGGATAGAGGAGTTTGGAAG-3′;

[0071] Downstream primer K8CpGR: 5'-TTTCCACAACACACAATTAAAATTC-3',

[0072] The length of the amplified product designed by the primers is 193 bp, and the above primers were used for detection of Chinese Merino fine-wool sheep and Suffolk coarse-wool sheep.

[0073] The sheep DNA to be tested was treated with bisulfite using a methylation kit, and the treated DNA was subjected to methylation-specific PCR amplification. The basic procedure was: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 62.5°C annealing for 40 s, 72°C extension for 45 s, 35 cycles, and 72°C extension for 7 min. The amplified product was detected by 2% agarose gel electrophoresis, and a single band was obtained. The amplified product size was 193 bp. The results are as follows Figure 4 The positive products were recovered by gel ligation into the cloning vector (pMD18-T) and sent to Hangzhou Qingke Biotechnology Co., Ltd. for cloning and sequencing.

[0074] Figure 4 The results of methylation-specific PCR amplification were consistent with the expected results, both of which were 193 bp.

[0075] Megalign software was used to align the methylated sequence obtained by sequencing with the original sequence, and the complete sequence was obtained after peak graph proofreading and splicing to confirm that it was the target fragment. Figure 5 Bisulfite can convert unmethylated cytosine (C) into uracil (U). If there is still a GC site in the sequence obtained by sequencing, it is determined that the site is methylated.

[0076] Figure 5 The results showed that the methylation of the KAP8 promoter region in coarse-wool sheep and fine-wool sheep was significantly different. The clone sequencing peak diagram showed that there were 3 methylation sites in the KAP8 gene regulatory region of Chinese Merino sheep, and only 1 methylation site in Suffolk sheep. Chinese Merino sheep were methylated at the 2nd, 3rd and 4th sites, while Suffolk sheep were only methylated at the 2nd site.

[0077] The online website MSR (http: / / www / msrcall.com / MSRcalcalate.aspx) was used to draw two different lollipop models of sheep KAP8 promoter methylation sites. Figure 6 shown.

[0078] Figure 6The results showed that there were significant differences in the methylation sites of the KAP8 gene between Chinese Merino sheep and Suffolk sheep. The incidence of CpG island methylation in fine-wool sheep was significantly higher than that in coarse-wool sheep, with an incidence rate of 3:1.

[0079] Bioinformatics analysis was performed on the three methylation sites in the methylated Chinese Merino sheep DNA. The prediction results of Alibaba2 online website showed that there was a Sp1 transcription factor binding region at the methylated site 4, and the sequence of -464 to -455 was CGCGCGCACG. Figure 7 As shown. Transcription factor Sp1 usually binds to the GC box (GGGGCGGGG) to regulate gene expression. According to the increase or loss of transcription factor binding sites in the DNA sequence after methylation modification, it plays a positive or negative regulatory role on gene expression. The GC sequence at -464 of the promoter of the fine-wool sheep KAP8 gene that binds to the transcription factor Sp1 disappears due to methylation modification, and cannot bind to DNA, thereby reducing gene expression. The preliminary results of gene expression detection and methylation detection indicate that the KAP8 gene affects gene expression through DNA methylation modification. Therefore, it is inferred that this regulatory pathway is one of the reasons that affect the thickness of wool.

[0080] Example 5 Application of KAP8 gene sequence DNA methylation detection

[0081] 10 2-month-old lambs and adult sheep of Chinese Merino fine wool sheep, 10 adult Chinese Merino meat multi-birth strains, Charolais sheep, Dorset sheep, and Duolang sheep were randomly selected from the experimental sheep farm of Xinjiang Academy of Agricultural Reclamation Sciences, and ear tissue skin was taken, and DNA was extracted according to the animal tissue DNA kit of Hangzhou Xinjing Biotechnology Co., Ltd. The methylation detection and analysis of the KAP8 gene regulatory region were performed with reference to the method of Example 4. The results showed that DNA methylation modification occurred in the KAP8 gene regulatory region of 2-month-old lambs and adult sheep of Chinese Merino fine wool sheep, and there were 3 sites, and DNA methylation modification occurred in the KAP8 gene regulatory region of the Chinese Merino meat multi-birth strain, and there were 2 sites, while the DNA of other sheep had only 1 site (18 / 30) or no DNA methylation modification (12 / 30) in the KAP8 gene regulatory region.

[0082] From the above examples, it can be seen that the present invention provides a primer pair for detecting the DNA methylation level of the KAP8 gene promoter in sheep, its application and methylation detection method. According to the method of the present invention, the DNA methylation level of the KAP8 gene promoter in sheep can be detected, and the DNA methylation level of the gene promoter can be used to evaluate the wool fineness of the breed of sheep, which can be used for molecular marker-assisted breeding to accelerate the breeding process.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of the primer pair shown in SEQ ID NO: 1-2 in determining the thickness of sheep wool, characterized in that: When the primer pair is used to determine the thickness of sheep wool, follow the steps below: (1) extracting DNA from in vitro sheep skin tissue, and performing methylation treatment on the DNA to obtain methylated genomic DNA to be tested; (2) Using the methylated genomic DNA to be tested as a template, performing PCR amplification using the primer pairs shown in SEQ ID NOs: 1-2, detecting the PCR amplification products, screening the positive products for sequencing, comparing the sequencing results, and determining the DNA methylation level of the promoter of the sheep KAP8 gene based on the comparison results, thereby determining the coarseness of the sheep wool; The ex vivo sheep skin tissue is sheep ear skin tissue and / or shoulder blade skin tissue; The methylation treatment is a bisulfite methylation treatment; The conditions for the PCR amplification are: Pre-denaturation at 94°C for 4.5-5.5 min; denaturation at 94°C for 29-35 s, annealing at 58-65°C for 38-50 s, extension at 72°C for 43-60 s, 34-40 cycles; then maintain at 72°C for 6.5-7.5 min; The size of the PCR amplification product is 193 bp; The detection in step (2) is agarose gel electrophoresis detection; The initial concentration of agarose during the agarose gel electrophoresis detection is 1.5-2.5 wt %; The sequencing is bidirectional sequencing or sequencing by connecting the positive product to an empty vector; The empty vector is pMD18-T; The alignment was performed using Megalign software.

2. The application of the method for detecting the DNA methylation level of the sheep KAP8 gene promoter in judging the thickness of sheep wool is characterized in that: The steps include: (1) extracting DNA from in vitro sheep skin tissue, and performing methylation treatment on the DNA to obtain methylated genomic DNA to be tested; (2) Using the methylated genomic DNA to be tested as a template, performing PCR amplification using the primer pairs shown in SEQ ID NOs: 1-2, detecting the PCR amplification products, screening the positive products for sequencing, comparing the sequencing results, and determining the DNA methylation level of the promoter of the sheep KAP8 gene based on the comparison results, thereby determining the coarseness of the sheep wool; The ex vivo sheep skin tissue is sheep ear skin tissue and / or shoulder blade skin tissue; The methylation treatment is a bisulfite methylation treatment; The conditions for the PCR amplification are: Pre-denaturation at 94°C for 4.5-5.5 min; denaturation at 94°C for 29-35 s, annealing at 58-65°C for 38-50 s, extension at 72°C for 43-60 s, 34-40 cycles; then maintain at 72°C for 6.5-7.5 min; The size of the PCR amplification product is 193 bp; The detection in step (2) is agarose gel electrophoresis detection; The initial concentration of agarose during the agarose gel electrophoresis detection is 1.5-2.5 wt %; The sequencing is bidirectional sequencing or sequencing by connecting the positive product to an empty vector; The empty vector is pMD18-T; The alignment was performed using Megalign software.

3. The use according to claim 1 or 2, characterized in that: The thickness of sheep wool is determined based on the DNA methylation level of the sheep KAP8 gene promoter; When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene was >2, it was determined to be a fine-wool sheep breed; When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene was ≤1, it was determined to be a coarse-wool sheep breed; When the number of DNA methylation modification sites in the promoter of sheep KAP8 gene = 2, it is judged to be a breed between fine-wool sheep and coarse-wool sheep.

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