Chicken SNP marker in promoter region of TRAF7 gene and its application in chicken breeding

Abstract

The application discloses a chicken TRAF7 gene promoter region SNP marker and application thereof in chicken breeding and belongs to the technical field of molecular genetics. The application finds that a SNP marker exists in the chicken TRAF7 gene promoter region through screening of SNPs between breeds, the nucleotide sequence of the SNP marker is shown as SEQ ID No. 1, and the 122th base from the 5' end of the sequence shown as SEQ ID No. 1 is a SNP site, the base of which is A or T. The SNPs genotype of Jining hundred-day chicken population is firstly correlated with the egg laying trait, and it is found that the individual with the TT ‑896 genotype corresponds to an earlier age of first egg laying; the individual with the TT ‑896 genotype corresponds to a higher maximum number of continuous egg laying, therefore, the molecular marker associated with the SNP site and the egg laying trait is detected, which is not only simple, fast and convenient, but also helps to breed a high-egg-yielding chicken breed and provides favorable help for breeding work.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics, specifically to a chicken TRAF7 gene promoter region SNP marker and its application in chicken breeding. Background Technology

[0002] The TRAF family is a class of multifunctional proteins and important adaptor proteins in the body's antiviral signaling pathways. In mammals, TRAFs can bind to multiple receptor families, including TNF receptors and TLRs / IL-1R (Toll-like / IL-1 receptor), to transduce upstream signals and activate multiple downstream signaling pathways, such as NF-κB (nuclear factor κB) and MAPK (mitogen-activated protein kinase). This, in turn, regulates important physiological processes such as immune responses, apoptosis, stress responses, and bone metabolism. TRAF7 was initially discovered as a novel protein interacting with MEKK3 in the TNFα-induced NF-κB signaling pathway (Bouwmeester et al., 2004). As an important signal adaptor protein and regulatory molecule, TRAF7 participates in multiple receptor-mediated signaling pathways, including TNFR and TLR2 (Zotti et al., 2011; Nakamura et al., 2003). Due to its unique RING domain, TRAF7 possesses E3 ubiquitin ligase activity (Bouwmeester et al., 2004).

[0003] TRAF7, an important molecule involved in regulating innate and adaptive immunity, is associated with the development of various diseases. Studies have found that in muscle cells, the myogenic regulatory factor MyoD1 (Myoblast determination protein 1) can bind to the TRAF7 promoter, regulating TRAF7 expression at the transcriptional level (Tsikitis et al., 2010). TRAF7 deficiency induces premature differentiation of muscle cells. In breast cancer, decreased TRAF7 levels hinder the ubiquitination and degradation of p53, leading to p53 accumulation in the cytoplasm and affecting its normal transcriptional activity, both contributing to cancer development. Therefore, decreased TRAF7 levels and p53 cytoplasmic accumulation can serve as predictive indicators of breast cancer development (Wang et al., 2013; Wu et al., 2012). Further studies have found that in venous endothelial cells, miR-126 directly acts on the 3'UTR of TRAF7, inhibiting TRAF7 expression at the posttranscriptional level. Endothelial cell apoptosis is an important factor in the pathogenesis of atherosclerosis and plaque rupture (Libby et al., 2011), and miR-126 inhibits apoptosis by reducing TRAF7 expression and ROS formation (Wang et al., 2015). TRAF7 acts as a tumor regulator in various cancers; 67% of malignant mesothelioma patients have TRAF7 deficiency in pleural effusion malignant cells. TRAF7 mutations mainly help improve the diagnosis, prognosis, and treatment of meningioma patients (Zotti et al., 2017; Sneddon et al., 2019; Klein et al., 2017). Our research group previously performed transcriptome sequencing on individual ovarian follicles with different FSHR expression levels in chickens. We found that the expression level of FSHR was similar in all individual ovarian follicles, but its expression level was significantly increased, except for selected ovarian follicles. According to previous studies, the mRNA level of the TRAF7 gene was also increased in ovarian follicles with high FSHR mRNA expression levels (Zhong Conghao, 2021).

[0004] Single nucleotide polymorphism (SNP) refers to the diversity of DNA sequences caused by variations in a single nucleotide at the genomic level. It is the most common type of heritable variation, abundant in number and rich in polymorphism. It includes variations such as single-base transitions, insertions, and deletions (Duan Fang, 2011). When the variation of a single nucleotide is greater than 1%, it is called a single nucleotide polymorphism; when it is less than 1%, this variation is usually called a genetic variation or mutation. Due to its strong genetic stability, SNPs are widely used in population genetics, disease-related gene mapping, and other research (Housman et al., 1998; Hu et al., 2016). In recent years, with the advancement of chicken genome sequencing technology, target genes are sequenced by searching chicken-related databases to screen for relevant SNP loci. Then, through statistical analysis, information on polymorphic sites of related genes is obtained (Liu Wenbo et al., 2011). The application of SNPs in poultry breeding is becoming increasingly widespread. By detecting SNP loci, many QTLs related to economic traits in poultry can be screened out, greatly shortening the breeding process (Li Ning et al., 2003). Three SNPs related to egg production in ACE were detected in 800 Taihang chickens. The study found that the ACE g.5066812A>C mutation was significantly associated with the egg production performance of Taihang chickens (P<0.05), and individuals with the g.5066812A>C mutation had a significantly increased egg production (Wang et al., 2022). In investigating the relationship between TAT gene polymorphism and Muscovy duck production performance, six SNP sites in the TAT gene (g.120G>T, g.122G>A, g.254G>A, g.270C>T, g.312G>A, and g.341C>A) were identified. Only g.254G>A and g.270C>T showed significant correlations with Muscovy duck egg production (Ju et al., 2023). GWAS analysis was used to analyze functional genes contributing to brown-shelled egg quality (Wolc et al., 2019). A single SNP (g.19942455C>G) was identified in the intron region of the chicken RBP4 gene, and this site showed significant associations with age at first laying, egg weight, and total egg production (Yin et al., 2013). In recent years, increasing research has found that alterations in SNPs in poultry genes can serve as potential genetic markers that can improve their production performance.

[0005] However, there are currently no reports on the correlation between SNP markers in the promoter region of the TRAF7 gene and egg production performance. Therefore, studying the SNPs in the 5′ promoter region of the TRAF7 gene can help identify meaningful molecular markers, provide a favorable theoretical basis for marker-assisted breeding, and facilitate the rapid breeding of early-laying and high-yielding breeds. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a SNP marker in the promoter region of the chicken TRAF7 gene and its application in chicken breeding. This invention has discovered that there is one SNP marker in the promoter region of the chicken TRAF7 gene that is associated with the egg production trait, which can be used for marker-assisted breeding of chickens.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a SNP marker for the promoter region of the chicken TRAF7 gene, wherein the nucleotide sequence of the SNP marker for the promoter region of the chicken TRAF7 gene is shown in SEQ ID No. 1; the 122nd base from the 5' end of the sequence shown in SEQ ID No. 1 is the SNP site, and its base is A or T. Specifically, as follows:

[0009] actgctcgca cttctcatcc gtgctggctc gtttacagct ttttgtttca tccctgttaa 60

[0010] taaatttttt tttgtaataa tctctgcggc tacggtggta ttttggctat tgccgcacac 120

[0011] a[a / t]cccggttg gttgaagtag cgtggttcct cgcgtacgca gagccaaagg tccgagccgg180tctgtggttt ctcgtgcgac gcacagaagg gacggaagca gaagttcgag tcagaaaact240ttattgaatc cgtaagctgt gaggtcagcc cggtacggcg gccccagggg agacacggag300gccgccaagc ggtgcgggcc gcacagcgcc gcgctcagcc ctccatcccc taccaccgcc360acgcgtaccc agcccggagc tcgcgaggag ccggggaagc gtgccgggcc ctcaggggag420caccccctgg ctctcgctgc ccctcacgcc gctgagattc agttcccgac aggtcctgag480gagagaaggc agcgcggtaa gagcgagccg caccgcccgg cccagcccca gcgcggggcc540gcgagcctca gccgtagcgc gtcagcagtg gccacggccg cccgccgccc tcagacgagg600tgtcagaaag gca

[0012] Note: "[a / t]" in the sequence represents an SNP site, which is represented by "n" in the sequence listing.

[0013] The SNP marker in the promoter region of the chicken TRAF7 gene is located 896 bp upstream of the transcription start site of the chicken TRAF7 gene (GenBank Accession NC_052545.1), and is named g.-896. The study found that this site is significantly associated with the egg production trait of chickens.

[0014] In a second aspect, the present invention provides the application of the above-mentioned chicken TRAF7 gene promoter region SNP marker in chicken genetic breeding.

[0015] In the above applications, chicken genetic breeding includes the selection and breeding of laying hen breeds with early onset of egg production, high egg production, and / or high maximum consecutive laying count.

[0016] Furthermore, in the above application, the chicken TRAF7 gene promoter region SNP marker is TT. -896 Individuals with this genotype exhibit egg-laying traits such as early onset of laying, high egg production, and a long consecutive laying period.

[0017] TT -896 An individual with a genotype is defined as having a TT genotype at the g.-896 ​​site of the TRAF7 gene promoter region.

[0018] A third aspect of the present invention provides primer pairs for detecting the SNP markers in the promoter region of the chicken TRAF7 gene, the nucleotide sequences of which are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively. Specifically:

[0019] P-TRAF7-F: 5'-ACTGCTCGCACTTCTCATCC-3'; (SEQ ID NO. 2)

[0020] P-TRAF7-R: 5'-TGCCTTTCTGACACCTCGTC-3'. (SEQ ID NO.3)

[0021] In a fourth aspect, the present invention provides a kit for detecting the above-mentioned chicken TRAF7 gene promoter region SNP markers, the kit comprising the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3.

[0022] A fifth aspect of the present invention provides the application of the above-mentioned primer pairs and / or kits in the assisted breeding of laying hen breeds; wherein the laying hen breed has laying traits such as early onset of egg production, high egg production, and / or high maximum consecutive laying count.

[0023] A sixth aspect of the present invention provides a method for identifying egg-laying traits in laying hens, comprising the following steps:

[0024] Using the genomic DNA of the laying hens to be tested as a template, PCR amplification was performed using the primer pairs shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the amplification products; the amplification products were sequenced, and the egg production traits of the laying hens were identified based on the sequencing results.

[0025] Specifically, if the sequencing result corresponds to the TT genotype at position 122 from the 5' end of the sequence shown in SEQ ID No. 1, then it is identified as having the egg-laying traits of early onset of laying, high egg production, and / or high consecutive laying count.

[0026] A seventh aspect of the present invention provides a method for assisting in the breeding of layer chicken breeds using the above-mentioned chicken TRAF7 gene promoter region SNP markers, comprising the following steps:

[0027] In the chicken population, those with the TT genotype were selected. -896 The rooster and the TT genotype -896 The hen, purebred to obtain TT -896Offspring of genotype; TT -896 The offspring of this genotype start laying eggs earlier than those of other genotypes, and have a higher number of eggs laid and a higher maximum number of consecutive lays.

[0028] The beneficial effects of this invention are:

[0029] This invention, through screening SNPs among breeds, discovered SNP sites in the promoter region of the chicken TRAF7 gene; and for the first time, it conducted an association analysis between the SNP genotypes and egg production traits of the Jining 100-day-old chicken population, finding that TT -896 Genotypes correspond to earlier age at onset of labor; TT -896 Genotype individuals correspond to higher maximum consecutive laying counts. Therefore, detecting molecular markers associated with this locus and the egg production trait is not only a simple and quick method, but also helps in the selection of high-producing egg-laying chicken breeds, providing valuable assistance for breeding work. Attached Figure Description

[0030] Figure 1 Electrophoresis image of fragments amplified by PCR using P-TRAF7-F / R primers.

[0031] Figure 2 Polymorphism in key regulatory regions of the TRAF7 gene promoter.

[0032] Figure 3 Polymorphism diagram of the chicken TRAF7 gene promoter region at position 896.

[0033] Figure 4 Sequence alignment of mutant vectors pGL3-(-896A) and pGL3-TRAF7.

[0034] Figure 5 The effect of SNPs on the transcriptional activity of the TRAF7 promoter. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] As mentioned earlier, the TRAF7 gene is a multifunctional gene. To further investigate the relationship between the TRAF7 gene and the breeding of laying hen breeds, the inventors used DNAMAN 6.0 software to analyze the TRAF7 gene sequences of different laying hen breeds, screened for polymorphic sites among breeds, and compared the screened polymorphic site information with the TRAF7 gene sequence on NCBI, found that there may be a g.-896A>T among different breeds.

[0037] Then, by analyzing the allele frequencies and genotype frequencies of candidate SNPs in different breed populations, and based on the genotypic statistics of 337 Jining 100-day-old chickens with production records, one-way ANOVA was used in IBM SPSS Statistics 27 software to perform association analysis between the genotype of each individual and the laying trait (AFE: age at first laying; E52: number of eggs laid at 52 weeks; and LCS: maximum consecutive laying). The results showed that the association analysis of the polymorphic locus g.-896A>T genotype with the laying trait was significantly correlated with age at first laying and maximum consecutive laying (P<0.05). The TT genotype at the g.-896A>T locus corresponded to an earlier age at first laying (AFE: 147.22 days) and a higher maximum consecutive laying (LCS: 42.11 eggs). This indicates that the homozygous genotype of this polymorphic locus is associated with the traits of age at first laying and maximum consecutive laying. Based on the fact that this polymorphic site can be used for the breeding of superior laying hen breeds, this invention is proposed.

[0038] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0039] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0040] Jinghong No. 1 laying hen (blood sample from Li Shijun's research group at Huazhong Agricultural University), Rizhao Langya chicken (Shandong Jihua Poultry Breeding Co., Ltd.), Zaozhuang Sunzhi chicken (Zaozhuang Fojiaoya Poultry Breeding Professional Cooperative), Hy-Line Brown commercial laying hen (Linxi Village, Daiyue District, Tai'an City, Shandong Province), and Jining 100-day chicken (Jining Datang 100-day chicken conservation farm).

[0041] Example 1: Identification and analysis of SNP markers in the promoter region of the chicken TRAF7 gene

[0042] 1. Test method:

[0043] Based on whole-genome sequencing results, multiple mutation sites were found in the promoter region of the chicken TRAF7 gene. In this example, several different breeds of laying hens were selected to screen and analyze SNP markers in the key promoter region of the chicken TRAF7 gene, as detailed below:

[0044] Thirty-four genomic DNA samples from each of the following chicken breeds were randomly selected: Jinghong chicken, Jining 100-day chicken, Zaozhuang Sunzhi chicken, Rizhao Langya chicken, and Hy-Line Brown commercial laying chicken. The key promoter region of the TRAF7 gene was amplified. After PCR amplification, the DNA was detected by 1% agarose gel electrophoresis. The gel was cut off and recovered and sent to BGI Genomics for forward and reverse sequencing. The sequencing sequences were then compared with the key promoter region of the TRAF7 gene on the NCBI website to preliminarily screen for SNP sites.

[0045] The primers used for amplification based on the published red junglefowl sequence (GenBank Accession NC_052545.1) are P-TRAF7-F and P-TRAF7-R, and the primer sequences are shown in Table 1.

[0046] The reaction mixture consisted of 25 μL of genomic DNA (50-100 ng), 12.5 μL of 2×Phanta MaxMaster Mix (Novizan), 1 μL each of forward and reverse primers (10 μM), and ddH2O to a final volume of 25 μL. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, annealing for 15 sec (annealing temperatures as shown in Table 1), 72℃ extension for 1 min 35 sec, for 34 cycles; after each cycle, a final extension was performed at 72℃ for 5 min.

[0047] Table 1: Primer sequences, annealing temperatures, and product lengths

[0048]

[0049] 2. Results and Analysis:

[0050] Gel electrophoresis results of amplified key promoter regions of the TRAF7 gene are as follows: Figure 1 As shown, the bands exhibit uniformity and are the correct size.

[0051] The sequencing results of genomic DNA from the blood of five chicken breeds—Hy-Line Brown, Jinghong, Zaozhuang Sunzhi, Rizhao Langya, and Jining 100-day chicken—were compared. The comparison results are as follows: Figure 2 As shown in the figure. The results indicate that the SNP site g.-896(A>T) exists in all different breeds of chickens.

[0052] The results of first-generation sequencing were analyzed for polymorphic sites using Chromas software, and the results are as follows: Figure 3 As shown, this site is a peak diagram of the reverse sequence, and two genotypes were obtained.

[0053] Example 2: Statistical analysis of SNP markers in the promoter region of the chicken TRAF7 gene in different breed populations

[0054] Based on the genotyping results of whole-genome sequencing and sequencing peak profiles, the genotype and allele frequencies of locus g.-896 ​​(A>T) were analyzed in the populations of Jinghong, Zaozhuang Sunzhi, Rizhao Langya, and Hy-Line Brown chickens. The chi-square test was used to determine whether each population conformed to Hardy-Weinberg equilibrium. The results are shown in Table 2.

[0055] Table 2: Genotype and allele frequency distribution of SNP markers in the chicken TRAF7 gene promoter region

[0056]

[0057] The results showed that the g.-896 ​​(A>T) locus met the Hardy-Weinberg equilibrium requirement in Langya chicken (P>0.05). The dominant allele for this locus was A in all four breeds.

[0058] Example 3: Association analysis of SNP markers in the promoter region of the chicken TRAF7 gene with age at first laying, number of eggs laid, and maximum consecutive laying number.

[0059] 1. Test materials

[0060] One-day-old Jining 100-day chickens were randomly selected from the Jining Datang 100-day Chicken Breeding Farm, wing tags were attached and recorded, and they were raised under the same environmental conditions. Production indicators for each chicken at various stages were recorded, such as age at first egg production, total egg production at 52 weeks of age, and longest consecutive laying period, resulting in a sample of 337 Jining 100-day chickens with production records. Approximately 2 mL of blood was collected from the ulnar vein and stored in the laboratory at -20°C.

[0061] 2. Test methods

[0062] 2.1 PCR amplification

[0063] Using the genome of Jining 100-day chickens with production performance records as a template, PCR amplification was performed. Primers are shown in Table 1. The reaction volume was 25 μL, including 1 μL of genomic DNA (50-100 ng), 12.5 μL of 2×Phanta Max Master Mix (Novizan), 1 μL each of forward and reverse primers (10 μM), and ddH2O to a final volume of 25 μL. The amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, annealing for 15 sec (annealing temperatures as shown in Table 1), 72℃ extension for 1 min 35 sec, for 34 cycles; after the cycles, a final extension was performed and incubated at 72℃ for 6 min.

[0064] PCR amplification products were subjected to agarose gel electrophoresis and then recovered and sequenced using an agarose gel recovery kit (Kangwei Century) (BGI Genomics, Qingdao).

[0065] 2.2 Haplotype Construction and Association Analysis

[0066] Statistical analysis of mutation sites was performed using DNAMAN and ChromasPro.

[0067] R software is used to count genotypes and genotype frequencies, while PHASE software is used for haplotype construction.

[0068] The correlation between polymorphic loci or diploids and egg production indicators (age at first laying, number of eggs laid, and maximum consecutive laying number) was analyzed using a general linear model in IBM SPSS Statistics 27 software. The mathematical model was Yi = μ + Gi + εi (where Yi is the phenotypic value, μ is the population mean, Gi is the effect value of genotype or diploid, and εi is the random error effect). LSD multiple comparison analysis was performed, and the results were expressed as mean ± standard error. The significance level was set at P < 0.05.

[0069] 3. Results and Analysis

[0070] In a population of 337 Jining 100-day-old chickens, the effect of the g.-896 ​​(A>T) site in the promoter region of the TRAF7 gene on age at first laying (AFE), total egg production at 52 weeks (E52), and maximum consecutive laying count (LCS) was analyzed. The results are shown in Table 4. Table 4: Association analysis of genotype at the g.-896 ​​site in the promoter region of the TRAF7 gene in Jining 100-day-old chickens with age at first laying (AFE), egg production at 52 weeks, and maximum consecutive laying count (LCS).

[0071]

[0072] Note: The values ​​in the table are the least squares mean ± standard error of age at first laying (AFE), total number of eggs laid at 52 weeks (E52), and maximum consecutive laying (LCS). There are no significant differences among the least squares means without the same letters (P < 0.05). * indicates a significant difference.

[0073] The results showed that the -896 locus was significantly associated with age at first laying (AFE) and maximum consecutive laying (LCS) (p<0.05). The TT genotype corresponded to an earlier age at first laying (AFE: 147.22 days), more total eggs laid at 52 weeks, and a higher maximum consecutive laying (LCS: 42.11).

[0074] Example 4: Effect of SNP markers in the promoter region of the chicken TRAF7 gene on gene expression

[0075] 1. Test materials

[0076] Three to five healthy Hy-Line Brown chickens at their peak egg-laying period were randomly collected from a poultry farm in Linxi Village, Tai'an City.

[0077] 2. Test Methods

[0078] 2.1 Construction of a mutant luciferase expression vector with a polymorphic site in the 5′ promoter region of the chicken TRAF7 gene

[0079] 1) Design of site-directed mutagenesis primers

[0080] Using the TRAF7 gene promoter region as a template, a mutation primer for the g.-896 ​​(A>T) site was designed. The primer sequence information is shown in Table 5.

[0081] Table 5: Primers for single point mutations

[0082]

[0083] 2) PCR amplification

[0084] The amplification reaction employed a high-fidelity enzyme. The reaction system (50 μL) included: 25 μL of 2×Phanta Max Master Mix (DyePlus), 2 μL each of forward and reverse primers, 2 μL of genomic DNA (50-100 ng), and ddH2O to a final volume of 50 μL. The reaction program was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s; annealing for 15 s; extension at 72℃ for 3 min 20 s; 34 cycles; and final extension at 72℃ for 6 min. PCR products were detected by 0.9% agarose gel electrophoresis at 220V for 15 min. Subsequently, the DNA fragment was recovered using an AxyPrep DNA Gel Extraction Kit (AXYGEN). The target fragment was ligated into a vector, transformed, and single-clone cultured overnight. Sequencing was then used to verify successful mutation.

[0085] 3) Preparation of endotoxin-free plasmids

[0086] The procedure was performed using the endotoxin-free high-purity plasmid miniprep kit from Adley Biotech, following the instructions.

[0087] The constructed vector plasmids pGL3-TRAF7 and pGL3-(-896T) were retransformed into competent cells. Single colonies were picked, cultured, and dual-luciferase expression vector plasmids were extracted using Adley Biotech's endotoxin-free plasmid large-scale extraction kit for primary cell transfection.

[0088] 2.2 Isolation of chicken follicle granulosa cells

[0089] 1) Isolation and culture of graded follicular granulosa cells

[0090] Hy-Line Brown commercial laying hens at peak egg production were selected and euthanized by exsanguination via the jugular vein. The abdomen was opened with sterile scissors, and the entire ovary was removed and placed in a beaker containing 5% penicillin-streptomycin phosphate buffer. Graded granulosa cells from follicles were aspirated using a disposable sterile syringe and transferred to a 15 mL centrifuge tube. The tube was centrifuged at 2,000 rpm for 5 minutes. The PBS buffer was discarded, and an appropriate amount of trypsin was added to the centrifuge tube. After mixing thoroughly, the tube was incubated in a 37°C water bath for 8–10 minutes for digestion. After the water bath, an appropriate amount of M199 medium was added to terminate the digestion. After digestion, the filtrate was filtered through a 200-mesh filter into a sterile beaker under a laminar flow hood. The filtrate was immediately transferred to a new 15 mL centrifuge tube and centrifuged at 2,000 rpm for 5 minutes. The supernatant was discarded, followed by washing with PBS buffer and centrifugation at 2,000 rpm for 5 minutes. The supernatant was discarded, and then an appropriate amount of M199 medium containing antibiotics and FBS was added. The cells were then resuspended using a pipette.

[0091] 2) Cell plating and culture

[0092] First, calculate the volume of cell suspension required for a 24-well plate. Add an equal volume of M199 culture medium to a centrifuge tube and mix thoroughly by pipetting. Add 800 μL of M199 culture medium containing antibiotics and FBS to the 24-well plate. You can first add 100 μL, 125 μL, 150 μL, and 200 μL of cell suspension to the first row of wells, respectively. After standing for 2–3 minutes, observe the cell density under a microscope and select an appropriate cell suspension density for subsequent plate formation. Place the culture plate with the added cell suspension in a 39°C CO2 incubator and incubate for 24 hours to continue subsequent experiments.

[0093] 2.3 Plasmid DNA Transfection Experiment

[0094] When the cell density reaches 75%–80%, transfection can be performed. Prepare the negative control plasmid (empty vector) and the constructed vector at a rate of 800 ng per well, and dilute them separately into an appropriate amount of antibiotic-free OPTI medium, and mix gently. Add the same volume of Plus as the plasmid, mix well, and incubate at room temperature for 5 min. Add an appropriate amount of LTX transfection reagent, invert to mix, and incubate at room temperature for 30 min. Remove the granulocytes from the incubator and wash them once with PBS. Add 500 μL of antibiotic-free medium to each well. Add 100 μL of the above transfection mixture to the washed cell wells, and gently shake the culture plate to mix it evenly. After culturing for 4–6 h, change the medium to 1 mL of fresh antibiotic-free M199 medium and continue culturing for 24–72 h. Then, harvest the cells for dual-luciferase activity detection.

[0095] 2.4 Dual-luciferase activity assay

[0096] First, prepare 1×PLB (100 μL per well), 1×stop solution (100 μL per well), and LAR II reagent. When the cells are almost fully colonized, harvest the cells, discard the culture medium, add 300 μL PBS to each well, wash the cells twice, then add 100 μL 1×PLB to each well, let stand for 10 min, scrape and pipette to mix, transfer to a 1.5 mL centrifuge tube, centrifuge at 13000 rpm for 2 min, and transfer 60 μL of supernatant to a new 1.5 mL centrifuge tube. Add 100 μL of LAR II to the centrifuge tube containing the supernatant, mix gently, and detect fluorescence activity using a microarray, recording the data. Add 100 μL of 1×stop solution to the tube, mix gently, and detect fluorescence activity using a microarray, recording the data.

[0097] 2.5 Statistical Analysis

[0098] Data were analyzed using t-tests for pairwise comparisons. Data were expressed as mean ± standard error. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0099] 3 Results and Analysis

[0100] Using the pGL3-TRAF7-3168 vector as a template, the site-directed mutagenesis vector A-896T at the -896 site was constructed. Figure 4 Chicken grade granulocytes were transfected with the empty vector and mutant vector, respectively, and dual-luciferase activity was measured after 24 hours. The results are as follows: Figure 5 As shown, the transcriptional activity of the TRAF7 gene was significantly increased by the upstream -896A mutation to T (P<0.01).

[0101] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A primer pair for detecting a SNP marker in the promoter region of the chicken TRAF7 gene, characterized in that The chicken TRAF7 The nucleotide sequence of the SNP marker in the promoter region of the gene is shown as SEQ ID No. 1; the base at position 122 from the 5' end of the sequence shown as SEQ ID No. 1 is the SNP site, which is A or T; The chicken genetic breeding is a selection of an egg-laying chicken variety with early onset of laying and / or a large number of maximum continuous laying; chicken TRAF7 The base of the SNP marker in the gene promoter region is TT genotype corresponding to early age of first laying and / or more maximum laying number of egg laying traits; The nucleotide sequences of the primer pair are shown in SEQ ID NO. 2 and SEQ ID NO. 3 respectively.

2. A kit for detecting chicken TRAF7 The use of a kit for SNP markers in the promoter region of the gene in the assisted selection of egg-laying chicken breeds, characterized by, The auxiliary selection of the egg-laying chicken variety is a selection of an egg-laying chicken variety with early onset of laying and / or a large number of maximum continuous laying; The chicken TRAF7 The nucleotide sequence of the SNP marker in the promoter region of the gene is shown as SEQ ID No. 1; the base at position 122 from the 5' end of the sequence shown as SEQ ID No. 1 is the SNP site, which is A or T; chickens TRAF7 The base of the SNP marker in the gene promoter region is TT genotype corresponding to early age of first laying and / or more maximum laying number of laying traits; The kit contains the primer pair shown in SEQ ID NO. 2 and SEQ ID NO.

3.

3. A method of identifying an egg laying trait in a laying hen, characterized in that, The method comprises the following steps: The genomic DNA of the egg-laying chicken to be tested is used as a template, and a primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3 is used for PCR amplification to obtain an amplification product; the amplification product is sequenced, and the laying performance of the egg-laying chicken is identified according to the sequencing result; If the sequencing result corresponds to the sequence shown in SEQ ID No. 1, and the genotype of the 122th base from the 5' end is TT, the egg-laying chicken is identified as having the laying performance of early onset of laying, a large number of laying and / or a large number of maximum continuous laying.

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