Application of DNA fragment in regulation and control of GHR gene expression quantity and bovine body size

Abstract

The invention belongs to the technical field of genetic breeding, and particularly relates to application of a DNA (deoxyribonucleic acid) fragment to regulation and control of GHR (growth hormone receptor) gene expression quantity and bovine body size, and a base sequence of the DNA fragment is shown as SEQ ID NO.12. According to the invention, SV detection and typing are carried out on individual genome sequencing data from different cattle varieties in the world, SV loci having significant selection in two differential populations are screened by a comparative genomics method, and SV variation located in a first intron region of a growth hormone receptor (GHR) is screened by combining gene functions annotated by SV, so that the SV variation is obtained. The influence of the SV on GHR expression is proved through analysis experiments such as a dual-fluorescein report system, it is proved that the SV can serve as a candidate molecular marker influencing the body type size, and an important target is provided for molecular breeding of beef cattle.

Description

Technical Field

[0001] This invention belongs to the field of genetic breeding technology, specifically relating to a DNA fragment in the regulation GHR Applications in gene expression levels and bovine body size. Background Technology

[0002] The beef cattle industry is crucial to the national economy and people's livelihood, but my country imports approximately 30% of its beef annually. The fundamental reason for this lies in the relatively late start of beef cattle breeding in my country. Domestic yellow cattle have long been used for draft purposes, resulting in significant differences in body size and meat production performance compared to European breeds. Large European beef cattle breeds can reach a slaughter weight of 1000 kg, while Chinese breeds only reach around 500 kg. Directly importing foreign breeds is an effective way to quickly improve the meat production performance of Chinese beef cattle. However, foreign breeds face adaptation issues in my country, with their meat production performance lower than those raised in their native environments. This leads to higher feeding costs for Chinese beef cattle and lower competitiveness against foreign breeds, thus limiting their access to the international beef market. Improving the slaughter size of Chinese beef cattle is an effective way to fundamentally solve the beef shortage in the Chinese market, reduce feeding costs, break free from the dependence on foreign breeding stock, and enhance competitiveness in the international market.

[0003] Genome and environment are two major factors determining animal phenotypes. Genomic variations, due to their stable inheritance to offspring, are considered effective molecular markers for early prediction of individual phenotypes. They are used not only as tools for early selection of target individuals but also as key targets for gene editing, thereby rapidly creating individuals with the desired traits. Therefore, screening for variations in the beef cattle genome that are associated with body size is of great significance for rapidly improving beef production performance.

[0004] Genomic variations include various types such as SNPs and SVs. SNPs are single-base variations, while SVs are considered to be large sequence variations ranging from 50 bp to 5 Mb in length, and they have a more dramatic effect on phenotypic regulation than SNPs. Current research focuses primarily on SNPs, and several SNPs associated with beef cattle body size have been identified. However, SVs have been studied relatively less due to their greater difficulty in identification and genotyping. In recent years, the rise of high-throughput sequencing technology and improvements in SV identification algorithms have made it possible to accurately screen for phenotypic SVs, making SVs more efficient targets for regulating animal phenotypes.

[0005] It is worth noting that, GHR The gene, short for Growth Hormone Receptor, is a growth hormone receptor gene. As an important candidate gene for body size regulation, it has been shown to be significantly associated with traits such as body weight, body length, and carcass weight in multiple cattle populations. Therefore, in-depth research into its gene pool is crucial. GHRSV variations in key gene regions, including [specific gene region name], will help reveal the genetic mechanisms underlying beef cattle body size. Summary of the Invention

[0006] The purpose of this invention is to provide a DNA fragment in regulation GHR The application of this DNA fragment in gene expression levels and cattle body size will serve as an important target in beef cattle breeding, providing necessary materials for cultivating high-efficiency beef cattle herds.

[0007] The technical solution adopted in this invention is: This invention provides a DNA fragment in regulation GHR Applications in gene expression levels and bovine body size, the base sequence of the DNA fragment is shown in SEQ ID NO.12.

[0008] Preferably, the regulation refers to at least one of the following: When the DNA fragment is present, upregulation GHR The level of gene expression in cells, which in turn increases the size of cattle; When the DNA fragment is missing, downregulation occurs. GHR The level of gene expression in cells, which in turn reduces the size of cattle.

[0009] Preferred, DNA fragment upregulation GHR The methods for measuring gene expression levels in cells are as follows: The sgRNA was ligated into a linearized Cas9 vector to obtain the sgRNA-Cas9 expression vector; the base sequence of the sgRNA is shown in SEQ ID NO.3; The DNA fragment was cloned into the PUC57 vector to obtain the Donor vector; The sgRNA-Cas9 expression vector and the Donor vector were co-transfected into cells and cultured to achieve upregulation of the aforementioned DNA fragment. GHR Gene.

[0010] Preferably, the process for preparing the sgRNA is as follows: A pair of primers were synthesized, and the sequences of the primers are shown in SEQ ID NO.4 and SEQ ID NO.5; Annealing the primers shown in SEQ ID NO.4 and SEQ ID NO.5 yields the sgRNA.

[0011] Preferably, the annealing procedure is as follows: 95℃ 10min, 65℃ 1h, 15℃ 2min.

[0012] Preferably, the Cas9 carrier is PHB-pSpCas9(BB)-2A-Puro(PX459).

[0013] Preferably, the restriction endonuclease used for linearizing the Cas9 vector is BpiI.

[0014] Preferably, the preparation process of the Donor carrier is as follows: The DNA fragment was ligated between the left and right homologous arms to obtain the Donor sequence; the sequence of the left homologous arm is shown in SEQ ID NO.14, and the sequence of the right homologous arm is shown in SEQ ID NO.15. The Donor sequence is cloned into the PUC57 vector to obtain the Donor vector.

[0015] Preferably, the mass ratio of the sgRNA-Cas9 expression vector to the Donor vector is 1:1.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a DNA fragment in regulation GHR Applications in gene expression levels and bovine body size, the base sequence of the DNA fragment is shown in SEQ ID NO. 12. This invention utilizes SV detection and genotyping based on individual genome sequencing data from different bovine breeds worldwide. Through comparative genomics, it screens for SV loci exhibiting significant selection in two differing populations, and combines this with gene function annotated by the SV loci to screen for loci located at the growth hormone receptor. GHR The variation of the SV in the first intron region was demonstrated through analysis experiments using a dual-luciferase reporter system, proving that this SV plays a crucial role in... GHR The influence of expression demonstrates that it can serve as a candidate molecular marker affecting body size, providing an important target for molecular breeding of beef cattle. Attached Figure Description

[0017] Figure 1 For F ST Manhattan plot of analysis and gene annotation.

[0018] Figure 2 The distribution of GHR-SV allele frequency and body size in different cattle populations.

[0019] Figure 3 For GHR-SV motif and functional enrichment analysis, A: motif analysis on GHR-SV; B: highest-scoring motif; C: GO functional enrichment analysis.

[0020] Figure 4Enhancer activity of GHR-SV sequence in different cells was detected. A: MDBK cells; B: HEK293T cells.

[0021] Figure 5 After knocking in mouse embryonic stem cells with GHR-SV sequence GHR The results of the tests are as follows: A: Gel electrophoresis results; B: qPCR results. Detailed Implementation

[0022] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0023] The inventive concept of this invention is as follows: Comparing the genomes of beef cattle of different sizes allows for effective screening of variant sites related to beef cattle body size. Currently, databases contain a large amount of whole-genome sequencing data from various cattle breeds worldwide, providing ample resources for rapidly screening for size-related variants in beef cattle.

[0024] Therefore, this invention utilizes SV detection and genotyping based on individual genome sequencing data from different cattle breeds worldwide. Through comparative genomics, SV loci exhibiting significant selection in two differing populations are screened. Combined with the gene functions annotated by these SV loci, loci located at the growth hormone receptor are identified. GHR The SV variation in the first intron region was denoted as GHR-SV, and analytical experiments, including those using a dual-luciferase reporter system, demonstrated that GHR-SV... GHR The impact of expression. When this GHR-SV exists, GHR Significantly upregulated gene expression corresponds to individuals exhibiting larger body size; while knocking out this GHR-SV sequence leads to... GHR Decreased expression and reduced individual body size.

[0025] GHR By regulating the growth hormone (GH) signaling pathway, it affects IGF-1 The expression of these molecules participates in bone growth and muscle development, thereby determining an individual's body size. GHR Higher expression levels of GH signaling lead to stronger GH signal transduction efficiency, faster growth and development, and larger individual body size. This invention demonstrates that the described SV sequence can serve as a candidate molecular marker affecting body size, providing an important target for molecular breeding of beef cattle.

[0026] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0027] The list of abbreviations for this invention is shown in Table 1.

[0028] Table 1. List of abbreviations for this invention Example 1 A DNA fragment in regulation GHR The applications of gene expression levels and bovine body size are as follows: 1. Materials and Methods.

[0029] 1.1 Data collection on different cattle breeds around the world.

[0030] This invention collected second-generation sequencing data from the NCBI public database, covering 82 cattle breeds worldwide, totaling 2407 samples, including data from Europe. Bos taurus Asia and Africa Bos taurus and Bos indicus It broadly represents the genetic diversity of cattle worldwide. NCBI Public Database: https: / / www.ncbi.nlm.nih.gov / sra / .

[0031] All raw data underwent quality control using FASTP to remove adapters, low-quality reads, and contaminated sequences, retaining clean reads for downstream alignment analysis.

[0032] 1.2 SV identification.

[0033] To obtain more comprehensive structural variant genotyping information, this invention employs a strategy combining multiple assembly alignment tools with population short-read sequencing data. Assembly data from 23 publicly available bovine genomes were collected and compared with the reference genome ARS-UCD1.2 to identify structural variants such as insertions and deletions. SV detection utilized three assembly-based methods: Svrefine v0.35, Assemblytics v1.2.1, and Minimap2 v2.25 combined with paftools. To improve breakpoint accuracy, all SV breakpoints obtained by the three methods were refined, and 100bp sequences upstream and downstream of each breakpoint were extracted and re-aligned to the local reference sequence. Precise breakpoints were then determined using mafft v7.487 and msa2vcf v1.0. Simultaneously, 92,518 high-quality deletion variants detected by WGS in 898 cattle from 57 breeds in previous studies were integrated to increase the genetic diversity of the genome map. Subsequently, all SVs were used to construct pan-genome maps based on the ARS-UCD1.2 reference genome using the vg v1.51.0 tool, and XG and GCSA indexes were generated using vg index. SVs are represented by bubble structures in the map, with different paths representing different alleles. Finally, map alignment and genotyping were performed on 2407 quality control samples. The cleaned reads were aligned to the map using vg giraffe, outputting GAM format files, and alignment records with alignment quality and base quality below 5 were removed. Coverage was then calculated using vg pack, and structural variation regions were identified using vg snarls. Finally, vg call was used to complete the SV genotyping of all samples.

[0034] 1.3 Signal Selection Analysis.

[0035] To reveal selection signals among different cattle populations, single-point FST calculations were performed on genotyped SV data using VCFtools v0.1.17 to assess the degree of genetic differentiation of each variant site across different populations. The top 1% of SVs by FST value were identified as significant selection variant sites, and genomic regions potentially affected by selection were screened. Gene annotation was performed on the identified significant selection SV regions using genome annotation information. Further gene functional enrichment analysis was conducted using DAVID.

[0036] Genome annotation information: ftp: / / ftp.ensembl.org / pub / relese-80 / gtf / bos_taurus; DAVID: https: / / david.ncifcrf.gov / .

[0037] 1.4 Verification of enhancer activity.

[0038] 1.4.1 Primer design.

[0039] Based on the GHR-SV enhancer region sequence chr20:32059312~32060523 found on the UCSC website, primers were designed to construct the dual-luciferase reporter vector pGL3-promoter-GHR-SV. The sequences of the upstream and downstream primers are shown in SEQ ID NO.1 and SEQ ID NO.2. The restriction enzyme site for both the upstream and downstream primers is XhoI.

[0040] SEQ ID NO.1: 5'-aacatttctctatcgataggtaccTTAAATTTTAAAACATTCTGGGAGACCCACCT-3'.

[0041] SEQ ID NO.2: 5'-gatgcagatcgcagatctcgagTTTTTTTTTTCTTCCAATTTTATTTTATTTTTAAACTTTACATAATTG-3'.

[0042] 1.4.2 PCR amplification reaction.

[0043] Genomic DNA was extracted from MDBK bovine kidney cells, and the GHR-SV enhancer region was amplified from the genomic DNA via PCR using I-5T 2× High-Fidelity Master Mix. The reaction mixture consisted of 25 μL of I-5T 2× High-Fidelity Master Mix, 2 μL of upstream primer, 2 μL of downstream primer, 500 ng of genomic DNA, and H2O to a final volume of 50 μL. The amplification program was as follows: pre-denaturation at 98℃ for 2 min; denaturation at 98℃ for 10 s, annealing at 60℃ for 10 s, extension at 72℃ for 30 s, for a total of 30 cycles; final extension at 72℃ for 2 min. Subsequently, the obtained PCR product was cloned upstream of the luciferase gene in the pGL3-promoter vector digested with restriction endonucleases XhoI and KpnI. After ligating the linearized pGL3-promoter vector according to the kit instructions, PCR-positive bacterial cultures were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results of the bacterial culture were consistent with the SV region sequence, indicating that the SV region was successfully obtained.

[0044] 1.4.3 Cell transfection.

[0045] Dual-luciferase activity assays were performed in MDBK and HEK293T cells cultured at 37°C in a 5% CO2 incubator. MDBK and HEK293T cells were seeded in 24-well plates, and transfection began when the cell density reached 60%. The reporter vector and pRL-TK Renilla luciferase control vector were co-transfected using Lipofectamine 3000. Specific procedures were as follows:

[0046] Configuration A: 25 μL DMEM medium per well + 500 ng pGL3-promoter-GHR-SV dual-luciferase reporter vector plasmid + 50 ng pRL-TK internal control plasmid + 1 μL P3000 TM Mix thoroughly by pipetting. System B: 25 μL DMEM medium + 1 μL Lipofectamine 3000 per well, mix thoroughly by pipetting, and let stand for 5 min. Mix System A and System B, let stand at room temperature for 15 min, then gently add 50 μL of the incubated mixture to each well, gently shake the culture plate, and place it in an incubator to continue culturing. After 12 h of transfection, determine whether to change the medium based on the cell condition. 48 h after transfection, collect cells for dual-luciferase activity assay.

[0047] 1.4.4 Dual-luciferase activity assay.

[0048] Cells were harvested 48 hours after transfection, and dual-luciferase activity was analyzed according to the Dual-Luciferase® Reporter Assay Systems kit instructions. (Using a 24-well plate as an example:)

[0049] (1) Prepare 1×PLB working solution, LAR II and 1×Stop&Glo® Reagent reagent respectively in the dark according to the instructions. After preparation, dispense and store in the dark at -80℃.

[0050] (2) Discard the culture medium, wash the cells twice with PBS, add 100 μL of the prepared 1×PLB solution to each well, and place on a shaker to fully lyse.

[0051] (3) Collect the fully lysed cell fluid into a 200 μL centrifuge tube and centrifuge at a short speed for later use. Prepare a 96-well plate and place it in an ELISA reader.

[0052] (4) Take 10 μL of cell lysis supernatant and place it in an enzyme-labeled plate. Then add 50 μL of LAR II and place it in an enzyme-labeled instrument to detect the fluorescence value of firefly luciferase. After reading, add 50 μL of 1×Stop&Glo® Reagent reagent to each well to detect the fluorescence value of Renida luciferase. This process should be carried out in the dark.

[0053] (5) Calculate the ratio of firefly luciferase to kidney luciferase activity, which is the relative activity of the enhancer.

[0054] 1.5. GHR-SV sequence knock-in mouse embryonic stem cells.

[0055] 1.5.1 Constructing the sgRNA-Cas9 expression vector.

[0056] The target specificity analysis was performed using the online website CCTop to select sgRNAs with high cleavage efficiency and good specificity. The sgRNA sequences are shown in SEQ ID NO.3, and the annealing primers are shown in Table 2.

[0057] SEQ ID NO.3: AACATTGGTGCACTGAACATTGG, with the bolded bases representing the PAM sequence.

[0058] CCTop: https: / / cctop.cos.uni-heidelberg.de:8043 / Table 2 sgRNA sequences and annealing primers Annealing primers were designed based on the sgRNA sequence. The upstream primer was the sgRNA target site sequence, and the downstream primer was the reverse complementary sequence of the sgRNA target site. Additionally, 5'-CACC-3' was added to the 5' end of the upstream sgRNA annealing primer, and 5'-AAAC-3' was added to the 5' end of the downstream sgRNA annealing primer.

[0059] sgRNA annealing system: 5 μL upstream primer, 5 μL downstream primer.

[0060] Annealing procedure: 95℃ for 10 min, 65℃ for 1 h, 15℃ for 2 min.

[0061] The annealed sgRNA was ligated into the PHB-pSpCas9(BB)-2A-Puro(PX459) vector digested with BpiI restriction enzyme. After ligation, the cells were transformed into DH5α competent cells, and single colonies were cultured overnight on LB medium. Single colonies were picked, and PCR-positive bacterial cultures were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were consistent with the corresponding sgRNA sequence, indicating successful construction of the sgRNA-Cas9 expression vector.

[0062] 1.5.2 Construction of Donor Vector: The repair DNA template plasmid was synthesized by Qingke and cloned into the PUC57 vector.

[0063] The sequence ~800bp upstream of the sgRNA target site in the genome was selected as the left homologous arm HA-L; the sequence ~800bp downstream of the sgRNA was selected as the right homologous arm HA-R. The bovine GHR-SV sequence to be knocked in was placed between the left and right homologous arms. The Donor sequence was amplified by PCR and cloned into the PUC57 vector. The sequence of GHR-SV is shown in SEQ ID NO.12; the sequence of the left homologous arm is shown in SEQ ID NO.14; and the sequence of the right homologous arm is shown in SEQ ID NO.15.

[0064] SEQ ID NO.14: 。

[0065] SEQ ID NO.15: TTCAGTGCACCAATGTTAATTAAAGAATAATTAATTGAGGAGCATACACTTAGATAATATGTTTTTTATTTGATTTAGTTCTCTCTCTCTCTGAGAAAATTTCCCAAGAAAGTGTTTGTTCTGTAGCCCAGAATGACCTTGAACTCCTCATCCTCTGGCTTCAGCCTCCAGTTGCTGAGTGCTCTAAATACAGATGTGCACCAGCATGCTCTAAATACAGATGTGCACCAGCATGCTTTGCCTGTTGGATGTTTTTTTAAATTATGTTATGATTGAGCTATTTCAGATTTTAAAAAAAGGTCATGAGTCAAGAAATGTTACAAAGGACCAAGTAGTTAGTGAAATACATATATGAGCAATACAAATGAAAAGGATGACTTCCCCGGTCTGGAATTTTGCTATGGTTCTTTTAACTACTCATGTAGTTAGAACATTCATGGTTGTCATTAGGACTAAATAACTCCACACAGAAGACTGTGAGACGAACATTTCACTATGTGAGTGACAGTCCTGTCCACACTAATACCACAGGAAGACAGTTGTGATCCATTCCTGTTGACAGAATTAATGTGCTGGGTATTAGACCTGTTCATGCAGCTTACCTTTTTGAAGTATGAAATTCAGCATTCTGTGATGTTTGATACATTCAAACTCCCTAAGGATAGAACGAAGAGCCTAAGGATAGCCTCAAAAAGAGTGTTCATGGGAATAGTTAACAGTGATAGCATTGAGAATTGTGATTGATTGTTTTTCAAATCATGTTTTGGAAAGACAGAAACCGATAGCATGGTAGAGGCCTCTG。

[0066] 1.5.3. The sgRNA-Cas9 expression vector and the Donor vector were electrotransferred into mouse embryonic stem cells.

[0067] (1) Take mouse ES cells in good growth condition, digest them with trypsin, gently pipette them to disperse them into single cells, and then transfer them into centrifuge tubes. Centrifuge at 300g for 5 min. After discarding the supernatant, resuspend the cells in 1 mL of sterile PBS and wash them once. Then wash them again with 1 mL of Opti-MEM. After centrifugation, discard the supernatant.

[0068] (2) Resuspend the cell pellet using Opti-MEM and count the cells to obtain a cell suspension. Take approximately 2 × 10⁻⁶ cells. 5 One cell per electroporation cycle. The electroporation buffer system uses Opti-MEM; no special electroporation buffer is required.

[0069] (3) Add the above cell suspension to a sterile EP tube, add 2 μg sgRNA-Cas9 expression vector and 2 μg Donor vector, and use Opti-MEM to make up to a total volume of 70 μL. Gently pipette to mix, avoiding the formation of bubbles.

[0070] (4) Transfer 60 μL of the mixture to an electroporation cuvette with a 2 mm gap and perform electroporation using a Super Electroporator NEPA21 Type II. This instrument has high transfection efficiency and good cell viability maintenance capabilities, making it suitable for ES cell electroporation without the need for a special electroporation buffer. Set the voltage to 220V and the pulse mode to single square wave. Immediately after electroporation, remove the cells and transfer them to 6-well plates pre-filled with 2 mL of complete culture medium, seeding one well for each reaction.

[0071] (5) Place the cells in a 37°C, 5% CO2 incubator and continue culturing. After about 12 hours, observe the cell status and replace with fresh complete culture medium to remove dead cells and residual debris.

[0072] (6) Antibiotics were added for screening 48 hours after electroporation. Since the sgRNA-Cas9 expression vector carries the puromycin resistance gene, 1 μg / mL puromycin was added to the culture medium for resistance screening for 48 hours. After screening, the medium was replaced with normal complete culture medium without antibiotics to prepare for subsequent single-clone selection and verification experiments.

[0073] 1.5.4 Monoclonal cell screening.

[0074] After screening for healthy mouse ES cells using Puromyci, single-clone plating was performed. The specific procedure was as follows:

[0075] Cells were digested with trypsin into centrifuge tubes, and cell density was calculated using a hemocytometer. Cells were seeded into 96-well plates using a serial dilution method, with 16 wells per group of cells. The plates were incubated at 37°C in a 5% CO2 incubator. After 5 days, the formation of monoclonal cells was observed under a microscope. The dilution was continued until an average of one cell per well was achieved. Morphologically normal and rapidly growing monoclonal cells were then selected for multiplication (one-to-three). Once sufficient cell expansion was achieved, stem cell DNA was extracted and identified by PCR.

[0076] 1.5.5 Detection of GHR-SV sequence knock-in in monoclonal cells.

[0077] Genomic DNA was extracted from the obtained monoclonal cells using a cell genomics extraction kit and then identified by PCR. The primer sequences used to detect GHR-SV sequence knock-in are shown in SEQ ID NO. 6 and SEQ ID NO. 7.

[0078] F primer, SEQ ID NO.6: GCAGAGACCTGACTTGACTTAGAGAC.

[0079] R primer, SEQ ID NO.7: CCTTGGAGACAGGTATTTATGGGTAC.

[0080] PCR reactions were performed using Takara high-fidelity enzyme PrimeSTAR® HS DNA Polymerase in a system of 5×PrimeSTAR Buffer (Mg 2+ Plus) 2 μL, 2.5 mM each dNTP Mixture 0.8 μL, 2.5 U / μl PrimeSTAR HS DNA Polymerase 0.1 μL, F primer 0.2 μL, R primer 0.2 μL, template DNA 100 ng, H2O to bring the total to 10 μL.

[0081] Reaction procedure: Pre-denaturation 98℃ for 2 min; denaturation 98℃ for 10 s, annealing 64℃ for 5 s, decreasing by 0.4℃ per cycle, extension 72℃ for 30 s, for a total of 36 cycles; final extension: 72℃ for 3 min. The PCR products were subjected to agarose gel electrophoresis to detect whether the GHR-SV sequence had been knocked in.

[0082] 1.6 qPCR analysis of mouse ES cells GHR Gene expression levels.

[0083] Total RNA was lysed and extracted from wild-type untreated WT, heterozygous (33), and homozygous (48) mouse ES cells using Trizol reagent, and cDNA was synthesized according to the TaKaRa reverse transcription kit instructions. The RNA was then detected by qPCR. GHR Gene expression levels.

[0084] qPCR amplification system: 5 μL SYBR Green PCR Master Mix, 0.2 μL each of forward and reverse primers, 2 μL cDNA template, and 2.6 μL RNA-free H2O.

[0085] qPCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ final extension for 10 min. Primer sequences for the qPCR experiment are shown in Table 3.

[0086] Table 3 qRT-PCR primer sequences Exploit 2 -ΔΔCT The relative expression levels of genes were calculated using a method. All experimental data are expressed as mean ± standard error, and the results were displayed as bar charts using GraphPad Prism 8.0 software. SPSS 24.0 statistical software was used to perform independent samples t-tests for significance analysis. P <0.01 indicates a highly significant difference. P <0.05 indicates a significant difference.

[0087] 2. Experimental Results and Analysis.

[0088] 2.1 Identification of SV.

[0089] This invention collected whole-genome sequencing (WGS) data from 2407 samples across 82 breeds and divided them into 13 populations based on geographical origin, fully representing the genetic diversity of cattle populations worldwide. Based on the constructed bovine pan-genome map, diploid-level genotyping was performed on structural variations across the entire genome, ultimately yielding 152,199 high-quality SVs.

[0090] 2.2 Signal Selection Analysis.

[0091] Based on structural variation data from two populations, European cattle and Central African zebu cattle, F... ST Analysis was conducted to assess genetic differentiation between populations. The results identified 1031 loci that served as significant differentiation signals between the two populations. Gene annotation revealed that these loci were located in the growth hormone receptor gene. GHR One SV site in the first intron region showed significant F STThe specific location of the SV site is chr20:32059312~32060523, which is denoted as GHR-SV for ease of description. The results are shown below. Figure 1 This indicates that this site may be subject to artificial selection and participate in the regulation of body size development. The base sequence of GHR-SV is shown in SEQ ID NO.12.

[0092] >chr20:32059312~32060523,SEQ ID NO.12:

[0093] 2.3 Relationship between GHR-SV and body size of different cattle herds around the world.

[0094] To further explore the relationship between GHR-SV and bovine body size, this invention calculated the allele frequency of GHR-SV in different populations. The results showed that GHR-SV exhibited a higher allele frequency in larger European cattle populations, such as WET, CSET, and NEAT, while its frequency was lower in relatively smaller southern Chinese and African zebu cattle populations, such as SCI and AI. Figure 2 This result suggests that GHR-SV may be related to the evolution of bovine body size phenotype.

[0095] 2.4 GHR-SV sequence functional prediction.

[0096] To investigate the potential regulatory function of the GHR-SV insert sequence, this invention performed motif analysis. Multiple potential conserved transcription factor binding sites were identified using MEME Suite, with the highest-scoring motif sequence shown in SEQ ID NO. 13. This motif appeared multiple times in the insert sequence, with extremely significant p-values, the lowest being 2.67 × 10⁻⁶. -15 This suggests that the motif possesses strong conservation and functional potential. Further GO functional enrichment analysis using the GoMo tool revealed that this motif is significantly associated with multiple transcriptional regulation-related biological processes, including transcription (GO:0006350), positive regulation of the RNA polymerase II promoter (GO:0045944) and negative regulation (GO:0000122), transcription factor complex (GO:0005667), and lung development (GO:0030324). Some GO terms exhibited high specificity, such as negative and positive regulation of the RNA polymerase II promoter. The GHR-SV region was enriched with high-confidence transcription factor binding sites, and these motifs were significantly associated with multiple core transcriptional regulatory functions, suggesting that this structural variation may participate in gene expression regulation as a cis-regulatory element. The above results are shown in [see attached table]. Figure 3 .

[0097] MEME Suite: http: / / meme-suite.org / meme / .

[0098] SEQ ID NO. 13: GVDGGAGDGGGKVKGRAGVWHHGGGA.

[0099] 2.5. GHR-SV sequence enhancer activity analysis.

[0100] The successfully cloned pGL3-promoter-GHR-SV luciferase reporter vector and pRL-TK plasmid were co-transfected into MDBK and HEK293T cells, and GHR-SV sequence enhancer activity was analyzed. The results showed that in MDBK cells, the luciferase activity of the pGL3-promoter-GHR-SV luciferase reporter vector was significantly higher than that of the empty vector. This result was further confirmed by the dual-luciferase activity assay of the pGL3-promoter-GHR-SV luciferase reporter vector in HEK293T cells. Figure 4 This means that the GHR-SV sequence has strong enhancer activity.

[0101] 2.6 GHR-SV sequence knockout pairs GHR The impact on gene expression.

[0102] The obtained monoclonal mouse ES cells were identified by PCR. The results showed that clones 31, 36, 37, 41, 43, 44, 46, 47, and 48 were homozygous cells with the GHR-SV sequence knock-in, while clone 33 was a heterozygous clone. To evaluate the effects of GHR-SV sequence knock-in... GHR The expression effect was investigated by collecting ES cells from WT group, heterozygous clone 33, and homozygous clone 48 mice in good growth condition for qPCR detection. The results showed that after knocking in the GHR-SV sequence, GHR Significantly increased expression P<0.05 See results Figure 5 .

Claims

1. A DNA fragment in regulation GHR Its application in gene expression levels and bovine body size is characterized by, The base sequence of the DNA fragment is shown in SEQ ID NO.

12.

2. The application as described in claim 1, characterized in that, The regulation refers to at least one of the following: When the DNA fragment is present, upregulation GHR The level of gene expression in cells, which in turn increases the size of cattle; When the DNA fragment is missing, downregulation occurs. GHR The level of gene expression in cells, which in turn reduces the size of cattle.

3. The application as described in claim 2, characterized in that, DNA fragment upregulation GHR The methods for measuring gene expression levels in cells are as follows: The sgRNA was ligated into a linearized Cas9 vector to obtain the sgRNA-Cas9 expression vector; the base sequence of the sgRNA is shown in SEQ ID NO.3; The DNA fragment was cloned into the PUC57 vector to obtain the Donor vector; The sgRNA-Cas9 expression vector and the Donor vector were co-transfected into cells and cultured to achieve upregulation of the aforementioned DNA fragment. GHR Gene.

4. The application as described in claim 3, characterized in that, The process for preparing the sgRNA is as follows: A pair of primers were synthesized, and the sequences of the primers are shown in SEQ ID NO.4 and SEQ ID NO.5; Annealing the primers shown in SEQ ID NO.4 and SEQ ID NO.5 yields the sgRNA.

5. The application as described in claim 4, characterized in that, The annealing procedure is as follows: 95℃ 10min, 65℃ 1h, 15℃ 2min.

6. The application as described in claim 3, characterized in that, The Cas9 carrier is PHB-pSpCas9(BB)-2A-Puro(PX459).

7. The application as described in claim 3, characterized in that, The restriction endonuclease used to linearize the Cas9 vector is BpiI.

8. The application as described in claim 3, characterized in that, The preparation process of the Donor carrier is as follows: The DNA fragment was ligated between the left and right homologous arms to obtain the Donor sequence; the sequence of the left homologous arm is shown in SEQ ID NO.14, and the sequence of the right homologous arm is shown in SEQ ID NO.

15. The Donor sequence is cloned into the PUC57 vector to obtain the Donor vector.

9. The application as described in claim 3, characterized in that, The mass ratio of the sgRNA-Cas9 expression vector to the Donor vector is 1:1.

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