Transcription factor capable of regulating igf2bp1 gene function and application thereof

Abstract

The application provides a transcription factor capable of regulating the function of an IGF2BP1 gene and an application, and researches find that a rs640807869 (C>G) site changes the expression efficiency of IGF2BP1 by affecting the binding of transcription factors SP1 and KLF4 to an IGF2BP1 DNA fragment; the site is significantly related to the chest circumference of Nanjiang yellow sheep and can be used as one of potential sites for breeding Nanjiang yellow sheep. SP1 can promote the proliferation and differentiation of goat MuSCs, and the function of KLF4 is opposite to that of SP1, and the function of the rs640807869 (C>G) site is closely related to the two transcription factors, and the proliferation and differentiation of goat MuSCs can be affected by regulating the two transcription factors.

Description

Technical Field

[0001] This invention relates to the field of poultry breeding technology, specifically to transcription factors that can regulate the function of the IGF2BP1 gene and their applications. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are sequence polymorphisms caused by changes in a single base in DNA. They are the most common type of heritable variation in organisms and the main form of DNA polymorphism. SNPs can play a direct role in changes in animal phenotypes by affecting amino acids (missense mutations), promoter activity, mRNA conformation, and translation efficiency. Currently, SNP genetic markers are widely used in livestock and poultry breeding, greatly improving economic efficiency.

[0003] Elucidating the mechanism by which this key gene regulates skeletal muscle growth and development is a core aspect of molecular genetic breeding research in meat sheep. IGF2BP1 is an important candidate gene affecting growth traits in livestock and poultry, but its specific molecular mechanism still needs further clarification. Regarding research on IGF2BP1 single nucleotide polymorphisms (SNPs), current studies in goats mainly focus on its second intron; research on SNPs in its upstream region and first intron has not been reported. Therefore, this invention aims to conduct genotype-phenotype association analysis on relevant regions and then detect the molecular function of related variant sites at the cellular level. This aims to improve the molecular regulatory network of goat muscle development and also provide a theoretical basis for the breeding of Nanjiang Yellow Goat. Summary of the Invention

[0004] This invention analyzes the relationship between IGF2BP1 genotype and phenotype, identifies phenotype-related variant sites, and then analyzes the function of these variant sites at the cellular level. This will contribute to a deeper understanding of the growth and development process of goat muscle tissue and provide a theoretical basis for molecular marker breeding of Nanjiang Yellow Goat. Therefore, it provides transcription factors that can regulate IGF2BP1 gene function and their applications.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides transcription factors that can regulate the function of the IGF2BP1 gene, wherein the transcription factors are SP1 and / or KLF4.

[0007] In a second aspect, the present invention provides the application of the transcription factor described in the first aspect in the preparation of reagents that promote the expression of the IGF2BP1 gene.

[0008] Thirdly, the present invention provides a reagent for promoting IGF2BP1 gene expression, containing transcription factors SP1 and / or KLF4.

[0009] Fourthly, the present invention provides the application of the IGF2BP1 gene expression promoting reagent described in the third aspect in the preparation of products that promote the proliferation and differentiation of goat skeletal muscle satellite cells or products that promote goat muscle development.

[0010] Fifthly, the present invention provides the use of reagents that enhance or promote the expression of transcription factor SP1 in the preparation of products that promote the proliferation and differentiation of goat skeletal muscle satellite cells or products that promote goat muscle development.

[0011] Preferably, the reagent is pEGFP-SP1, and the method for constructing pEGFP-SP1 includes:

[0012] Step 1, design homologous primers;

[0013] Step 2: Using goat cDNA as a template, amplify the target fragment SP1 using homologous primers;

[0014] Step 3: Linearize the pEGFP-N1 vector;

[0015] Step 4: Purify the target fragment amplification product obtained in Step 2 and the linearized vector obtained in Step 3, and then ligate the target fragment amplification product and the linearized vector.

[0016] Step 5: Transform the ligation product into competent E. coli cells and culture them. Then, pick single colonies and use the bacterial solution as a template to perform PCR amplification of the target fragment, and screen out positive bacterial solutions.

[0017] Step 6: Extract plasmids from the positive bacterial solution.

[0018] Preferably, in step 1, the homologous primers have sequences as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0019] Preferably, in step 3, the pEGFP-N1 vector is linearized using two restriction endonucleases, XhoⅠ and BamHI.

[0020] In a sixth aspect, the present invention provides the use of reagents that reduce or inhibit the expression of transcription factor KLF4 in the preparation of products that promote the proliferation and differentiation of goat skeletal muscle satellite cells or products that promote goat muscle development.

[0021] Preferably, the reagent is si-KLF4, having the sequence shown in SEQ ID NO.22.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention reveals that the rs640807869(C>G) locus alters the expression efficiency of IGF2BP1 by affecting the binding of transcription factors SP1 and KLF4 to the IGF2BP1 DNA fragment. This locus is significantly correlated with chest circumference in Nanjiang Yellow Sheep and can be considered a potential breeding site for this breed. SP1 promotes the proliferation and differentiation of goat muscle cells (MuSCs), while KLF4 has the opposite function. The function of the rs640807869(C>G) locus is closely related to these two transcription factors; by regulating these two transcription factors, the proliferation and differentiation of goat MuSCs can be affected. This invention elucidates the molecular mechanism related to IGF2BP1 from the perspective of DNA sequence variation, further refining the molecular regulatory network of goat muscle development and providing a theoretical basis for the breeding of Nanjiang Yellow Sheep. Attached Figure Description

[0024] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0025] Figure 1 The results of fluorescence activity detection for different dual-luciferase vectors in Example 2 of this invention are shown. The Basic vector does not contain a promoter, while the Promoter vector does. The bar chart represents the mean ± standard error of at least three replicates, with *p < 0.05 and **p < 0.01. Pairs with one identical label letter are considered insignificant, while pairs with different label letters are considered significant. Lowercase letters represent p < 0.05.

[0026] Figure 2 To verify the binding of the transcription factor at the rs640807869 (C>G) site in Example 3 of this invention, the following data are presented: A. Effects of different treatments on the fluorescence activity of the dual-luciferase vector; B. Numbers in B, C, and F represent the fold change in luciferase activity of different genotype vectors; GL. Effects of SP1 and KLF4 on IGF2BP1. The bar charts represent the mean ± standard error of at least three replicates, *p<0.05, **p<0.01.

[0027] Figure 3This invention presents Example 4 of the study, illustrating the effect of SP1 on the proliferation and differentiation of goat MuSCs. The study includes: A. SP1 expression in different goat tissues; B. SP1 expression during the proliferation and differentiation of goat MuSCs; C. The effect of SP1 overexpression on proliferation markers; D. EdU assay, with red representing EdU-positive cells (scale bar: 400 μm); E. The effect of SP1 interference on myotubes during MuSC differentiation, using MYHC immunofluorescence assay (scale bar: 400 μm); F. The proportion of EdU-positive cells to the total number of cells; G. The effect of SP1 overexpression on the OD value of CCK-8 treated cells; and H. The effect of SP1 interference on differentiation markers. The bar chart represents the mean ± standard error of at least three replicates. *p<0.05, **p<0.01; any identical label letter indicates no significant difference, while different label letters indicate significant differences. Lowercase letters represent p<0.05.

[0028] Figure 4 This invention presents Example 5 of the study, illustrating the effect of KLF4 on the proliferation and differentiation of goat MuSCs. The study includes: A. KLF4 expression in different goat tissues; B. KLF4 expression during the proliferation and differentiation of goat MuSCs; C. Effect of KLF4 overexpression on proliferation marker genes; D. EdU assay, with red representing EdU-positive cells (scale bar: 400 μm); E. Effect of KLF4 interference on myotubes during MuSC differentiation, using MYHC immunofluorescence assay (scale bar: 400 μm); F. The proportion of EdU-positive cells to the total number of cells; G. Effect of KLF4 overexpression on the OD value of CCK-8 treated cells; H. Effect of KLF4 interference on differentiation marker genes. The bar chart represents the mean ± standard error of at least three replicates. *p<0.05, **p<0.01; a single identical label letter indicates no significant difference, while different label letters indicate significant differences. Lowercase letters represent p<0.05.

[0029] Figure 5 The possible mechanism by which the rs640807869 site affects IGF2BP1 expression. Detailed Implementation

[0030] In the description of this invention, it should be noted that unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0031] In the embodiments of the present invention, the animal tissues and cells were sourced as follows: (1) The goat tissues used in the experiment were collected from Chengdu Ma sheep, including the heart, liver, spleen, lung, kidney, and longissimus dorsi muscle 1 day after birth, and the longissimus dorsi muscle 45 days after embryonic period. All samples were stored at -80℃ after collection, and all operations were strictly carried out in accordance with the experimental animal operation specifications of Sichuan Agricultural University (Sichuan Agricultural University

[2014] No. 18). (2) The goat skeletal muscle satellite cells (MuSCs) used in the experiment were independently isolated and obtained by our laboratory. The simplified method was to cut the collected longissimus dorsi muscle tissue blocks of newborn lambs into pieces, then perform the first digestion with 0.1% type I collagenase and 0.1% type Ⅱ collagenase, and then perform the second digestion with 0.25% trypsin. Finally, the MuSCs were separated and purified by centrifugation and differential adhesion. The expression of Pax7 and the differentiation capacity (MYHC) of the obtained cells were then detected to determine whether the cells were goat skeletal muscle satellite cells. The successfully identified MuSCs cells were stored in liquid nitrogen for later use.

[0032] In the embodiments of this invention, all experimental samples (n=348, female=217, male=131) used for growth morphology data determination were sourced from the Nanjiang Yellow Sheep Breeding Farm in Sichuan Province and were raised according to the feeding and management requirements for Nanjiang Yellow Sheep. 5 mL of jugular venous blood was collected from the sheep using blood collection tubes containing EDTA anticoagulant and stored at -20°C for later use. The birth weight of the Nanjiang Yellow Sheep and their body weight, height, body length, and chest circumference at different ages (4 months, 6 months, 12 months, and 18 months) were measured using conventional methods.

[0033] In embodiments of the present invention, the cell transfection reagent is used. For the transfection assay, strictly follow the instructions. Dilute the plasmid with P3000 and Lip3000 separately and incubate for 5 min. Then, mix the two dilutions and incubate for 15 min before adding to the cell culture medium for transfection. Adjust the post-transfection treatment time according to experimental requirements. RNA extraction is performed 48 h post-transfection using the Trizol assay, and the dual-luciferase reporter assay is performed 36-72 h post-transfection.

[0034] In an embodiment of the present invention, the cell proliferation capacity was detected using the CCK-8 cell proliferation kit. The experiment was conducted according to the instructions. Cells were seeded in 96-well cell culture plates. After processing the cells according to the experimental requirements, 10 μL of CCK-8 reagent was added to each well. The cells were incubated at 37°C in the dark for 1-4 hours, and the absorbance was measured at 450 nm using a microplate reader. The results were plotted as a cell growth curve with culture time on the ordinate and OD value on the ordinate.

[0035] In this embodiment of the invention, the EdU cell proliferation kit was used to detect cell proliferation capacity, and the experimental procedure was performed according to the instruction manual. After treating the cells according to the experimental requirements, diluted EdU solution was added to the culture medium and incubated for 2 hours. After incubation, the culture medium was discarded, the cells were washed three times with PBS, and then fixed with 4% paraformaldehyde at room temperature for 10 minutes. The fixative was discarded, and cell permeation solution was added to treat the cells at 4°C for 10 minutes. The permeation solution was discarded, and chromogenic solution was added for colorimetric reaction. Cell nuclei were then stained using Hoechst 33342. Finally, the cells were detected using an inverted fluorescence microscope, and the total number of cells and the number of EdU-positive cells were statistically analyzed using ImageJ.

[0036] In the embodiments of the present invention, the primer information for RT-qPCR is shown in Table 1:

[0037] Table 1 Quantitative Primer Information

[0038]

[0039] Note: GAPDH is used as an internal reference gene in RT-qPCR to correct for differences between samples and ensure the accuracy of the relative expression levels of the target gene.

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] In this embodiment, SNPs were scanned in the first intron and upstream of the IGF2BP1 gene of 348 Nanjiang yellow sheep experimental population, and verified by pooled PCR sequencing. A total of 6 SNP sites were identified: rs640807869, rs635680028, rs653131898, rs640899771, rs644725642, and rs669926820. Among them, rs640807869 (C>G) is located in the first intron of the IGF2BP1 gene, and rs635680028 (G>A), rs653131898 (A>T), rs640899771 (C>T), rs644725642 (C>T), and rs669926820 (G>T) are located 2.4-5.7 kb upstream of the IGF2BP1 gene.

[0043] In the embodiments of the present invention, the primer information for mixed-pool PCR is shown in Table 2:

[0044] Table 2 Primer information for mixed-tank PCR

[0045]

[0046] In this embodiment, the Sequenom MassArray system genotyping technology was used to genotype 348 Nanjiang Yellow Sheep experimental populations. The blood genomic DNA of all samples was genotyped by Beijing Fuyu Biotechnology Co., Ltd. The genotyping data was imported into Assay, the sample table was entered, plate was built, samples were spotted, and Mass ARRAY analysis was performed. The raw data and genotyping map were obtained using TYPER 4.0 software, and the integrity and correctness of the data files were checked.

[0047] Microsoft Excel 2016 software was used to calculate the gene frequency, genotype frequency, polymorphism information content (PIC), homozygosity (Ho), heterozygosity (He), and effective allele number (Ne) of each SNP locus in the Nanjiang Yellow Sheep experimental samples.

[0048] (1) Allele frequency and genotype frequency

[0049] The formula for calculating each SNP is as follows:

[0050] Frequency of allele C: p = (2N) CC +N CD ) / (2(N CC +N CD +N DD ));

[0051] Frequency of allele D: q = (2N) DD +N CD ) / (2(N CC +N CD +N DD ));

[0052] Frequency of genotype CC: D = N CC / (N CC +N CD +N DD );

[0053] Frequency of genotype CD: H = N CD / (N CC +N CD +N DD );

[0054] Frequency of genotype DD: R = N DD / (N CC +N CD +NDD );

[0055] Where, N CC N CD N DD These represent the number of individuals with genotypes CC, CD, and DD in the population, respectively.

[0056] (2) Polymorphic Information Content (PIC): Where n represents the number of alleles, P i P represents the frequency of the i-th allele. j This represents the frequency of the j-th allele.

[0057] (3) Homozygosity (H) o ), heterozygosity (H) e ) and effective allele number (N) e ): H e =1-H o N e =1 / H o Where n represents the number of alleles, P i This represents the frequency of the i-th allele.

[0058] The results are shown in Table 3.

[0059] Table 3. Population genetic parameters and HW equilibrium test of 6 SNPs in the Nanjiang Tibetan gazelle population.

[0060]

[0061]

[0062] Note: χ2>0.05 indicates that the site conforms to Hardy-Weinberg equilibrium.

[0063] Association analysis of growth traits: SAS 9.4 software was used to perform association analysis between genotypes at each locus and growth traits of the Nanjiang Yellow Sheep. GLM and Y chromosomes were used in SAS 9.4. ijk =μ+G i +S j +P k +G i ×S j +e ijk , where Y ijk G represents the measured value of growth traits; μ is the population mean; G i Genotype effect; S j For gender effect; P k This is due to the batch effect; G i ×S j The interaction effect between sex and genes; e ijkTo account for random error, all results are expressed as least squares mean (LSM) ± SEM. Specifically, association analysis was performed on six SNPs of IGF2BP1 with birth weight, weight at six months of age (BW-6), body length (BL-6), height (BH-6), and chest circumference (CC-6) of Nanjiang Yellow Sheep (n=348) (Table 3-3). The results showed that the rs644725642 (C>T) locus was significantly associated with birth weight in Nanjiang Yellow Sheep. Among them, the birth weight of individuals with the CT genotype was significantly higher than that of individuals with the CC genotype (P<0.05), while the other SNPs were not significantly associated with birth weight in Nanjiang Yellow Sheep; the rs640807869 (C>G) locus... Individuals with the CC genotype at six months of age had significantly higher chest circumference than those with the CG genotype (P<0.05); individuals with the TT genotype at the rs653131898 (A>T) locus had significantly higher body weight at six months of age than those with the AT genotype (P<0.05), while individuals with the AT genotype had significantly lower chest circumference at six months of age than the other two genotypes (P<0.05); individuals with the CC genotype at the rs640899771 (C>T) locus had significantly higher chest circumference at six months of age than the other two genotypes (P<0.05). No significant association was found between the six SNPs and the body length and height of the Nanjiang gazelle at six months of age. The results are shown in Tables 4-7.

[0064] Table 4. Effects of different SNP genotypes of Nanjiang Yellow Sheep on birth weight and growth traits at six months of age.

[0065]

[0066]

[0067] Note: BW represents body weight, BL represents body length, BH represents body height, and CC represents chest circumference. The numbers following the letters represent age in months, for example, BW-6 represents the weight of a six-month-old infant. Different uppercase letters between different genotypes of the same SNP indicate highly significant differences (P<0.01), different lowercase letters indicate significant differences (P<0.05), and identical letters or the absence of any letter indicates no significant difference (P>0.05).

[0068] Table 5. Effects of different SNP genotypes on growth traits of two-month-old Nanjiang Yellow Goats

[0069]

[0070] Table 6. Effects of different SNP genotypes on growth traits of 12-month-old Nanjiang Yellow Goats

[0071]

[0072]

[0073] Table 7. Effects of different SNP genotypes on growth traits of 18-month-old Nanjiang Yellow Goats

[0074]

[0075] The results above show that the six SNPs are significantly correlated with the growth traits of the Nanjiang Tibetan gazelle.

[0076] Example 2

[0077] DNA fragments from different genotypes were inserted into dual-luciferase reporter vectors. The Basic vector was used to verify promoter activity, and the Promoter vector was used to verify enhancer activity. The template was DNA from different genotypes of Nanjiang Yellow Goat. The linearization restriction sites for both vectors were Kpn I and Xho I, and the antibiotic resistance in LB medium was Amp. The samples were then transfected into 293T and goat MuSCs cells to examine the effects of SNPs on the activity of potential promoters and enhancers before and after mutation.

[0078] For the rs640899771(C>T) site, the fragment containing this site exhibits certain promoter activity. In both cell types, the mutant promoter activity (Promoter-T) was significantly lower than the wild-type (Promoter-G) (p<0.01). This fragment also exhibits enhancer activity. In 293T cells, the mutant enhancer activity (Enhancer-T) was significantly lower than the wild-type (Enhancer-G) (p<0.01), while in goat MuSCs, the difference was not significant (p>0.05). Figure 1 (A)

[0079] For the rs644725642(C>T) site, the fragment containing this site has certain promoter activity. In both cell types, the promoter activity (Promoter-T) of the mutant type was significantly higher than that of the wild type (Promoter-G) (p<0.01). The fragment containing this site also has enhancer activity. In 293T cells, the enhancer activity (Enhancer-T) of the mutant type was significantly lower than that of the wild type (Enhancer-G) (p<0.05). In goat MuSCs, there was no significant difference between the two (p>0.05).

[0080] For the rs669926820 (G>T) site ( Figure 1In 293T cells, the promoter activity of the mutant (C) showed no significant difference before and after mutation (p>0.05), while the enhancer activity (Enhancer-T) of the mutant was significantly higher than that of the wild-type (Enhancer-G) (p<0.01). In goat MuSCs, the promoter activity (Promoter-T) of the mutant was significantly lower than that of the wild-type (Promoter-G) (p<0.05), but the enhancer activity of the two was not significantly different (p>0.05). Figure 1 (B)

[0081] For the rs653131898(A>T) site, the mutant enhancer activity (Enhancer-T) was significantly lower than the wild-type (Enhancer-A) in both cell types (p<0.01). Only in 293T cells was the mutant promoter activity (Promoter-T) significantly lower than the wild-type (Promoter-A) (p<0.01).

[0082] For the rs640807869 (C>G) site ( Figure 1 The fragment containing this site showed remarkably consistent behavior in both cell types. In both 293T and goat MuSCs cells, the mutant promoter activity (Promoter-G) was significantly lower than the wild-type (Promoter-C) (p<0.01), and similarly, the mutant enhancer activity (Enhancer-G) was significantly lower than the wild-type (Enhancer-C) (p<0.01). Figure 1 (D).

[0083] The rs653131898(A>T) and rs640899771(C>T) sites are strongly linked and located close to each other on the chromosome. A fragment containing both sites was inserted into a dual-luciferase reporter vector and transfected into 293T and MuSCs, respectively, to investigate the combined effect of these two sites on promoter and enhancer activity. The results showed that only the rs653131898(A>T) site mutation (Promoter-AT) fragment exhibited the highest promoter activity in both cell types (p<0.05). Figure 1 In 293T cells, the enhancer activity of the fragment with mutants at both sites (Enhancer-TT) was significantly reduced compared to the fragment with wild-types at both sites (Enhancer-CC) (p<0.05). Figure 1 In MuSCs, there was no significant difference between fragments containing different genotypes at the two loci (p>0.05). Figure 1These results indicate that the rs653131898 (A>T) and rs640899771 (C>T) sites can jointly affect the promoter and enhancer activities of this fragment, and there may be a synergistic effect between them.

[0084] The rs635680028 (G>A) site showed no significant change in transcription factor binding after prediction.

[0085] Example 3

[0086] Based on the experimental results of Examples 1 and 2, rs640807869(C>G) is located in the first intron of the IGF2BP1 gene, is significantly associated with the growth traits of Nanjiang Yellow Sheep, and causes significant changes in dual-luciferase activity, making it a potential breeding site for Nanjiang Yellow Sheep. Therefore, in this embodiment of the invention, transcription factor prediction was performed on the rs640807869(C>G) site. The upstream and downstream 15bp sequences of this SNP were selected as references. The transcription factor binding sites of rs640807869 in Nanjiang Yellow Sheep were predicted using the JASPAR 2022 database (https: / / jaspar.genereg.net / ). Transcription factor binding sites with a score higher than 8.5 were selected. The results showed that the site containing rs640807869(C>G) can bind to SP1. After mutation, the binding score of this site to SP1 increased, and a new site binding to KLF4 was added.

[0087] Functional validation of these two transcription factors at the rs640807869 (C>G) site was performed, and the effects of transcription factors SP1 and KLF4 on IGF2BP1 were investigated, as detailed below:

[0088] SP1 and KLF4 were overexpressed and interfered with in goat MuSCs, respectively. Then, dual-luciferase reporter vectors containing DNA fragments of different genotypes were transfected into cells to investigate the effects of SP1 and KLF4 on the activity of dual-luciferase at different genotypes of the rs640807869 (C>G) site.

[0089] Overexpression was performed using RT-qPCR, and the primers used are shown in Table 1. The pEGFP-SP1 and pEGFP-KLF4 overexpression vectors were constructed using the homologous recombination kit (Vazyme), and the steps are as follows:

[0090] Step 1, Homologous primer design: Based on the SP1 and KLF4 gene mRNA CDS region sequences (excluding stop codons) on NCBI, homologous primers were designed using the Vazyme online experimental tool by selecting single-fragment cloning and double-enzyme digestion linearized vectors (as shown in Table 1).

[0091] Step 2, Target Fragment Amplification: Using goat cDNA as a template, using... Max DNA Polymerase (Takara) is a high-fidelity enzyme used to amplify the target fragment using homologous primers. The reaction system and amplification procedure should be followed according to the instructions for the high-fidelity enzyme reagent.

[0092] Step 3, Vector linearization: The pEGFP-N1 vector was linearized using two restriction endonucleases, XhoⅠ and BamHI, and the reaction was performed according to the restriction endonuclease instructions.

[0093] Step 4, Purification of the target fragment and linearized vector: Perform gel electrophoresis on the reaction products of the above two steps, cut out the target band for gel recovery, and follow the instructions of the gel recovery kit (Omega) for the purification process.

[0094] Step 5, Ligation: Ligate the target fragment and the linearized vector, following the reaction conditions specified in the homologous recombination kit (Vazyme);

[0095] Step 6, Transformation: The ligation product was transformed into E. coli competent cells (DH5-α), and the cells were inoculated onto LB solid medium containing antibiotic (Kana) using the dilution plate method and grown overnight at 37°C.

[0096] Step 7, picking single colonies: Pick single colonies from the plate and inoculate them into LB liquid medium containing antibiotic (Kana), and incubate at 37°C until the medium becomes turbid;

[0097] Step 8, bacterial culture PCR: PCR amplification of the target fragment is performed using bacterial culture as a template, and positive bacterial cultures are screened by gel electrophoresis;

[0098] Step 9, Sequencing verification: Sanger sequencing (Sangon) is performed on the positive bacterial culture for identification. If successful, the bacterial culture is propagated and cultured.

[0099] Step 10, Plasmid Extraction: After propagation, the bacterial culture is used for plasmid extraction. The steps are as per the plasmid extraction instructions (Omega).

[0100] RT-qPCR experiments were performed using ChamQ SYBR qPCR Master Mix (Vazyme). The reaction volume was 10 μL: 5 μL ChamQ SYBR qPCR Master Mix, 0.4 μL upstream primer, 0.4 μL downstream primer, 0.8 μL cDNA, and 3.4 μL ddH2O. The amplification program was: 95℃ for 2 min; 95℃ for 5 s, Tm 10 s, 39 cycles, 65℃~95℃, 0.5℃ / 30 s. Results were based on 2... -ΔΔCt The method calculates the relative expression level of genes.

[0101] The interfering siRNAs were designed and synthesized by Ribobio, with the following sequences: si-SP1: CAGGTCAGATACAGATCAT (SEQ ID NO.21), si-KLF4: GCAGCTTCAGCTATCCAAT (SEQ ID NO.22). RNA was extracted using the standard Trizol method, and reverse transcription was performed using RNAiso Plus (Takara) lysis buffer and an RNA reverse transcription kit (Vazyme).

[0102] The results showed that overexpression of SP1 significantly increased the promoter activity of the mutant type (Promoter-Mutant-G) (p<0.01), and was higher than that of the wild type (Promoter-Wild-C). Figure 2 (A) and overexpression of KLF4 had no effect on the promoter activity of either (A). Figure 2 (B) Regarding enhancer activity, after SP1 overexpression, the mutant (Enhancer-Mutant-G) showed lower activity than the wild-type (Enhancer-Wild-C) (p<0.05 or p<0.01). Figure 2 (C), but the fold difference between the two decreased from 0.54 to 0.38; after interfering with SP1, the enhancer activities of the two tended to be consistent (p>0.05). Figure 2 (D). After overexpression of KLF4, the fluorescence activity of wild-type (Enhancer-Wild-C) and mutant (Enhancer-Mutant-G) tended to be consistent. Figure 2 Interference with KLF4 had no effect on the fluorescence activity trends of either drug, but the fold difference between them decreased from 0.56 to 0.52. Figure 2 (F).

[0103] To further investigate the effects of these two transcription factors on IGF2BP1, overexpression and interference of these two transcription factors were performed in goat MuSCs, respectively, using the same methods as described above. Changes in IGF2BP1 expression levels were detected by RT-qPCR. The results showed that overexpression of SP1 (pEGFP-SP1) significantly increased the expression level of IGF2BP1 (p<0.01). Figure 2 Interference with SP1 (siSP1) significantly reduced IGF2BP1 expression levels (p<0.05). Figure 2 (H). Meanwhile, overexpression of KLF4 (pEGFP-KLF4) significantly reduced IGF2BP1 expression levels (p<0.01). Figure 2In contrast, KLF4 interference (siKLF4) significantly increased IGF2BP1 expression (p<0.05). Figure 2 (Middle K). Furthermore, the effects of both on IGF2BP1 are accompanied by a dose-dependent effect, from... Figure 2 As can be seen from Figure I, with the increase of SP1 expression level, the expression level of IGF2BP1 also gradually increased (p<0.05 or p<0.01); with the gradual increase of KLF4 expression level, the expression level of IGF2BP1 gradually decreased (p<0.05 or p<0.01). Figure 2 (Middle L).

[0104] In summary, transcription factor SP1 can promote the expression of IGF2BP1, while transcription factor KLF4 can inhibit the expression of IGF2BP1. The two have opposite functions, and their effect on IGF2BP1 is dose-dependent, which suggests that these two transcription factors may affect the expression of IGF2BP1 through competitive inhibition.

[0105] Example 4

[0106] This embodiment investigated the effect of transcription factor SP1 on the proliferation and differentiation of MuSCs, as detailed below:

[0107] RT-qPCR experiments were performed on the heart, liver, spleen, lungs, kidneys, and longissimus dorsi muscle of Chengdu Ma sheep 1 day after birth, using the same method as in Example 3. The results showed that SP1 was expressed at a high level in the spleen and lungs, at a moderate level in the liver and kidneys, and at a low level in the heart and longissimus dorsi muscle. Figure 3 (A) As cell differentiation progresses, the expression level of SP1 gradually increases, and the expression level during the differentiation phase is significantly higher than that during the proliferation phase (p<0.05). Figure 3 (B)

[0108] Overexpression of SP1 during the proliferation phase of MuSCs showed by RT-qPCR that overexpression of SP1 (pEGFP-SP1) significantly increased the expression of IGF2BP1 (p<0.01) and the proliferation marker gene PCNA (p<0.05). Figure 3 (C). CCK-8 assay results showed that, compared with the control group (pEGFP-N1), overexpression of SP1 (pEGFP-SP1) significantly increased the absorbance of MuSCs cells in the 24H, 48H, and 72H proliferation phases (p<0.05 or p<0.01). Figure 3 (G). EdU assay results showed that overexpression of SP1 (pEGFP-SP1) significantly increased the number of EdU-positive cells (p<0.01). Figure 3 (D and F). The above results indicate that overexpression of SP1 can promote the proliferation of MuSCs.

[0109] To investigate the effect of SP1 on MuSC differentiation, SP1 was interfered with during the MuSC differentiation period. RT-qPCR results showed that after SP1 interference (siSP1), the expression level of IGF2BP1 was significantly reduced (p<0.05), and the expression levels of differentiation marker genes MYOD, MYOG, and MYHC were also significantly reduced (p<0.01). Figure 3 The results of the MYHC immunofluorescence assay showed that interference with SP1 (siSP1) reduced the number of myotubes and decreased the myocyte fusion rate. Figure 3 (E). These results indicate that interfering with SP1 can suppress the differentiation of MuSCs.

[0110] Therefore, SP1 promotes IGF2BP1 expression and can also promote the proliferation and differentiation of goat MuSCs.

[0111] Example 5

[0112] This example investigated the effect of transcription factor KLF4 on the proliferation and differentiation of MuSCs, as detailed below:

[0113] KLF4 expression in different tissues of Chengdu Ma sheep was detected in the heart, liver, spleen, lungs, kidneys, and longissimus dorsi muscle on day 1 after birth. Results showed that KLF4 expression was highest in the lungs, moderate in the spleen, and low in the heart, liver, kidneys, and longissimus dorsi muscle. Figure 4 (A) As cell differentiation progresses, the expression level of KLF4 gradually increases, and the expression level during the differentiation phase is significantly higher than that during the proliferation phase (p<0.05). Figure 4 (B)

[0114] Overexpression of KLF4 during the proliferation phase of MuSCs showed by RT-qPCR that overexpression of KLF4 (pEGFP-KLF4) significantly reduced the expression of IGF2BP1 (P<0.01) and the proliferation marker genes PCNA and mki67 (P<0.05). Figure 4 (C). CCK-8 assay results showed that, compared with the control group (pEGFP-N1), overexpression of KLF4 (pEGFP-KLF4) reduced the absorbance of MuSCs cells in the 48H and 72H proliferation phases, but the difference was not significant (p>0.05). Figure 4 (G). EdU assay results showed that overexpression of KLF4 (pEGFP-KLF4) significantly reduced the number of EdU-positive cells (p<0.01). Figure 4 (D and F). The above results indicate that overexpression of KLF4 can inhibit the proliferation of MuSCs.

[0115] To investigate the effect of KLF4 on MuSC differentiation, KLF4 was interfered with during the MuSC differentiation period. RT-qPCR results showed that after KLF4 interference (siKLF4), the expression level of IGF2BP1 was significantly increased (p<0.05), and the expression levels of differentiation marker genes MYOD, MYOG, and MYHC were also significantly increased (p<0.05 or p<0.01). Figure 4 The results of the MYHC immunofluorescence assay showed that interference with KLF4 (siKLF4) increased the number of myotubes and improved the myocyte fusion rate. Figure 4 (E). These results indicate that interfering with KLF4 can promote the differentiation of MuSCs.

[0116] Therefore, KLF4 can inhibit IGF2BP1 expression and also inhibit the proliferation and differentiation of goat MuSCs.

[0117] In summary, this invention hypothesizes that the first intron of the IGF2BP1 gene in the Nanjiang Tibetan gazelle population contains the rs640807869 (C>G) mutation, which is significantly correlated with chest circumference in Nanjiang Tibetan gazelles. This mutation site causes alterations in the binding of transcription factors SP1 and KLF4 (e.g., ...). Figure 5 (As shown). Among them, SP1 can promote the proliferation and differentiation of goat MuSCs, while KLF4 has the opposite function. The function of the rs640807869 (C>G) site is closely related to these two transcription factors. By regulating these two transcription factors, the proliferation and differentiation of goat MuSCs can be affected.

[0118] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementations of the invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the invention, and these all fall within the protection scope of the invention. Therefore, the protection scope of this invention should be determined by the appended claims.

Claims

1. Application of reagents that enhance or promote the expression of transcription factor SP1 in the preparation of products that promote the proliferation and differentiation of goat skeletal muscle satellite cells or promote goat muscle development; The reagent is pEGFP-SP1, and the method for constructing pEGFP-SP1 includes: Step 1, design homologous primers; Step 2: Using goat cDNA as a template, amplify the target fragment SP1 using homologous primers; Step 3: Linearize the pEGFP-N1 vector; Step 4: Purify the target fragment amplification product obtained in Step 2 and the linearized vector obtained in Step 3, and then ligate the target fragment amplification product and the linearized vector. Step 5: Transform the ligation product into competent E. coli cells and culture them. Then, pick single colonies and use the bacterial solution as a template to perform PCR amplification of the target fragment, and screen out positive bacterial solutions. Step 6: Extract plasmids from the positive bacterial solution.

2. The application according to claim 1, characterized in that, In step 1, the sequences of the homologous primers are shown in SEQ ID NO.5 and SEQ ID NO.

6.

3. The application according to claim 1, characterized in that, In step 3, the pEGFP-N1 vector is linearized using two restriction endonucleases, Xho I and BamH I.

4. The application of a reagent that reduces or inhibits the expression of transcription factor KLF4 in the preparation of products that promote the proliferation and differentiation of goat skeletal muscle satellite cells or products that promote goat muscle development; the reagent is si-KLF4, the sequence of which is shown in SEQ ID NO.22.

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