Transcription factor capable of regulating IGF2BP1 gene function and application

Abstract

The invention provides a transcription factor capable of regulating and controlling functions of an IGF2BP1 gene and application of the transcription factor, and the research finds that an rs640807869 (Cgt; G) site changes the expression efficiency of the IGF2BP1 by influencing the combination of transcription factors SP1 and KLF4 with 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. Wherein the SP1 can promote proliferation and differentiation of goat MuSCs, the function of the KLF4 is opposite to that of the SP1, the rs640807869 (Cgt, G) site exerts functions and is closely related to the two transcription factors, and proliferation and differentiation of goat MuSCs can be affected by regulating the two transcription factors.

Description

Technical Field

[0001] The invention relates to the technical field of poultry breeding, and in particular to a transcription factor capable of regulating the function of an IGF2BP1 gene and an application thereof. Background Art

[0002] Single nucleotide polymorphisms (SNPs) refer to sequence polymorphisms caused by changes in a single base in DNA. They are the most common heritable variation in organisms and the main manifestation 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. At present, SNPs genetic markers are widely used in livestock and poultry production and breeding, greatly improving economic benefits.

[0003] Analyzing the mechanism of this key gene regulating skeletal muscle growth and development is the core content of molecular genetic breeding research in meat sheep. IGF2BP1 is an important candidate gene that affects the growth traits of livestock and poultry, but the specific molecular mechanism needs to be further clarified. Regarding the research level of IGF2BP1 single nucleotide polymorphism, at present, the research in goats is mainly focused on its second intron, and the research on SNPs in its upstream and first intron has not been reported. Therefore, the present invention intends to conduct genotype and phenotype association analysis on the relevant regions, and then detect the molecular function of the relevant variant sites at the cellular level, aiming to improve the molecular regulatory network of goat muscle development, and also provide a certain theoretical basis for the breeding of Nanjiang yellow sheep. Summary of the invention

[0004] The present invention analyzes the relationship between IGF2BP1 genotype and phenotype, clarifies the variant sites related to the phenotype, and then analyzes the function of the variant sites at the cellular level, which will help to deeply understand the growth and development process of goat muscle tissue, and at the same time provide a certain theoretical basis for the molecular marker breeding of Nanjiang yellow sheep. Therefore, a transcription factor and application that can regulate the function of the IGF2BP1 gene are provided.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a transcription factor capable of regulating the function of the IGF2BP1 gene, wherein the transcription factor is SP1 and / or KLF4.

[0007] In a second aspect, the present invention provides use of the transcription factor described in the first aspect in preparing a reagent for promoting IGF2BP1 gene expression.

[0008] In a third aspect, the present invention provides an agent for promoting IGF2BP1 gene expression, comprising transcription factor SP1 and / or KLF4.

[0009] In a fourth aspect, the present invention provides use of the reagent for promoting IGF2BP1 gene expression described in the third aspect in the preparation of a product for promoting proliferation and differentiation of goat skeletal muscle satellite cells or a product for promoting muscle development of goats.

[0010] In a fifth aspect, the present invention provides the use of an agent for enhancing or promoting the expression of transcription factor SP1 in the preparation of a product for promoting the proliferation and differentiation of goat skeletal muscle satellite cells or a product for promoting goat muscle development.

[0011] Preferably, the reagent is pEGFP-SP1, and the construction method of pEGFP-SP1 comprises:

[0012] Step 1, design homologous primers;

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

[0014] Step 3, linearize the pEGFP-N1 vector;

[0015] Step 4, purifying the target fragment amplification product obtained in step 2 and the linearized vector obtained in step 3, and then connecting the target fragment amplification product and the linearized vector;

[0016] Step 5, transforming the ligation product into competent E. coli cells and culturing them, then picking monoclonal colonies, using the bacterial solution as a template to perform PCR amplification on the target fragment, and screening out positive bacterial solutions;

[0017] Step 6, extracting plasmid 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 an agent for reducing or inhibiting the expression of transcription factor KLF4 in the preparation of a product for promoting the proliferation and differentiation of goat skeletal muscle satellite cells or a product for promoting 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 present invention has the following beneficial effects:

[0023] The present invention found that the rs640807869 (C>G) site changed the expression efficiency of IGF2BP1 by affecting the binding of transcription factors SP1 and KLF4 to the IGF2BP1 DNA fragment; this site is significantly correlated with the chest circumference of Nanjiang yellow sheep and can be used as one of the potential sites for breeding Nanjiang yellow sheep. 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. The present invention analyzes the molecular mechanism related to IGF2BP1 from the aspect of DNA sequence variation, further improves the molecular regulatory network of goat muscle development, and also provides a certain theoretical basis for the breeding of Nanjiang yellow sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0025] Figure 1 The results of fluorescence activity detection of different dual luciferase vectors in Example 2 of the present invention, wherein the Basic vector does not contain a promoter and the Promoter vector contains a promoter. The bar graph represents the mean ± standard error of at least three replicates, *p<0.05, **p<0.01. Any number with the same letter indicates no significant difference, any number with different letters indicates significant difference, and lowercase letters represent p<0.05.

[0026] Figure 2 This is the verification of the binding of transcription factors at the rs640807869 (C>G) site in Example 3 of the present invention, wherein AF. Effects of different treatments on the fluorescence activity of the dual-luciferase vector, and the numbers in B, C, and F represent the fold change of the luciferase activity of different genotype vectors; GL. Effects of SP1 and KLF4 on IGF2BP1. The bar graph represents the mean ± standard error of at least three replicates, *p<0.05, **p<0.01.

[0027] Figure 3The effect of SP1 on the proliferation and differentiation of goat MuSCs in Example 4 of the present invention, wherein A. the expression of SP1 in different tissues of goats; B. the expression of SP1 during the proliferation and differentiation of goat MuSCs; C. the effect of overexpression of SP1 on proliferation markers; D. EdU test, red represents EdU positive cells (scale bar: 400 μm); E. the effect of interfering with SP1 on myotubes in the differentiation period of MuSCs, MYHC immunofluorescence test (scale bar: 400 μm); F. the proportion of EdU positive cells to the total number of cells; G. the effect of overexpression of SP1 on the OD value of CCK-8 treated cells; H. the effect of interfering with SP1 on differentiation markers. The bar graph represents the mean ± standard error of at least three repetitions, *p<0.05, **p<0.01; any with the same letter means no significant difference, any with different letters means significant difference, and lowercase letters represent p<0.05.

[0028] Figure 4 The effect of KLF4 on the proliferation and differentiation of goat MuSCs in Example 5 of the present invention, wherein A. the expression of KLF4 in different tissues of goats; B. the expression of KLF4 during the proliferation and differentiation of goat MuSCs; C. the effect of overexpression of KLF4 on proliferation marker genes; D. EdU test, red represents EdU positive cells (scale bar: 400 μm); E. the effect of interfering with KLF4 on myotubes in the differentiation period of MuSCs, MYHC immunofluorescence test (scale bar: 400 μm); F. the proportion of EdU positive cells to the total number of cells; G. the effect of overexpression of KLF4 on the OD value of CCK-8 treated cells; H. the effect of interfering with KLF4 on differentiation marker genes. The bar graph represents the mean ± standard error of at least three replicates, *p<0.05, **p<0.01; all with one identical letter are not significantly different, all with different letters are significantly different, and lowercase letters represent p<0.05.

[0029] Figure 5 This is the possible mechanism by which rs640807869 site affects IGF2BP1 expression. DETAILED DESCRIPTION

[0030] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0031] In the embodiments of the present invention, the sources of animal tissues and cells are 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 at 1 day after birth and the longissimus dorsi muscle at 45 days of embryonic period. All samples were stored at -80°C after collection, and all operations were strictly carried out in accordance with the Sichuan Agricultural University Experimental Animal Operation Specifications (Sichuan Agricultural University

[2014] No. 18). (2) The goat skeletal muscle satellite cells (MuSCs) used in the experiment were obtained by our laboratory. The brief method was to cut the longissimus dorsi muscle tissue blocks collected from newborn lambs, and then use 0.1% type I collagenase and 0.1% type ⅠⅠ collagenase for the first digestion, and then use 0.25% trypsin for the second digestion, and finally separate and purify MuSCs by centrifugation and differential adhesion method. Then, the expression of Pax7 in the obtained cells and whether they have differentiation ability (MYHC) were detected to determine whether the cells are goat skeletal muscle satellite cells. The successfully identified MuSCs cells were stored in liquid nitrogen for future use.

[0032] In the embodiment of the present invention, all test samples (n=348, female=217, male=131) for growth shape data determination in the present invention are from Sichuan Nanjiang Yellow Sheep Original Farm, and are raised in accordance with the Nanjiang Yellow Sheep breeding and management requirements. 5 mL of test sheep jugular vein blood was collected using a blood collection tube containing EDTA anticoagulant and stored at -20°C for standby use. The birth weight of Nanjiang Yellow Sheep and the weight, height, body oblique length and chest circumference at different periods (4 months, 6 months, 12 months and 18 months) were measured by conventional methods.

[0033] In an embodiment of the present invention, the cell transfection reagent is used 3000, the specific experimental operation was carried out strictly according to the instructions, the plasmid used for transfection was diluted with P3000 and Lip3000 respectively and incubated for 5 minutes, and then the two dilutions were mixed and incubated for 15 minutes before adding to the cell culture medium for transfection. The time of post-transfection treatment of cells was adjusted according to the experimental requirements. RNA extraction was performed using the Trizol method 48 hours after transfection, and the dual luciferase reporter assay was performed 36-72 hours after transfection.

[0034] In the embodiment of the present invention, the cell proliferation ability was detected using a cell proliferation kit CCK-8. The test was carried out according to the instructions, the cells were plated in a 96-well cell culture plate, and the cells were treated according to the test requirements. 10ul CCK-8 reagent was added to each well, and the absorbance was measured at 450nm using an ELISA instrument after incubation at 37°C in the dark for 1-4h. The results were plotted with the culture time as the ordinate and the OD value as the ordinate to draw a cell growth curve.

[0035] In the embodiment of the present invention, an EdU cell proliferation kit is used to detect cell proliferation ability, and the test steps are carried out in accordance with the instructions. After treating the cells according to the test requirements, a diluted EdU solution is added to the culture medium and incubated for 2 hours; after the incubation, the culture medium is discarded, the cells are rinsed 3 times with PBS, and then 4% paraformaldehyde is added to fix the cells at room temperature for 10 minutes; the fixative is discarded, and the cells are treated with a cell permeabilization solution at 4°C for 10 minutes; the permeabilization solution is discarded, and a color development solution is added for color development reaction; Hoechst 33342 is used for cell nucleus staining; finally, an inverted fluorescence microscope is used for detection, and the total number of cells and the number of EdU positive cells are counted using imageJ.

[0036] In an embodiment 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 for RT-qPCR to correct differences between samples and ensure the accuracy of the relative expression of the target gene.

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1

[0042] In this example, SNPs were scanned in the first intron and upstream of the IGF2BP1 gene of 348 Nanjiang yellow sheep experimental groups, and mixed pool PCR sequencing was used for verification, and a total of 6 SNP sites were identified: rs640807869, rs635680028, rs653131898, rs640899771, rs644725642 and rs669926820, among which rs640807869 (C>G) was 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) were located 2.4-5.7Kb upstream of the IGF2BP1 gene.

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

[0044] Table 2 Pooled PCR primer information

[0045]

[0046] In this example, Sequenom MassArray system genotyping technology was used to genotype 348 Nanjiang yellow sheep in the experimental group. The blood genomic DNA of all samples was handed over to Beijing Fuyu Biotechnology Co., Ltd. for typing. The typing data was imported into Assay, sample tables were input, plates were built, samples were spotted, and Mass ARRAY analysis was performed. TYPER 4.0 software was used to obtain raw data and genotyping diagrams, and the integrity and correctness of the data files were checked.

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

[0048] (1) Allele frequency and genotype frequency

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

[0050] The 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] Among them, N CC 、N CD 、N DD Represents the number of individuals with genotype CC, CD, and DD in the population, respectively.

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

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

[0058] The results are shown in Table 3.

[0059] Table 3 Population genetic parameters and HW equilibrium test of six SNPs in Nanjiang yellow sheep population

[0060]

[0061]

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

[0063] Growth trait association analysis: SAS 9.4 software was used to analyze the association between the genotypes of each locus and the growth traits of Nanjiang yellow sheep. GLM was used in SAS 9.4. ijk =μ+G i +S j +P k +G i ×S j +e ijk , where Y ijk is the growth trait measurement value; μ is the population mean; G i is the genotype effect; S j is the gender effect; P k is the batch effect; G i ×S j is the interaction effect of sex and gene; ijkThe results are expressed as the least square mean (LSM) ± SEM. Specifically, the six SNPs of IGF2BP1 were associated with the birth weight (Birth Weight), six-month-old body weight (BW-6), body oblique length (BL-6), body 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) site was significantly associated with the birth weight of Nanjiang yellow sheep, among which the birth weight of individuals with CT genotype was significantly higher than that of individuals with CC genotype (P<0.05), and the other sites were not significantly associated with the birth weight of Nanjiang yellow sheep; the rs640807869 (C>G) site was significantly associated with the birth weight of Nanjiang yellow sheep. The six-month-old chest circumference of individuals with CC genotype was significantly higher than that of individuals with CG genotype (P<0.05); the six-month-old weight of individuals with TT genotype at rs653131898 (A>T) was significantly higher than that of individuals with AT genotype (P<0.05), while the six-month-old chest circumference of individuals with AT genotype was significantly lower than that of the other two genotypes (P<0.05); the six-month-old chest circumference of individuals with CC genotype at rs640899771 (C>T) was significantly higher than that of the other two genotypes (P<0.05). The six SNPs were not significantly associated with the body length and height of Nanjiang yellow sheep at six months of age. The results are shown in Tables 4 to 7:

[0064] Table 4 Effects of different SNP genotypes on birth weight and growth traits at six months of age in Nanjiang yellow sheep

[0065]

[0066]

[0067] Note: BW stands for body weight, BL stands for body length, BH stands for body height, CC stands for chest circumference, and the number behind stands for age in months, for example, BW-6 stands for weight at six months of age. Different capital letters between different genotypes of the same SNP indicate extremely significant differences (P<0.01), different lowercase letters indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>0.05)

[0068] Table 5 Effects of different SNP genotypes of Nanjiang yellow sheep on growth traits at two months of age

[0069]

[0070] Table 6 Effects of different SNP genotypes of Nanjiang yellow sheep on growth traits at 12 months of age

[0071]

[0072]

[0073] Table 7 Effects of different SNP genotypes of Nanjiang yellow sheep on growth traits at 18 months of age

[0074]

[0075] From the above results, it can be seen that the six SNPs are significantly correlated with the growth traits of Nanjiang yellow sheep.

[0076] Example 2

[0077] The DNA fragments of different genotypes were inserted into the dual luciferase reporter vector. The Basic vector was used to verify the promoter activity, and the Promoter vector was used to verify the enhancer activity. The template was the DNA of Nanjiang yellow sheep of different genotypes. The linearization restriction sites of the two vectors were Kpn I and Xho I, and the resistance in LB medium was Amp. Then, the vectors were transfected into 293T and goat MuSCs cells to detect the effects of SNPs on the possible promoter and enhancer activities before and after mutation.

[0078] For the rs640899771 (C>T) site, the fragment where the site is located has a certain promoter activity. In both cells, the promoter activity of the mutant type (Promoter-T) is significantly lower than that of the wild type (Promoter-G) (p<0.01); the fragment where the site is located also has enhancer activity. In 293T cells, the enhancer activity of the mutant type (Enhancer-T) is significantly lower than that of the wild type (Enhancer-G) (p<0.01). In goat MuSCs, the difference between the two is not significant (p>0.05) ( Figure 1 (A).

[0079] For the rs644725642 (C>T) site, the fragment where the site is located has a certain promoter activity. In both cell types, the promoter activity of the mutant type (Promoter-T) is significantly higher than that of the wild type (Promoter-G) (p<0.01); the fragment where the site is located also has enhancer activity. In 293T cells, the enhancer activity of the mutant type (Enhancer-T) is significantly lower than that of the wild type (Enhancer-G) (p<0.05). In goat MuSCs, the difference between the two is not significant (p>0.05).

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

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

[0082] For rs640807869 (C>G) Figure 1 E), the fragment where the site is located in the two cells showed surprisingly consistent performance. Whether in 293T or goat MuSCs cells, the promoter activity of the mutant (Promoter-G) was significantly lower than that of the wild type (Promoter-C) (p<0.01). Similarly, the enhancer activity of the mutant (Enhancer-G) was significantly lower than that of the wild type (Enhancer-C) (p<0.01) ( Figure 1 (middle D).

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

[0084] The rs635680028 (G>A) site had no obvious changes in transcription factor binding after prediction.

[0085] Example 3

[0086] Combined with the relevant test results of Example 1 and Example 2, rs640807869 (C>G) is located in the first intron of the IGF2BP1 gene, which is significantly related to the growth traits of Nanjiang yellow sheep, and will cause significant changes in dual luciferase activity, which can be used as one of the potential sites for breeding of Nanjiang yellow sheep. Therefore, in an embodiment of the present invention, transcription factor prediction is performed for the rs640807869 (C>G) site, and the 15bp base sequences upstream and downstream of the SNP are selected as references. The transcription factor binding sites of rs640807869 in Nanjiang yellow sheep are predicted according to the JASPAR 2022 database (https: / / jaspar.genereg.net / ), and the transcription factor binding sites with a score higher than 8.5 are selected. The result is: the site where rs640807869 (C>G) is located can bind to SP1. After mutation, the site has an increased score for binding to SP1, and a new site for binding to KLF4 is added.

[0087] The rs640807869 (C>G) site was functionally verified around these two transcription factors, and the effects of transcription factors SP1 and KLF4 on IGF2BP1 were investigated as follows:

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

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

[0090] Step 1, homologous primer design: According to the CDS region sequence of SP1 and KLF4 gene mRNA on NCBI (excluding the stop codon), single fragment cloning and double enzyme linearization vector were selected in the Vazyme online experimental tool to design homologous primers (as shown in Table 1);

[0091] Step 2, target fragment amplification: using goat cDNA as template, Max DNA Polymerase (Takara) high-fidelity enzyme, amplify the target fragment with homologous primers, and refer to the instructions for the high-fidelity enzyme reagent for the reaction system and amplification procedure;

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

[0093] Step 4, purification of target fragment and linearized vector: The reaction products of the above two steps were subjected to gel electrophoresis, and the target band was cut out for gel recovery. The purification process was referred to the instructions of the gel recovery kit (Omega);

[0094] Step 5, ligation: ligate the target fragment and the linearized vector. The reaction conditions refer to the homologous recombination kit (Vazyme);

[0095] Step 6, transformation: transform the ligation product into E. coli competent cells (DH5-α), inoculate it on LB solid medium containing antibiotics (Kana) by dilution plate coating method, and grow at 37°C overnight;

[0096] Step 7, picking monoclonal colonies: picking monoclonal colonies on the plate, inoculating them into LB liquid culture medium containing antibiotics (Kana), and culturing at 37° C. until the culture medium becomes turbid;

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

[0098] Step 9, sequencing verification: Sanger sequencing (Sangon) is performed on the positive bacterial solution, and if successful, the bacterial solution is expanded and seeded;

[0099] Step 10, plasmid extraction: After expansion, the bacterial solution is used for plasmid extraction, and the steps refer to the plasmid extraction instruction manual (Omega).

[0100] RT-qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme). The reaction used a 10ul system, ChamQ SYBR qPCR Master Mix 5ul, upstream primer 0.4ul, downstream primer 0.4ul, cDNA 0.8ul, ddH 2 O 3.4ul. Amplification program: 95℃2min; 95℃5s, Tm 10s, 39cycles, 65℃~95℃, 0.5℃ / 30s. The results were based on 2 -ΔΔCtMethods The relative expression levels of genes were calculated.

[0101] Interference siRNA was designed and synthesized by Ruibo Biotechnology, and the sequences are as follows: si-SP1: CAGGTCAGATACAGATCAT (SEQ ID NO.21), si-KLF4: GCAGCTTCAGCTATCCAAT (SEQ ID NO.22). RNA was extracted using the conventional Trizol method, and RNAiso Plus (Takara) lysis buffer was used, and RNA reverse transcription kit (Vazyme) was used for RNA reverse transcription.

[0102] The results showed that overexpression of SP1 could significantly increase the promoter activity of the mutant (Promoter-Mutant-G) (p<0.01) and was higher than that of the wild type (Promoter-Wild-C) ( Figure 2 Overexpression of KLF4 had no effect on the promoter activity of the two genes ( Figure 2 Middle B). For enhancer activity, after overexpression of SP1, the mutant type (Enhancer-Mutant-G) was lower than the wild type (Enhancer-Wild-C) (p<0.05 or p<0.01) ( Figure 2 C), but the difference between the two was reduced 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 Middle D). After overexpression of KLF4, the fluorescence activity of the wild type (Enhancer-Wild-C) and the mutant type (Enhancer-Mutant-G) became consistent ( Figure 2 Interference with KLF4 had no effect on the fluorescence activity change trend of the two, but the difference between the two was reduced from 0.56 to 0.52 ( Figure 2 Middle F).

[0103] To further explore the effects of these two transcription factors on IGF2BP1, these two transcription factors were overexpressed and interfered in goat MuSCs, respectively, using the same method as above, and the 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 G), interference with SP1 (siSP1) can significantly reduce the expression level of IGF2BP1 (p<0.05) ( Figure 2 Meanwhile, overexpression of KLF4 (pEGFP-KLF4) could significantly reduce the expression level of IGF2BP1 (p<0.01) ( Figure 2J), the results of interfering with KLF4 (siKLF4) were the opposite, significantly increasing the expression of IGF2BP1 (p<0.05) ( Figure 2 Moreover, the effects of both on IGF2BP1 were also accompanied by a dose-dependent effect, as shown by Figure 2 As shown in Figure I, with the increase of SP1 expression level, the expression of IGF2BP1 also gradually increased (p<0.05 or p<0.01); with the gradual increase of KLF4 expression level, the expression 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 effects on IGF2BP1 are accompanied by a dose-dependent effect, which indicates that these two transcription factors may affect the expression of IGF2BP1 through competitive inhibition.

[0105] Example 4

[0106] This example investigates the effect of transcription factor SP1 on the proliferation and differentiation of MuSCs, as follows:

[0107] The heart, liver, spleen, lung, kidney and longissimus dorsi muscle of Chengdu Ma sheep were selected for RT-qPCR test 1 day after birth. The method was the same as that in Example 3. The results showed that SP1 was expressed at a high level in spleen and lung, at a medium level in liver and kidney, and at a low level in heart and longissimus dorsi muscle ( Figure 3 As cell differentiation progressed, the expression level of SP1 gradually increased, and the expression level in the differentiation stage was significantly higher than that in the proliferation stage (p<0.05) ( Figure 3 (middle B).

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

[0109] To explore the effect of SP1 on MuSCs differentiation, SP1 was interfered during the differentiation period of MuSCs. RT-qPCR results showed that after interfering with SP1 (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 Middle H), MYHC immunofluorescence assay results showed that the number of myotubes decreased and the myocyte fusion rate decreased after interfering with SP1 (siSP1) ( Figure 3 These results indicate that interference with SP1 can inhibit the differentiation of MuSCs.

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

[0111] Example 5

[0112] This example investigates the effect of transcription factor KLF4 on the proliferation and differentiation of MuSCs, as follows:

[0113] The heart, liver, spleen, lung, kidney and longissimus dorsi muscle of Chengdu Ma sheep were selected at 1 day after birth to detect the expression of KLF4 in different tissues of goats. The results showed that the expression level of KLF4 was the highest in the lung, with a moderate level of expression in the spleen, and low expression in the heart, liver, kidney and longissimus dorsi muscle ( Figure 4 As cell differentiation progressed, the expression level of KLF4 gradually increased, and the expression level in the differentiation stage was significantly higher than that in the proliferation stage (p<0.05) ( Figure 4 (middle B).

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

[0115] To explore the effect of KLF4 on MuSCs differentiation, KLF4 was interfered during the differentiation period of MuSCs. RT-qPCR results showed that after interference with KLF4 (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 Middle H), MYHC immunofluorescence assay results showed that the number of myotubes increased and the myocyte fusion rate increased after interfering with KLF4 (siKLF4) ( Figure 4 These results indicate that interfering with KLF4 can promote the differentiation of MuSCs.

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

[0117] In summary, the present invention speculates that the rs640807869 (C>G) mutation in the first intron of the IGF2BP1 gene in the Nanjiang yellow sheep population is significantly correlated with the chest circumference of the Nanjiang yellow sheep. This mutation site causes a change in the binding of transcription factors SP1 and KLF4 (such as Figure 5 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 the embodiments of the present invention have been shown and described, it is understood that a person of ordinary skill in the art may make various changes, modifications, substitutions and variations to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the attached claims and their equivalents. The above-described embodiments only express several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that a person of ordinary skill in the art may make several variations and improvements without departing from the concept of the present invention, and these all belong to the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the attached claims.

Claims

1. A transcription factor capable of regulating the function of the IGF2BP1 gene, characterized in that: The transcription factor is SP1 and / or KLF4.

2. Use of the transcription factor according to claim 1 in preparing a reagent for promoting IGF2BP1 gene expression.

3. A reagent for promoting IGF2BP1 gene expression, characterized in that: Contains transcription factors SP1 and / or KLF4.

4. Use of the IGF2BP1 gene expression promoting agent according to claim 3 in the preparation of a product for promoting the proliferation and differentiation of goat skeletal muscle satellite cells or a product for promoting goat muscle development.

5. Use of reagents for enhancing or promoting the expression of transcription factor SP1 in the preparation of products for promoting the proliferation and differentiation of goat skeletal muscle satellite cells or products for promoting goat muscle development.

6. The use according to claim 5, characterized in that: The reagent is pEGFP-SP1, and the construction method of pEGFP-SP1 includes: Step 1, design homologous primers; Step 2, using goat cDNA as a template and homologous primers to amplify the target fragment SP1; Step 3, linearize the pEGFP-N1 vector; Step 4, purifying the target fragment amplification product obtained in step 2 and the linearized vector obtained in step 3, and then connecting the target fragment amplification product and the linearized vector; Step 5, transforming the ligation product into competent E. coli cells and culturing them, then picking monoclonal colonies, using the bacterial solution as a template to perform PCR amplification on the target fragment, and screening out positive bacterial solutions; Step 6, extracting plasmid from the positive bacterial solution.

7. The use according to claim 6, characterized in that In step 1, the homologous primers have sequences as shown in SEQ ID NO.5 and SEQ ID NO.

6.

8. The use according to claim 6, characterized in that In step 3, the pEGFP-N1 vector is linearized using two restriction endonucleases, Xho Ⅰ and BamH Ⅰ.

9. Use of an agent for reducing or inhibiting the expression of transcription factor KLF4 in the preparation of a product for promoting the proliferation and differentiation of goat skeletal muscle satellite cells or a product for promoting goat muscle development.

10. The use according to claim 9, characterized in that The reagent is si-KLF4, having the sequence shown in SEQ ID NO.22.

Images