A bovine FTO gene promoter mutation site and its application

Abstract

The present invention provides a bovine FTO gene promoter mutation site and its application, belonging to the fields of biotechnology and genetic engineering. This mutation site is located in the promoter region of the FTO gene on chromosome 18 of the bovine genome, specifically, the A base at the -33 base position upstream of the transcription start site is mutated to the G base (chromosome coordinate: Chr:22040490); this mutation significantly increases the expression level of the FTO gene and significantly enhances the differentiation ability of intramuscular fat precursor cells; the FTO gene promoter mutation site provided by the present invention can not only increase the gene expression level, but also promote the differentiation of intramuscular fat precursor cells, providing a solid technical foundation for the cultivation of new beef cattle breeds and the improvement of intramuscular fat content.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and genetic engineering, and specifically relates to a bovine FTO gene promoter mutation site and its application. Background Art

[0002] The intramuscular fat content of beef cattle is one of the important indicators for measuring the quality of beef, directly affecting the tenderness, flavor and taste of the meat. With the continuous improvement of consumers' requirements for meat quality, more and more attention has been paid to the accumulation and distribution of intramuscular fat in beef cattle breeding. However, traditional beef cattle breeding methods usually rely on selection and mating, which is both time-consuming and laborious, and the breeding cycle is relatively long. Therefore, how to accelerate the improvement of beef cattle traits and increase the intramuscular fat content has become an important issue in the field of beef cattle breeding.

[0003] The FTO gene (Fat Mass and Obesity-associated gene) is one of the important genes known to be associated with fat metabolism and obesity. Studies have shown that the FTO gene plays a key role in the process of fat accumulation and metabolism, especially in obesity and fat storage. The high expression of the FTO gene can promote fat accumulation and affect the body fat distribution. Therefore, how to regulate the expression of the FTO gene in beef cattle to enhance the accumulation of intramuscular fat has become a research hotspot in the fields of genetic engineering and molecular breeding.

[0004] The CRISPR / Cas9 gene editing technology has been widely used in the fields of animal gene function research and trait improvement. At present, most gene editing focuses on the coding region of the genome, and this method often destroys the overall structure of the gene. By designing targets for editing in the gene promoter or UTR (untranslated region) region, the expression of the gene can be precisely regulated without changing the coding region of the gene. For the FTO gene, increasing its expression level can effectively promote the differentiation of adipocytes and enhance the accumulation of intramuscular fat, providing new possibilities for the improvement of beef cattle quality. Therefore, developing an efficient editing strategy for the bovine FTO gene promoter region has become a potential solution to increase the intramuscular fat content of beef cattle. Summary of the Invention

[0005] By precisely editing the FTO gene promoter region, the present invention screened out the bovine FTO gene promoter mutation site, successfully increased the expression of the FTO gene, and significantly enhanced the differentiation ability of intramuscular fat precursor cells, specifically as follows:

[0006] A bovine FTO gene promoter mutation site, which is located in the promoter region of the FTO gene on chromosome 18 of the bovine genome. Specifically, the A base at the -33 base position upstream of the transcription start site is mutated to the G base, and the chromosome coordinate is Chr: 22040490. The mutation site is obtained by screening after editing the promoter region of the bovine FTO gene by the CRISPR / Cas9 technology.

[0007] Further, the editing site region for editing the promoter region of the bovine FTO gene by the CRISPR / Cas9 technology is a DNA sequence containing 20 deoxyribonucleotides as shown in SEQ ID NO: 1. The deoxyribonucleotide sequence is located at the 26th to 45th base positions upstream of the promoter of the FTO gene on chromosome 18 of the bovine genome, and the chromosome coordinate is Chr: 22040478 - 22040497.

[0008] In a second aspect, the present invention provides a mutant. The mutation site of the mutant is located in the promoter region of the FTO gene on chromosome 18 of the bovine genome. Specifically, the A base at the -33 base position upstream of the transcription start site is mutated to the G base, and the chromosome coordinate is Chr: 22040490; the mutant increases the expression level of the bovine FTO gene.

[0009] In a third aspect, the present invention provides an application of the bovine FTO gene promoter mutation site for enhancing the differentiation ability of intramuscular fat precursor cells.

[0010] In a fourth aspect, the present invention provides an application of the bovine FTO gene promoter mutation site for increasing the expression level of the bovine FTO gene and promoting the differentiation of intramuscular fat precursor cells, thereby being used for the cultivation of new beef cattle breeds or improving the intramuscular fat content of beef cattle.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] Without changing the overall structure of the FTO gene, the present invention introduces an A→G mutation in the promoter region through the CRISPR / Cas9 technology, increases the expression level of the FTO gene, and enhances the differentiation ability of intramuscular fat precursor cells. The present invention provides a safe and precise gene regulation method, providing a technical basis for the cultivation of new beef cattle breeds with high fat content. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the position of the editing site of the FTO gene promoter on chromosome 18 of the bovine genome.

[0014] Figure 2They are different mutants generated after CRISPR / Cas9 editing. Among them, A is the summary graph after mutant site identification, and B is the Sanger sequencing graph of the mutant site;

[0015] Figure 3 It is the detection of the activities of different mutants. Among them, A represents the luciferase activity detected after the promoter regions of different mutants are constructed into the dual-luciferase reporter; B represents the expression levels of FTO protein in the wild type (WT) and T1 mutant;

[0016] Figure 4 It is the induction of adipogenic differentiation of the wild type (WT), T1 mutant, T2 mutant and T3 mutant to detect lipid accumulation in the cells. Specific implementation manners

[0017] The present invention will be described below in conjunction with specific embodiments. It should be noted that these embodiments are exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the technical details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0018] For the molecular biology test methods not specifically described in the following embodiments, they are all carried out according to the specific methods described in "Molecular Cloning: A Laboratory Manual" (Third Edition) by J. Sambrook, or according to the operation instructions of the kits and product manuals.

[0019] Example 1 Construction of the sgRNA-Cas9 expression vector for editing the target site of the FTO gene promoter

[0020] 1. Main reagents and sources

[0021] In the following embodiments, the experimental materials and reagents used include: pSpCas9 is purchased from Addgene, Escherichia coli competent cells E.co1i DH5α and plasmid extraction kit are purchased from Beijing TransGen Biotech Co., Ltd., BsaⅠ restriction endonuclease, T4 DNA ligase, and Master Taq mix reagents are all purchased from TaKaRa, DMEM medium, Opti-MEM medium and fetal bovine serum FBS are purchased from Gibco, and Lipofectamine3000 is purchased from Invitrogen. Primer synthesis and sequencing are completed by Shanghai Sangon Biological Engineering Co., Ltd.

[0022] Target primers:

[0023] T1F: SEQ ID NO.2;

[0024] T1R: SEQ ID NO.3.

[0025] 2. Operating steps

[0026] First step: The target primers anneal into double strands.

[0027] Centrifuge the upstream and downstream primers to the bottom of the tube first. Then add 0.5×TE to dilute the primers to 100 μmol / L. Then add 1 μL of the upstream and downstream primers to the PCR tube respectively, and add 98 μL of sterile water to make up to 100 μL. Then perform annealing according to the program of 90 °C for 30 s, and then cool and anneal at room temperature to obtain double-stranded sgRNA.

[0028] Second step: Linearize the plasmid and ligate it with double-stranded sgRNA.

[0029] 20 μL restriction enzyme reaction system:

[0030] 1 μg of SpCas9 vector, 2 μL of 10×FastDigest Buffer, 1 μL of BsaI enzyme (10 U / μL), make up to 20 μL system with enzyme-free water.

[0031] React at 37 °C for 30 min, then perform 1% gel recovery, dissolve with enzyme-free water and detect the concentration. Then insert the double-stranded sgRNA prepared in the first step into the linearized SpCas9 vector, with the ratio: 1 μL of linearized SpCas9 vector, 3 μL of annealed double-stranded sgRNA, 5 μL of SolutionI. Then make up to 10 μL with double-distilled water, mix well and ligate at 37 °C for 2 hours to obtain the ligation product.

[0032] Then add the ligation product to 50 μL of competent DH5α. Gently flick and mix, then place on ice for 30 min, then heat shock at 42 °C for 90 s, then ice bath for 5 min. First add 1 mL of antibody-free LB medium to resuscitate for 45 min, then coat on the plate containing ampicillin antibody, pick monoclonal colonies and continue to culture. The sgRNA-SpCas9 plasmid is successfully constructed. Send the sgRNA-SpCas9 plasmid to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing to detect whether the ligation is correct. The sequencing primer is the universal primer U6 to confirm that the sgRNA is correctly inserted into the vector.

[0033] Figure 1The figure shows the schematic position of the editing site of the FTO gene promoter on chromosome 18 of the bovine genome. In the figure, Chr:18 represents chromosome 18, E1-E9 represent exons, TGG represents the sgRNA recognition site, the editing region is located at the 26th to 45th base positions upstream of the FTO gene promoter, the chromosome coordinates are Chr:22040478-22040497, and (G) represents the mutated base.

[0034] From Figure 1 It can be seen that in the present invention, by precisely designing sgRNA to target the FTO gene promoter region, the CRISPR / Cas9 technology is used to introduce an A→G mutation in this region, specifically at the -33 base position upstream of the transcription start site.

[0035] Example 2: Cultivation, transfection and expansion culture of bovine preadipocytes

[0036] First, bovine preadipocytes are resuscitated in a 39 °C water bath and inoculated into a 6-well cell culture dish. The culture medium is DMEM medium containing 15% fetal bovine serum and 1% double antibody, and the culture conditions are 5% CO 2 , 39 °C, saturated humidity. When the cell confluence reaches about 85%, transfection can be carried out. In the experiment, Lipofectamine3000 liposome is used for transfection. 7.5 μL of Lipofectamine 3000 reagent is fully mixed and diluted with 125 μL of Opti-MEM medium. Then, 5 μg of sgRNA-SpCas9 plasmid is diluted with 250 μL of Opti-MEM medium. After adding 10 μL of P3000 reagent, it is fully mixed. The above-diluted Lipofectamine 3000 reagent is added to the tube containing the diluted sgRNA-SpCas9 plasmid without mixing, and it is left standing in the dark for 15 min. Then, the mixture is added to the cells, and the cells are incubated at 37 °C for 2-4 days. During this period, the fresh medium is changed every 2 days. Then, single clones are selected into a 96-well cell culture plate, with one single clone cell cluster in each well. After the confluence is cultured to 70%-80%, all the cells in each well are collected and transferred to a 48-well cell culture dish for continued culture. By analogy, the single clones are transferred to a 6-well cell culture dish for continued culture for standby. This cell is the mutant monoclonal cell obtained after editing.

[0037] Example 3: Genotype identification of monoclonal cells

[0038] Extract the DNA of wild-type (WT) and mutant monoclonal cells for PCR targeted amplification. The amplification site is 400 bp upstream and downstream of the transcription start site. Primer sequences:

[0039] FTO-T1-F: SEQ ID NO.4;

[0040] FTO-T1-R: SEQ ID NO.5.

[0041] PCR amplification system:

[0042] Premix Taq 10 uL, FTO-T1-F 1.25 uL, FTO-T1-R 1.25 uL, gDNA 50 ng, ddH 2 0 supplemented to a 20 μL system;

[0043] PCR amplification conditions:

[0044] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 59°C for 45 s, extension at 72°C for 1 min, 32 cycles, and final extension at 72°C for 5 min.

[0045] Afterwards, the PCR products of the mutant monoclonal cells were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were compared with the gene sequence of the bovine FTO-T1 region to identify the genotype of each monoclonal cell. The genotype identification results ( Figure 2 ) Figure 2 A is the summary diagram after mutant site identification, Figure 2 B is the Sanger sequencing diagram of the mutant site: Figure 2 In A, the pink box represents the sgRNA targeting sequence, the blue letters represent the sgRNA recognition sites, and the red letters and dotted lines represent point mutations, single-base insertions, and fragment deletions respectively; compared with the wild type (WT), three mutants were identified. Among them, the T1 mutant has a mutation from A to G at Chr: 22040490, the T2 mutant has an insertion of T base at Chr: 22040495, and the T3 mutant has a 10-base deletion in the region of Chr: 22040478 - 22040487. Figure 2 In B, the red box represents the sgRNA targeting sequence.

[0046] Example 4 Detection of FTO activity and expression level

[0047] Amplify 1000 bases upstream of the transcription start site in the wild type (WT) and its T1 mutant, T2 mutant, and T3 mutant, and then ligate them upstream of the firefly luciferase gene in the pGL3-basic vector to construct plasmids containing different promoters for detecting promoter activity.

[0048] The amplification primers are as follows:

[0049] FTO pro-pGL3-F: SEQ ID NO.6;

[0050] FTO pro-pGL3-R: SEQ ID NO.7;

[0051] The sgRNA-SpCas9 plasmid was transfected into 293T cells using Lipofectamine 3000. After 24 hours of transfection, the luciferase activity was measured using a dual-luciferase kit (Promega), and the Renilla luciferase activity was used as an internal reference to detect the activities of different mutants. Figure 3 This is the FTO activity assay.

[0052] Subsequently, the FTO expression levels of the T1 mutant and the wild type (WT) were detected by Western Blot. The experimental procedure is as follows:

[0053] After digesting the wild type (WT) and the T1 mutant, they were washed 3 times with PBS. After the last centrifugation, cell lysis was performed using RIPA buffer containing protease and phosphatase inhibitors. The lysis reaction was carried out on ice for 40 minutes, followed by centrifugation for 30 minutes to collect the supernatant, and the protein concentration was measured using a BCA kit. Then, the proteins were separated using a 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk powder and then incubated with the primary antibody overnight at 4°C. After washing, the membrane was incubated with the HRP-labeled secondary antibody at room temperature for 1 hour. The relative density of each band was normalized using GAPDH as an internal reference, and visualization and analysis were performed using the Tanon 5200 system. Figure 3 Shown are the FTO activity assay and the expression level detection graphs. Among them, 3A represents the luciferase activity detected after constructing the promoter regions of different mutants into the dual-luciferase reporter, and the activity of the T1 mutant is the highest; 3B represents the FTO protein expression levels in the wild type (WT) and the T1 mutant, with GAPDH as the protein internal reference, and the FTO expression level in the T1 mutant is higher than that in the WT group.

[0054] Example 5 Detection of adipogenic differentiation of FTO mutant cells

[0055] The T1 mutant, T2 mutant, T3 mutant, and wild type (WT) in Example 3 were subjected to adipogenic differentiation to detect the effects of different FTO mutations on adipogenic differentiation. When the confluence of the above cells reached 90%, the growth medium was discarded, and the cells were washed 3 times with PBS. Then, adipogenic induction medium was added for induction, and the medium was changed every 72 hours. During adipogenic differentiation induction, the morphological changes such as the size of lipid droplets in the cytoplasm after adipogenic induction were observed at all times and photographed. Subsequently, Bodipy staining was performed.

[0056] Dissolve 10 mg of Bodipy powder in 7.63 mL of DMSO solution to prepare a 5 mM stock solution. After complete dissolution, aliquot and store at -20°C in the dark and dry. Dilute to 10 μM before use. Take the above cells for staining. After discarding the culture medium, gently and quickly wash with PBS 3 times, 5 minutes each time. Then fix with 4% paraformaldehyde for 30 minutes, adding 1 ml per well. After discarding the fixative, wash with PBS 3 times. Stain with the Bodipy fluorescent probe staining solution diluted to 10 μM at room temperature for 30 minutes. After discarding the staining solution, wash with PBS 3 times. Stain the cell nuclei with DAPI staining solution for 10 - 15 minutes. After discarding the staining solution, wash with PBS 3 times. Then add a small amount of PBS and take pictures under fluorescence microscopy.

[0057] Figure 4 Shown are the adipogenic differentiation detections of T1 mutant, T2 mutant, T3 mutant, and wild type (WT). Among them, green is stained with the Bodipy probe, representing lipid droplets, and blue is stained with DAPI, representing cell nuclei. The scale bar is 100 μm. The results show that the adipogenic differentiation degree of the T1 mutant is the highest, indicating that the Chr: 2204049A→G mutation can improve the adipogenic differentiation ability.

[0058] The research of the present invention shows that in the FTO promoter region of the bovine genome, the Chr: 2204049A→G mutation can increase the expression of the FTO gene and promote the differentiation of intramuscular fat precursor cells, providing an effective technical means for the cultivation of new beef cattle breeds or the improvement of intramuscular fat content in beef cattle.

[0059] The above has described the preferred embodiments of the present invention in detail, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention.

Claims

1. A mutant, characterized in that: The gene sequence of the mutant is shown in SEQ ID NO.8, and the mutation site of the mutant is located in the FTO gene promoter region on chromosome 18 of the bovine genome, where the A base at the position of -33 bases upstream of the transcription start site is mutated to the G base, and the chromosome coordinate is Chr: 22040490; the mutant increases the expression level of the bovine FTO gene; the mutation site is screened after editing in the bovine FTO gene promoter region using CRISPR / Cas9 technology; The editing site region of the bovine FTO gene promoter region edited by the CRISPR / Cas9 technology is a DNA sequence as shown in SEQ ID NO: 1 containing 20 deoxyribonucleotides, and the deoxyribonucleotide sequence is located at the 26th to 45th base positions upstream of the FTO gene promoter on chromosome 18 of the bovine genome, and the chromosome coordinates are Chr: 22040478-22040497.

2. The use of a mutant according to claim 1, characterized in that: Used to enhance the differentiation ability of intramuscular adipocytes.

3. The use of a mutant according to claim 1, characterized in that: It is used to increase the expression of the cattle FTO gene and promote the differentiation of intramuscular fat precursor cells, so as to be used for breeding new beef cattle breeds or increasing the intramuscular fat content of beef cattle.

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