An enhancer that regulates chicken PGC-1α gene expression and its application

Abstract

This invention discloses an enhancer that regulates chicken PGC-1α gene expression and its applications, belonging to the field of bioengineering. The nucleotide sequence of the enhancer is shown in SEQ ID NO: 1. This invention further discloses biomaterials containing this enhancer and a method for preparing cell lines with the enhancer knocked out. A dual-luciferase reporter gene system confirmed its significant activity in DF1 and ICP2 cells. Knocking out this enhancer downregulates PGC-1α gene expression in chicken preadipocytes, significantly promoting cell differentiation and lipid droplet formation. This enhancer can be used to prepare biological agents that regulate PGC-1α gene expression levels and chicken preadipocyte differentiation. It can also be applied to the genetic improvement of chicken fat traits and to the preparation of models for studying related gene function and cell differentiation mechanisms. This invention provides new targets and technical support for improving chicken fat traits.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to an enhancer that regulates the expression of the chicken PGC-1α gene and its application. Background Technology

[0002] Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α, encoded by the PPARGC1A gene) is a transcriptional coactivator that interacts with various transcription factors, thereby participating in a wide range of biological processes. These processes include mitochondrial biosynthesis, adaptive thermogenesis, energy homeostasis, skeletal muscle fiber type conversion, and adipose tissue development. PGC-1α is widely distributed in various organs and tissues of mammals, but is mainly expressed in mitochondrial-rich tissues such as the heart, skeletal muscle, and adipose tissue. PGC-1α transgenic mice exhibit obesity resistance or insulin sensitivity phenotypes. Adipose tissue-specific PGC-1α overexpression transgenic mice show improved mitochondrial biosynthesis and respiration, while fasting reduces blood glucose, blood pressure, and fibrosis, and increases oxygen consumption. PGC-1α knockout mice have significantly higher amounts of epididymal white adipose tissue and total abdominal adipose tissue than wild-type mice. Therefore, targeted regulation of PGC-1α gene expression levels may be a potential means of controlling lipogenesis in animals.

[0003] Enhancers are short DNA sequences, approximately 100-1000 bp, that regulate gene expression by enriching various transcription factors and are not limited by distance or direction. Enhancers have the following characteristics: (1) Active enhancers usually bind to transcription factors; (2) The chromatin flanking active enhancers usually has histone modifications, among which the enhancer region commonly has three histone modifications: H3K27ac, H3K4me1, and H3K27me3; (3) Enhancers usually lack nucleosomes and are sensitive to DNase I; (4) During enhancer recruitment, mediators and cofactors form chromatin loops close to their target promoters; (5) In addition, active enhancers recruit RNAPII for unidirectional or bidirectional transcription to produce enhancer RNA (eRNA), which helps regulate the expression of target genes.

[0004] Chickens are important economic animals and also crucial model organisms in fields such as developmental biology and immunology. However, while long-term genetic selection in the modern broiler industry has significantly improved growth rate and meat yield, it has also led to the negative problem of excessive abdominal fat accumulation. Excessive fat accumulation not only reduces feed conversion efficiency and meat quality but also severely impacts the economic benefits of poultry farms and reduces consumer willingness to buy. Therefore, in-depth research into the molecular mechanisms of broiler fat generation and development, thereby effectively reducing excessive abdominal fat deposition and breeding new low-fat broiler breeds, is crucial for the sustainable development of the broiler breeding industry. Given the importance of PGC-1α, editing its coding region could lead to complete loss of PGC-1α gene function or affect normal growth and development in animals. Previous studies have identified a potential enhancer sequence in the second intron region of the PGC-1α gene. Therefore, the potential feasibility of regulating PGC-1α gene expression levels through enhancers lays a theoretical foundation for poultry breeding. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a chicken PGC-1α gene enhancer and to provide a cell model in which the enhancer is knocked out.

[0006] The technical solution adopted in this invention is: an enhancer that regulates the expression of the chicken PGC-1α gene, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0007] Furthermore, the present invention also provides a biological material comprising the enhancer described above for regulating chicken PGC-1α gene expression, wherein the biological material is recombinant DNA, a vector, or engineered bacteria.

[0008] This invention also provides a method for preparing a cell line based on the knockout of an enhancer that regulates chicken PGC-1α gene expression, comprising the following steps:

[0009] S1: Design a targeted knockout mechanism to regulate the upstream sgRNA1 and downstream sgRNA2 of the chicken PGC-1α gene enhancer, and ligate the DNA sequences corresponding to the upstream sgRNA1 and downstream sgRNA2 into the PX458 vector, wherein the sequences of the upstream sgRNA1 and downstream sgRNA2 are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

[0010] S2: The vector obtained in step S1 was co-transfected into chicken preadipocytes ICP2, and positive cells were screened to obtain cell lines with PGC-1α gene enhancer knockout.

[0011] Another object of the present invention is to disclose the application of an enhancer that regulates chicken PGC-1α gene expression as described above in the preparation of a biological agent for regulating chicken PGC-1α gene expression levels.

[0012] Furthermore, the biological agent is used to upregulate chicken PGC-1α gene expression, and the biological agent contains the enhancer or a substance that promotes the activity of the enhancer.

[0013] Furthermore, the biological agent is used to downregulate chicken PGC-1α gene expression, and the biological agent contains a substance that inhibits the activity of the enhancer or a nucleic acid sequence capable of knocking out the enhancer.

[0014] Another object of the present invention is to disclose the application of an enhancer that regulates chicken PGC-1α gene expression as described above in the preparation of a biological agent for regulating chicken preadipocyte differentiation.

[0015] Furthermore, the biological agent is used to inhibit the differentiation of chicken preadipocytes, and the biological agent contains the enhancer or a substance that promotes the activity of the enhancer.

[0016] Furthermore, the biological agent is used to promote the differentiation of chicken preadipocytes, and the biological agent contains a substance that inhibits the activity of the enhancer or a nucleic acid sequence capable of knocking out the enhancer.

[0017] Another objective of this invention is to disclose the application of an enhancer that regulates chicken PGC-1α gene expression as described above in the genetic improvement of chicken fat traits.

[0018] Furthermore, by enhancing the activity of the enhancer, chicken PGC-1α gene expression is increased, thereby inhibiting chicken preadipocyte differentiation and improving chicken fat traits.

[0019] Furthermore, by inhibiting the activity of the enhancer or knocking out the enhancer, the expression of the chicken PGC-1α gene is reduced, thereby promoting the differentiation of chicken preadipocytes and improving chicken fat traits.

[0020] Another objective of this invention is to disclose the application of an enhancer that regulates chicken PGC-1α gene expression as described above in the preparation of cell or animal models for studying chicken PGC-1α gene function and chicken preadipocyte differentiation mechanism.

[0021] This invention offers the following technical effects and advantages: It provides a novel enhancer regulating chicken PGC-1α gene expression. The dual-luciferase reporter gene system confirmed that this enhancer significantly enhances its activity in DF1 and ICP2 cells. Functional verification at the chicken preadipocyte level showed that knocking out the PGC-1α gene enhancer significantly promoted preadipocyte differentiation, revealing for the first time the role of the PGC-1α gene enhancer in adipocyte development. Subsequent RT-PCR analysis revealed that knocking out the PGC-1α gene enhancer significantly reduced PGC-1α gene expression levels. These results indicate that the PGC-1α gene enhancer is a regulatory element for PGC-1α gene expression. This invention identifies a novel target for regulating chicken adipocyte growth and development, providing a powerful resource for the genetic improvement of important economic traits in chickens. Furthermore, the chicken PGC-1α gene enhancer knockout cell line constructed in this invention can provide technical support for the future preparation of PGC-1α gene enhancer knockout chickens. Attached Figure Description

[0022] Figure 1 Plasmid map of the PGC-1α gene enhancer cloned into the PGL3-Basic vector;

[0023] Figure 2 To verify the enhancer activity diagram of the PGC-1α gene enhancer in chicken DF1 cells and ICP2 cells;

[0024] Figure 3 Physical map of the location of the relevant sequences for the PGC-1α gene enhancer knockout;

[0025] Figure 4 The plasmid map of the PX458 vector;

[0026] Figure 5 To identify PGC-1α gene enhancer knockout cell lines using PCR technology;

[0027] Figure 6 The figure shows the results of detecting the level of PGC-1α gene mRNA expression suppressed by the PGC-1α gene enhancer knockout.

[0028] Figure 7 The diagram illustrates the inhibition of chicken preadipocyte differentiation by knocking out the PGC-1α gene enhancer. In the diagram, A shows the results of ICP2 cell differentiation induced by oleic acid; B shows the colorimetric quantitative results of lipid deposition extraction in A; and C shows the mRNA expression of PPARγ, AdipoQ, C / EBPα, and LPL genes detected by RT-PCR. Detailed Implementation

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0030] Example 1

[0031] 1. Cloning and activity analysis of the PGC-1α gene enhancer sequence, including the following steps:

[0032] 1.1 Using chicken whole blood DNA as a template, the PGC-1α gene enhancer sequence (shown in SEQ ID NO: 1) was amplified by PCR using primers PE-F (SEQ ID NO: 2) and PE-R (SEQ ID NO: 3). Subsequently, the pGL3-promoter vector was used as a substrate and double-digested with BamHI and SalI restriction enzymes. Then, using Vazyme's One Step Cloning Kit, the double-stranded DNA fragment containing the PGC-1α enhancer sequence was cloned downstream of the reporter gene in the pGL3-promoter vector. The constructed plasmid map is shown below. Figure 1 As shown.

[0033] 1.2 The constructed PGC-1α gene enhancer reporter gene vector was transfected into DF1 and ICP2 cells, respectively, and simultaneously into the internal control pRL-TK vector. Forty-eight hours after transfection, the results were analyzed using the Luciferase Assay System (Promega) kit according to the manufacturer's instructions. The experimental results are as follows: Figure 2 As shown, compared with the empty pGL3-promoter vector, the reporter gene activity was significantly increased after the insertion of the PGC-1α gene enhancer, indicating that the PGC-1α gene enhancer sequence (SEQ ID NO: 1) has enhancer activity.

[0034] 2. The method for constructing a single-clonal cell line with chicken PGC-1α gene enhancer knockout includes the following steps:

[0035] 2.1 Construction of the PX458-sgRNA vector: sgRNA1 targeting the upstream and downstream sgRNA2 of the chicken PGC-1α gene enhancer were designed, with specific physical locations as follows... Figure 3 As shown. The designed sgRNA was annealed and complemented, then ligated to the BbsI restriction site of the PX458 vector. The PX458 vector map is shown below. Figure 4 As shown in the figure. The nucleotide sequences corresponding to sgRNA1 and sgRNA2 are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0036] 2.2. PX458-sgRNA1 and PX458-sgRNA2 were co-transfected into ICP2 cells. Forty-eight hours after transfection, positive monoclonal cells were screened using flow cytometry and the cells were further cultured. Cell genomes were extracted using a rapid DNA extraction and detection kit (Tiangen) for PCR amplification to identify cell genotypes.

[0037] 2.3. Using the cell genome as a template, PCR amplification was performed using primers PGC-1α-F (SEQ ID NO: 6) and PGC-1α-R (SEQ ID NO: 7). The results are as follows: Figure 5 As shown, when using the genomes of cell lines #2, #10, and #11 as templates, amplification yielded target bands of approximately 310 bp. This indicates that we successfully obtained three cell lines with PGC-1α gene enhancer knockout.

[0038] 2.4. Expression analysis of chicken PGC-1α gene in chicken preadipocytes. Total RNA was extracted from wild-type ICP2 cells and cells with PGC-1α gene enhancer knockout, and reverse transcribed into cDNA. Using cDNA as a template, it was added to a 96-well plate for quantitative PCR according to the kit instructions, and the reaction was carried out in a quantitative PCR chamber. The results are shown below. Figure 6 As shown, compared with wild-type ICP2 cells, the mRNA expression level of the PGC-1α gene in the successfully constructed PGC-1α gene enhancer knockout cells was significantly downregulated, demonstrating that the PGC-1α gene enhancer has a significant regulatory effect on the PGC-1α gene.

[0039] 2.5. When wild-type ICP2 cells and PGC-1α gene enhancer knockout cells reach 60% confluence, replace the complete culture medium with induction differentiation medium containing 200 μM sodium oleate. Change the induction differentiation medium daily during the differentiation process. Collect cells after 24, 48, and 72 hours of differentiation for Oil Red O staining. Figure 7 As shown in Figure A, both wild-type ICP2 cells and cells with the PGC-1α gene enhancer knocked out exhibited lipid droplet formation. The results of Oil Red O extraction colorimetric analysis are as follows: Figure 7 B showed that lipid droplet accumulation in cells with the PGC-1α gene enhancer knocked out was significantly higher than in wild-type ICP2 cells. Total RNA was extracted from cells at different time points and reverse transcribed into cDNA. Using cDNA as a template, it was added to a 96-well plate for quantitative PCR according to the kit instructions and placed in a quantitative PCR incubator. The results are as follows: Figure 7 As shown in Figure C, compared with wild-type ICP2 cells, the expression levels of PPARγ, AdipoQ, C / EBPα, and LPL were significantly upregulated in the successfully constructed PGC-1α gene enhancer knockout cells, demonstrating that PGC-1α gene enhancer knockout promotes chicken preadipocyte differentiation and lipid droplet formation.

Claims

1. An enhancer that regulates the expression of the chicken PGC-1α gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

1.

2. A biomaterial, characterized in that: The invention comprises an enhancer that regulates chicken PGC-1α gene expression as described in claim 1, wherein the biological material is recombinant DNA, a vector, or engineered bacteria.

3. A method for preparing a cell line based on the enhancer knockout of the chicken PGC-1α gene as described in claim 1, comprising the following steps: S1: Design a method to target and knock out the upstream sgRNA1 and downstream sgRNA2 of the chicken PGC-1α gene enhancer, and ligate the DNA sequences corresponding to upstream sgRNA1 and downstream sgRNA2 into the PX458 vector, respectively. The sequences of the upstream sgRNA1 and downstream sgRNA2 are shown in SEQ ID NO: 4 and SEQ ID NO: 5; S2: The vector obtained in step S1 was co-transfected into chicken preadipocytes ICP2, and positive cells were screened to obtain cell lines with PGC-1α gene enhancer knockout.

4. The use of an sgRNA capable of knocking out the enhancer as described in claim 1 in the preparation of a biological agent for downregulating chicken PGC-1α gene expression levels, characterized in that: The sequence of the sgRNA is shown in SEQ ID NO:4 and SEQ ID NO:

5.

5. The application as described in claim 4, characterized in that, The biological agent mentioned is a biological agent that promotes the differentiation of chicken preadipocytes.

6. An application of an sgRNA capable of knocking out the enhancer as described in claim 1 to downregulate the expression level of the chicken PGC-1α gene in the genetic improvement of chicken fat traits, wherein knocking out the enhancer reduces the expression of the chicken PGC-1α gene, thereby promoting the differentiation of chicken preadipocytes and improving chicken fat traits, characterized in that: The sequence of the sgRNA is shown in SEQ ID NO: 4 and SEQ ID NO:

5.

7. The application of an sgRNA capable of knocking out the enhancer as described in claim 1 in the preparation of cell or animal models for studying the function of the chicken PGC-1α gene and the differentiation mechanism of chicken preadipocytes, characterized in that: The sequence of the sgRNA is shown in SEQ ID NO:4 and SEQ ID NO:5.

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