Enhancer for regulating chicken PGC-1alpha gene expression and application thereof

Abstract

The invention discloses an enhancer for regulating and controlling chicken PGC-1alpha gene expression and application of the enhancer, and belongs to the field of bioengineering. The nucleotide sequence of the enhancer is shown as SEQ ID NO: 1. The invention further discloses a biological material containing the enhancer and a preparation method of a cell strain with the enhancer knocked out. A dual-luciferase reporter gene system proves that the enhancer has remarkable activity in DF1 and ICP2 cells, the expression of the PGC-1alpha gene in chicken preadipocytes can be reduced by knocking out the enhancer, and cell differentiation and lipid droplet generation are remarkably promoted. The enhancer can be used for preparing a biological preparation for regulating and controlling the expression level of the PGC-1alpha gene and regulating and controlling chicken preadipocyte differentiation, can also be applied to genetic improvement of chicken fat traits and can be used for preparing a model for researching related gene functions and cell differentiation mechanisms. The invention provides a new target and technical support for chicken fat character improvement.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and particularly relates to an enhancer for regulating chicken PGC-1α gene expression and an application thereof. Background Art

[0002] Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α, encoded by the PPARGC1A gene) is a transcriptional coactivator that interacts with multiple transcription factors and participates in a wide range of biological processes. These processes include mitochondrial biogenesis, adaptive thermogenesis, maintenance of energy homeostasis, skeletal muscle fiber type transitions, and adipose tissue development. PGC-1α is widely distributed in various organs and tissues of mammals and is primarily expressed in mitochondria-rich tissues such as the heart, skeletal muscle, and adipose tissue. PGC-1α transgenic mice exhibit obesity-resistant or insulin-sensitive phenotypes. Adipose tissue-specific PGC-1α overexpression in transgenic mice exhibits improved mitochondrial biogenesis and respiration, as well as decreased fasting blood glucose, blood pressure, and fibrosis, and increased oxygen consumption. PGC-1α knockout mice have significantly higher epididymal white adipose tissue and abdominal total adipose tissue mass than wild-type mice. Therefore, targeted regulation of PGC-1α gene expression may be a potential means of controlling adipogenesis in animals.

[0003] Enhancers are short DNA sequences, approximately 100-1000 bp, that regulate gene expression by recruiting multiple transcription factors and are not restricted by distance or direction. Enhancers have the following characteristics: (1) Active enhancers are often bound by transcription factors; (2) Chromatin flanking active enhancers often contains histone modifications, among which three histone modifications, H3K27ac, H3K4me1, and H3K27me3, are common in enhancer regions; (3) Enhancers often lack nucleosomes and are sensitive to DNase I; (4) Enhancers recruit mediators and auxiliary factors to form chromatin loops close to their target promoters; (5) In addition, active enhancers recruit RNAPII to transcribe unidirectionally or bidirectionally to produce enhancer RNA (eRNA), which helps regulate the expression of target genes.

[0004] Chickens are important economic animals and models for research in developmental biology and immunology. However, in the modern broiler industry, while long-term genetic selection has significantly improved broiler growth and meat production, it has also led to the negative issue 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 chicken farming and reduces consumer purchases. Therefore, in-depth research on the molecular mechanisms of fat production and development in broilers is crucial for the sustainable development of the broiler breeding industry, aiming to effectively reduce excessive abdominal fat deposition and develop new low-fat broiler breeds. Given the importance of PGC-1α, editing the coding region to completely disable PGC-1α gene function could compromise normal growth and development. Previous studies have identified a potential enhancer sequence within the second intron of the PGC-1α gene. Therefore, the potential feasibility of regulating PGC-1α gene expression through enhancers provides a theoretical foundation for poultry breeding. Summary of the Invention

[0005] The technical problems to be solved by the present invention are: providing a chicken PGC-1α gene enhancer; and providing a cell model for knocking out the enhancer.

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

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

[0008] The present 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 upstream sgRNA1 and downstream sgRNA2 for targeted knockout regulation of the chicken PGC-1α gene enhancer, and connect the DNA sequences corresponding to upstream sgRNA1 and downstream sgRNA2 to the PX458 vector, respectively. The sequences of upstream sgRNA1 and downstream sgRNA2 are shown in SEQ ID NO: 4 and SEQ ID NO: 5;

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

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

[0012] Further, the biological preparation for up-regulating chicken PGC-1α gene expression comprises the enhancer or a substance promoting the activity of the enhancer.

[0013] Further, the biological preparation for down-regulating chicken PGC-1α gene expression comprises a substance inhibiting the activity of the enhancer or a nucleic acid sequence capable of knocking out the enhancer.

[0014] Another object of the present application is to disclose the use of the enhancer for regulating chicken PGC-1α gene expression in the preparation of a biological preparation for regulating chicken preadipocyte differentiation.

[0015] Further, the biological preparation for inhibiting chicken preadipocyte differentiation comprises the enhancer or a substance promoting the activity of the enhancer.

[0016] Further, the biological preparation for promoting chicken preadipocyte differentiation comprises a substance inhibiting the activity of the enhancer or a nucleic acid sequence capable of knocking out the enhancer.

[0017] Another object of the present application is to disclose the use of the enhancer for regulating chicken PGC-1α gene expression in the genetic improvement of chicken fat traits.

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

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

[0020] Another object of the present application is to disclose the use of the enhancer for regulating chicken PGC-1α gene expression in the preparation of a cell model or an animal model for studying the function of chicken PGC-1α gene and the mechanism of chicken preadipocyte differentiation.

[0021] The present invention has the following technical effects and advantages: The present invention provides a novel enhancer for regulating chicken PGC-1α gene expression. Using a dual-luciferase reporter gene system, the enhancer was confirmed to significantly enhance activity in DF1 and ICP2 cells. Functional validation at the chicken preadipocyte level revealed that knocking out the PGC-1α gene enhancer significantly promoted chicken preadipocyte differentiation, revealing for the first time the role of the PGC-1α gene enhancer in fat development. Subsequent RT-PCR analysis revealed that knocking out the PGC-1α gene enhancer significantly reduced PGC-1α gene expression levels. These results demonstrate that the PGC-1α gene enhancer is a regulatory element for PGC-1α gene expression. The present invention identifies a novel target for regulating chicken fat growth and development, providing a powerful resource for genetically improving important economic traits in chickens. Furthermore, the cell line constructed by the present invention, which regulates the chicken PGC-1α gene enhancer knockout, can provide technical support for the future development of PGC-1α gene enhancer knockout chickens. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0024] Figure 3 A physical map to locate the relevant sequences for knocking out the PGC-1α gene enhancer;

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

[0026] Figure 5 Figure 3. PGC-1α gene enhancer knockout cell line identified using PCR technology;

[0027] Figure 6 This is the result of detecting the mRNA expression of PGC-1α gene by knocking out the enhancer of PGC-1α gene;

[0028] Figure 7 Figure 3: Knockout of the PGC-1α gene enhancer inhibits the differentiation of chicken preadipocytes. A shows the results of inducing ICP2 cell differentiation using oleic acid. B shows the colorimetric quantitative results of lipid deposition extraction in A. C shows the mRNA expression of PPARγ, AdipoQ, C / EBPα, and LPL genes detected by RT-PCR. DETAILED DESCRIPTION

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

[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, primers PE-F (SEQ ID NO: 2) and PE-R (SEQ ID NO: 3) were used to PCR amplify the PGC-1α gene enhancer sequence (shown in SEQ ID NO: 1). Subsequently, the pGL3-promoter vector was used as a substrate and double-digested with BamHI and SalI endonucleases. The double-stranded DNA fragment containing the PGC-1α enhancer sequence was then cloned downstream of the reporter gene in the pGL3-promoter vector using Vazyme's One Step Cloning Kit. The constructed plasmid map is shown in the figure. Figure 1 shown.

[0033] 1.2. The constructed PGC-1α gene enhancer reporter gene vector was transfected into DF1 and ICP2 cells respectively, and the internal control pRL-TK vector was also transfected. 48 hours after transfection, the Luciferase Assay System (Promega) kit was used for detection according to the instructions. The experimental results are shown in the figure below. Figure 2 As shown, compared with the empty pGL3-promoter vector, the reporter gene activity was significantly increased after the PGC-1α gene enhancer was inserted, indicating that the PGC-1α gene enhancer sequence (SEQ ID NO: 1) has enhancer activity.

[0034] 2. A method for constructing a monoclonal cell line with a knockout of the chicken PGC-1α gene enhancer comprises the following steps:

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

[0036] 2.2, Co-transfect ICP2 cells with PX458-sgRNA1 and PX458-sgRNA2, and after 48 hours of transfection, screen positive monoclonal cells by flow cytometry, and continue to expand the culture. Use a rapid DNA extraction detection kit (Tiangen) to extract the cell genome for PCR amplification to identify the cell genotype.

[0037] 2.3, Use the cell genome as a template, and use primers PGC-1a-F (SEQ ID NO: 6) and PGC-1a-R (SEQ ID NO: 7) to perform PCR amplification. The results are shown in Figure 5 , when the #2, #10, and #11 cell strains are used as templates, a 310 bp band of interest is obtained. This shows that we have successfully obtained three PGC-1a gene enhancer knockout cells.

[0038] 2.4, Analysis of the expression amount of chicken PGC-1a gene in chicken preadipocytes. Extract total RNA from wild-type ICP2 cells and PGC-1a gene enhancer knockout cells, and reverse transcribe it into cDNA. Add the cDNA template to the quantitative PCR 96-well plate according to the kit instructions, and place it in the quantitative PCR machine for reaction. The results are shown in Figure 6 , compared with wild-type ICP2 cells, the mRNA expression amount of PGC-1a gene in the successfully constructed PGC-1a gene enhancer knockout cells is significantly down-regulated, proving that the PGC-1a gene enhancer has a significant regulatory effect on the PGC-1a gene.

[0039] 2.5, When the wild-type ICP2 cells and PGC-1a gene enhancer knockout cells reach 60% confluence, replace the complete culture medium with an induction differentiation medium containing 200 μM sodium oleate for culture, and replace the new induction differentiation medium every day during the induction and differentiation process. Collect the cells at 24, 48, and 72 hours of differentiation, and perform oil red O staining. As shown in Figure 7 A, both wild-type ICP2 cells and PGC-1a gene enhancer knockout cells have lipid droplets. The oil red O extraction colorimetric results are shown in Figure 7 B, the lipid droplet accumulation in PGC-1a gene enhancer knockout cells is significantly higher than that in wild-type ICP2 cells. Extract total RNA from cells at different time points, and reverse transcribe it into cDNA. Add the cDNA template to the quantitative PCR 96-well plate according to the kit instructions, and place it in the quantitative PCR machine for reaction. The results are shown in Figure 7 C, compared with wild-type ICP2 cells, the expression amount of PPARy, AdipoQ, C / EBPalpha, and LPL in the successfully constructed PGC-1a gene enhancer knockout cells is significantly up-regulated, proving that PGC-1a gene enhancer knockout promotes chicken preadipocyte differentiation and promotes lipid droplet formation.

Claims

1. An enhancer for regulating chicken PGC-1α gene expression, characterized in that: The nucleotide sequence thereof is shown in SEQ ID NO:

1.

2. A biomaterial, characterized in that: The enhancer for regulating chicken PGC-1α gene expression as claimed in claim 1 is included, and the biological material is recombinant DNA, a vector or an engineered bacterium.

3. A method for preparing a cell line with a knockout of the enhancer that regulates chicken PGC-1α gene expression according to claim 1, comprising the following steps: S1: Design the upstream sgRNA1 and downstream sgRNA2 of the chicken PGC-1α gene enhancer for targeted knockout regulation, and connect the DNA sequences corresponding to the upstream sgRNA1 and downstream sgRNA2 to 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 is co-transfected into chicken preadipocytes ICP2, positive cells are screened, and a cell line with PGC-1α gene enhancer knockout is obtained.

4. Use of the enhancer for regulating chicken PGC-1α gene expression as claimed in claim 1 in the preparation of a biological agent for regulating the expression level of chicken PGC-1α gene.

5. The use according to claim 4, characterized in that The biological preparation is used to upregulate the expression of the chicken PGC-1α gene, and the biological preparation contains the enhancer or a substance that promotes the activity of the enhancer; or the biological preparation is used to downregulate the expression of the chicken PGC-1α gene, and the biological preparation contains a substance that inhibits the activity of the enhancer or a nucleic acid sequence that can knock out the enhancer. 6 . Use of the enhancer for regulating chicken PGC-1α gene expression as claimed in claim 1 in the preparation of a biological agent for regulating chicken preadipocyte differentiation.

7. The use according to claim 6, characterized in that The biological preparation is used to inhibit the differentiation of chicken preadipocytes, and the biological preparation contains the enhancer or a substance that promotes the activity of the enhancer; or the biological preparation is used to promote the differentiation of chicken preadipocytes, and the biological preparation contains a substance that inhibits the activity of the enhancer or a nucleic acid sequence that can knock out the enhancer.

8. Use of the enhancer for regulating chicken PGC-1α gene expression as claimed in claim 1 in genetic improvement of chicken fat traits.

9. The use according to claim 8, characterized in that By enhancing the activity of the enhancer, the expression of the chicken PGC-1α gene is increased, thereby inhibiting the differentiation of chicken preadipocytes and improving the fat traits of chickens; or 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 the fat traits of chickens.

10. Use of the enhancer for regulating chicken PGC-1α gene expression according to claim 1 in preparing a cell model or animal model for studying the function of chicken PGC-1α gene and the differentiation mechanism of chicken preadipocytes.

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