Application of ELN gene in regulating proliferation of skeletal muscle satellite cells and feed utilization

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to application of ELN gene in regulating proliferation of skeletal muscle satellite cells and feed utilization rate. The present application finds that the ELN gene can regulate the proliferation of skeletal muscle satellite cells, and specifically, increasing the expression amount of the ELN gene and / or ELN protein can promote the proliferation of skeletal muscle satellite cells, and decreasing the expression amount of the ELN gene and / or ELN protein can inhibit the proliferation of skeletal muscle satellite cells. Based on this, the meat production performance and feed conversion rate of meat livestock can be improved by increasing the expression amount of the ELN gene and / or ELN protein, and a new breed of meat livestock with improved meat production performance can be bred.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of the ELN gene in regulating skeletal muscle satellite cell proliferation and feed utilization. Background Technology

[0002] The ELN (elastin) gene encodes elastin, a core component of elastic fibers in the extracellular matrix. This protein provides tissues and organs with the ability to stretch and rebound. The expression of this gene is precisely regulated by various transcription factors and signaling pathways, thereby affecting the development and homeostasis of the cardiovascular system and skin. The ELN gene is also closely linked to a variety of diseases; it is considered a key connective tissue structural protein, and its loss of function can lead to severe genetic syndromes. Current research on the ELN gene, both domestically and internationally, mainly focuses on human and mouse models, with less research on ruminant ELN genes, and more emphasis on economic traits.

[0003] Skeletal muscle cells appear as alternating light and dark bands under a microscope, hence the name "striated muscle." Composed of thousands of muscle cell groups covered and connected by connective tissue, they account for approximately 40% of a mammal's body weight and are the primary site of metabolic activity, playing a crucial role in regulating energy metabolism homeostasis. Bovine skeletal muscle satellite cells are adult stem cells attached to the sarcolemma and basement membrane of bovine skeletal muscle fibers. They typically remain dormant when the muscle is at rest. When the muscle is injured, subjected to weight-bearing, or stimulated by exercise, these cells are rapidly activated, proliferating, differentiating, and fusing together. They not only repair damaged muscle fibers but also add new nuclei to existing muscle fibers. Promoting the proliferation of bovine skeletal muscle satellite cells can enhance the growth and repair capacity of muscle tissue, thereby improving muscle growth efficiency and lean meat percentage in cattle. This allows cattle to more effectively convert ingested feed nutrients into valuable muscle tissue, ultimately resulting in improved feed utilization phenotype, making it an important target for muscle development research in animal husbandry. Currently, the relationship between the ELN gene and skeletal muscle cells is unclear. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides the application of the ELN gene in regulating skeletal muscle satellite cell proliferation and feed utilization. This invention discovers that the ELN gene can regulate skeletal muscle satellite cell proliferation, thereby improving meat production traits and feed conversion ratio in meat-producing livestock.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of the ELN gene and / or ELN protein in regulating the proliferation of skeletal muscle satellite cells.

[0006] Preferably, the regulation is as follows: increasing the expression level of ELN gene and / or ELN protein promotes skeletal muscle satellite cell proliferation, and decreasing the expression level of ELN gene and / or ELN protein inhibits skeletal muscle satellite cell proliferation.

[0007] Preferably, promoting skeletal muscle satellite cell proliferation includes enhancing the proliferative capacity of skeletal muscle satellite cells and / or increasing the expression levels of skeletal muscle satellite cell proliferation markers; The inhibition of skeletal muscle satellite cell proliferation includes reducing the proliferative capacity of skeletal muscle satellite cells and / or reducing the expression levels of skeletal muscle satellite cell proliferation markers; The skeletal muscle satellite cell proliferation markers are skeletal muscle satellite cell proliferation marker genes and / or marker proteins.

[0008] Preferably, the skeletal muscle satellite cell proliferation marker genes include one or more of the CCND1 gene, CDK1 gene, KI67 gene, and PCNA gene; the skeletal muscle satellite cell proliferation marker proteins include one or more of the CCND1 protein, CDK1 protein, KI67 protein, and PCNA protein.

[0009] Preferably, the skeletal muscle satellite cells comprise bovine skeletal muscle satellite cells.

[0010] This invention provides applications of the ELN gene and / or ELN protein in one or more of the following aspects: 1) Regulating meat production traits in meat-producing livestock; 2) Regulating feed conversion efficiency in meat-producing livestock; 3) Improving meat-producing livestock breeds.

[0011] Preferably, the improvement of meat livestock breeds includes improving the meat production traits of meat livestock; increasing the expression level of ELN gene and / or ELN protein to improve the meat production traits of meat livestock, and decreasing the expression level of ELN gene and / or ELN protein to decrease the meat production traits of meat livestock.

[0012] Preferably, increasing the expression level of the ELN gene and / or ELN protein improves the feed conversion efficiency of meat livestock, while decreasing the expression level of the ELN gene and / or ELN protein reduces the feed conversion efficiency of meat livestock.

[0013] Preferably, the livestock for meat production includes beef cattle.

[0014] This invention provides a transgenic cattle with improved meat production traits, wherein the transgenic cattle are beef cattle that overexpress the ELN gene.

[0015] Beneficial effects: This invention provides the application of the ELN gene and / or ELN protein in regulating skeletal muscle satellite cell proliferation. This invention discovers that the ELN gene can regulate skeletal muscle satellite cell proliferation, specifically by increasing the expression levels of the ELN gene and / or ELN protein to promote skeletal muscle satellite cell proliferation, and by decreasing the expression levels of the ELN gene and / or ELN protein to inhibit skeletal muscle satellite cell proliferation. Based on this, increasing the expression levels of the ELN gene and / or ELN protein can improve meat production traits and feed conversion ratios in meat-producing livestock, leading to the development of new meat-producing livestock breeds with improved meat production traits. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The results show the effect of ELN gene overexpression on the mRNA levels of proliferation marker genes in bovine skeletal muscle satellite cells; where A represents the relative expression level of ELN; and B represents the relative mRNA expression levels of CCND1, CDK1, KI67, and PCNA genes. Figure 2 The results show the effect of interfering with ELN gene expression on the mRNA levels of proliferation marker genes in bovine skeletal muscle satellite cells; where A represents the relative expression level of ELN; and B represents the relative mRNA expression levels of CCND1, CDK1, KI67, and PCNA genes. Figure 3 The results show the efficiency of ELN overexpression; where A represents the results of Western blot and B represents the relative expression level of ELN protein. Figure 4 To interfere with the efficiency detection results of ELN; where A is the result of Western blot and B is the result of the relative expression level of ELN protein; Figure 5 The effect of ELN overexpression on the protein levels of proliferation marker genes in bovine skeletal muscle satellite cells was investigated. In this study, A represents the results of Western blot, and B represents the relative expression levels of CCND1, CDK6, KI67, and PCNA proteins. Figure 6 To investigate the effect of interference ELN on the protein levels of proliferation marker genes in bovine skeletal muscle satellite cells; where A represents the results of Western blot, and B represents the relative expression levels of CCND1, CDK6, KI67, and PCNA proteins; Figure 7 Results of CCK-8 assay to detect the effects of ELN overexpression (A) or ELN interference (B) on the proliferation of bovine skeletal muscle satellite cells; Figure 8The results show the effect of EdU detection on the proliferation of bovine skeletal muscle satellite cells; where A is a representative image of EdU-positive cell count; B is the EdU detection result. Figure 9 The results show the effect of EdU detection on the proliferation of bovine skeletal muscle satellite cells; where A is a representative image of EdU-positive cell counts; and B is the EdU detection result. in, express P <0.05, express P <0.01, express P <0.001. Detailed Implementation

[0018] This invention provides the application of the ELN gene and / or ELN protein in regulating the proliferation of skeletal muscle satellite cells.

[0019] In one implementation, the regulation involves: increasing the expression levels of the ELN gene and / or ELN protein to promote skeletal muscle satellite cell proliferation, and decreasing the expression levels of the ELN gene and / or ELN protein to inhibit skeletal muscle satellite cell proliferation. In one implementation, the reagent for increasing the expression levels of the ELN gene and / or ELN protein can be a plasmid overexpressing the ELN gene, and the backbone vector for overexpressing the ELN gene can be a pcDNA3.1(+) vector. In another implementation, the reagent for decreasing the expression levels of the ELN gene and / or ELN protein can be siRNA, and the siRNA can be siRNA with nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 2.

[0020] As one implementation, promoting skeletal muscle satellite cell proliferation includes enhancing the proliferative capacity of skeletal muscle satellite cells and / or increasing the expression levels of skeletal muscle satellite cell proliferation markers; The inhibition of skeletal muscle satellite cell proliferation includes reducing the proliferative capacity of skeletal muscle satellite cells and / or reducing the expression levels of skeletal muscle satellite cell proliferation markers; The skeletal muscle satellite cell proliferation markers are skeletal muscle satellite cell proliferation marker genes and / or marker proteins.

[0021] In one embodiment, the skeletal muscle satellite cell proliferation marker genes include one or more of the CCND1 gene, CDK1 gene, KI67 gene, and PCNA gene; the skeletal muscle satellite cell proliferation marker proteins include one or more of the CCND1 protein, CDK1 protein, KI67 protein, and PCNA protein.

[0022] In one embodiment, the skeletal muscle satellite cells include bovine skeletal muscle satellite cells.

[0023] This invention discovers that the ELN gene can regulate the proliferation of skeletal muscle satellite cells. Specifically, increasing the expression levels of the ELN gene and / or ELN protein promotes skeletal muscle satellite cell proliferation, while decreasing the expression levels inhibits it. Furthermore, promoting the proliferation of bovine skeletal muscle satellite cells enhances the growth and repair capabilities of muscle tissue, thereby improving muscle growth efficiency and lean meat percentage. This allows cattle to more effectively convert ingested feed nutrients into valuable muscle tissue, ultimately resulting in improved feed utilization. Based on this, increasing the expression levels of the ELN gene and / or ELN protein can improve meat production traits and feed conversion ratios in meat-producing livestock, leading to the development of new breeds of meat-producing livestock with improved meat production traits.

[0024] Based on the above advantages, the present invention provides applications of the ELN gene and / or ELN protein in one or more of the following aspects: 1) Regulating meat production traits in meat-producing livestock; 2) Regulating feed conversion efficiency in meat-producing livestock; 3) Improving meat-producing livestock breeds.

[0025] In one implementation, the improvement of meat livestock breeds includes improving the meat production traits of meat livestock; increasing the expression levels of the ELN gene and / or ELN protein to improve the meat production traits of meat livestock, and decreasing the expression levels of the ELN gene and / or ELN protein to decrease the meat production traits of meat livestock.

[0026] As one implementation method, increasing the expression level of the ELN gene and / or ELN protein improves the feed conversion efficiency of meat livestock, while decreasing the expression level of the ELN gene and / or ELN protein reduces the feed conversion efficiency of meat livestock.

[0027] In one implementation, the livestock for meat production includes beef cattle.

[0028] This invention provides a transgenic cattle with improved meat production traits, wherein the transgenic cattle are beef cattle that overexpress the ELN gene.

[0029] To further illustrate the present invention, the application of the ELN gene provided by the present invention in regulating skeletal muscle satellite cell proliferation and feed utilization is described in detail below with reference to embodiments and accompanying drawings, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1 1. Materials and Methods The primary bovine skeletal muscle satellite cells used in this experiment were obtained from Xiamen Yimo Biotechnology Co., Ltd.; the EdU cell proliferation assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the Lipofectamine 3000 transfection reagent was purchased from Thermo Scientific; and the ELN, CCND1, and TUBULIN antibodies were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0031] 2. Cell Culture Remove the cells from liquid nitrogen and dissolve them by shaking in a 37°C constant temperature water bath; transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant; resuspend the cell pellet and transfer it to a 10cm culture dish, removing air bubbles; place it in a 37°C, 5% CO2 cell incubator for culture.

[0032] Once the cells have reached 80%-90% confluence, discard the culture medium, gently wash with PBS, discard the PBS, add trypsin to the cell culture flask, and incubate at 37°C and 5% CO2 for 2 minutes to digest the cells. After observing cell floating, add an equal volume of culture medium to stop the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge, and discard the supernatant. Add culture medium to the centrifuge tube to resuspend the cells, and seed the cell suspension into new culture dishes at a specific ratio, then incubate at 37°C and 5% CO2 for growth.

[0033] The culture medium formula is as follows: 89% DMEM-F12 + 10% FBS + 1% penicillin and streptomycin antibiotics.

[0034] 3. Cell transfection The pcDNA3.1(+) overexpression vector for ELN (Gene ID: 28078) was synthesized by Shanghai Sangon Biotech Co., Ltd.; the siRNA (si-NC, si-ELN) was synthesized by Suzhou Gemma Gene Co., Ltd., and the sequences are as follows: si-NC: 5'-CCGCUAAAGCAGCCAAAUUTT-3', SEQ ID NO.1; si-ELN: 5'-AAUUUGGCUGCUUUAGCGGTT-3', SEQ ID NO.2; Bovine skeletal muscle satellite cells in good growth condition were seeded into six-well plates, and transfection experiments were performed when the cell density was about 60%.

[0035] The preparation method for the overexpression vector mixture solution is as follows: Reagent A: 125 μl Opti-MEM + 5 μl P3000 Reagent + 2.5 μg plasmid pcDNA3.1 (+) or pcDNA3.1 (+) plasmid overexpressing ELN; Reagent B: 125 μl Opti-MEM + 5 μl Lipofectamine 3000 Reagent; When preparing reagents A and B, handle them gently and slowly. Add A to B and mix. Let stand at room temperature for 15 minutes. The method for preparing the siRNA mixed solution is as follows: Reagent A: 125 μl Opti-MEM + 5 μl siRNA; Reagent B: 125 μl Opti-MEM + 5 μl Lipofectamine 3000 Reagent; When preparing reagents A and B, handle them gently and slowly. Add A to B and mix. Let stand at room temperature for 15 minutes. The mixed solution was added dropwise to six-well plates containing culture medium, mixed by cross-shaped shaking, and incubated in a 37°C, 5% CO2 incubator. Cell RNA was collected after 24 hours, and cell protein was collected after 48 hours.

[0036] 4. Effects of ELN on mRNA levels of proliferation marker genes in bovine skeletal muscle satellite cells Total RNA was isolated from bovine skeletal muscle satellite cells using Trizol reagent. Subsequently, 1.5 μg of total RNA was reverse transcribed into cDNA using the ABScript Neo RTMaster Mi× for qPCR with gDNA Remover kit. RT-qPCR analysis was performed using the ChamQSYBR qPCR Master Mix (Low RO×Premixed) real-time fluorescence kit. −ΔΔCt The relative expression levels of the gene were quantified using a quantitative method, and normalized using GAPDH as an internal reference gene. The primer sequences, reaction system, and procedure are shown in Tables 1-3.

[0037] Table 1 RT-qPCR primer sequences

[0038] Table 2 RT-qPCR reaction system

[0039] Table 3 RT-qPCR reaction procedure

[0040] The results are as follows Figure 1 and Figure 2 As shown, overexpression of ELN significantly upregulated the expression levels of CCND1, CDK1, KI67, and PCNA mRNAs; while interference with ELN expression significantly downregulated the expression levels of CCND1, CDK1, KI67, and PCNA mRNAs. This indicates that overexpression of ELN can promote the proliferation of bovine skeletal muscle satellite cells, while interference with ELN can inhibit the proliferation of bovine skeletal muscle satellite cells.

[0041] 5. Effects of ELN on the protein levels of proliferation marker genes in bovine skeletal muscle satellite cells Bovine skeletal muscle satellite cells overexpressing or interfering with ELN were lysed at low temperature in RIPA with added PMSF for 20 min, collected in 1.5 ml centrifuge tubes, centrifuged at 13000g for 15 min at 4 °C, and 150 μl of supernatant was taken. 50 μl of protein loading buffer was added, mixed, and briefly centrifuged. The protein sample was denatured at 100 °C for 10 min and then placed on ice. Gel electrophoresis was performed using 10% SDS-PAGE to separate proteins according to molecular weight, and the separated proteins were then transferred to a PVDF membrane. The PVDF membrane with protein blots was blocked with rapid blocking buffer for 10 min and incubated overnight at 4 °C with the corresponding primary antibody. After washing with 1×TBST containing Tween-20, the PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 1 h. The membrane was placed in a TANON-5200SF chemiluminescence imaging system, and developing solution was dropped onto the membrane to cover it. After standing for 1 min, images were taken and stored. The gray values ​​of the bands were analyzed using ImageJ software.

[0042] Overexpression or interference with ELN efficiency results are as follows Figure 3 and Figure 4 As shown, the WB (Western blot) detection results are as follows: Figure 5 and Figure 6 As shown, overexpression of ELN significantly upregulated the protein expression levels of bovine skeletal muscle satellite cell proliferation-related genes CCND1, CDK6, KI67, and PCNA; while interference with ELN significantly downregulated the protein expression levels of these genes. This indicates that overexpression of ELN can promote the proliferation of bovine skeletal muscle satellite cells, while interference with ELN can inhibit their proliferation.

[0043] 6. CCK-8 assay to detect the effect of ELN on the proliferation of bovine skeletal muscle satellite cells Bovine skeletal muscle satellite cells were seeded into 96-well plates, overexpressed or interfered with ELN, and 10 μl of CCK-8 reagent and 90 μl of complete culture medium were added to each well at 24, 48, 72 and 96 hours after transfection. After incubation for 1 h, the absorbance at 450 nm was measured.

[0044] CCK8 test results are as follows Figure 7 As shown, compared with the control group, the proliferation capacity of bovine skeletal muscle satellite cells was significantly increased after ELN overexpression and significantly decreased after ELN interference.

[0045] 7. EdU assay to detect the effect of ELN on the proliferation of bovine skeletal muscle satellite cells Bovine skeletal muscle satellite cells were passaged into 24-well plates with three biological replicates per group. After incubation in 10 μmol / L EdU medium for 2 h, the proliferation status of bovine skeletal muscle satellite cells was assessed using an EdU cell proliferation assay kit. Images were captured under a fluorescence inverted microscope (Leica), and the results were analyzed using ImageJ software.

[0046] Representative images and EdU detection results of EdU-positive cell counts after overexpression or interference with ELN are shown below. Figure 8 and Figure 9 As shown in the figure, compared with the control group, the proliferation capacity of bovine skeletal muscle satellite cells was significantly upregulated after ELN overexpression and significantly downregulated after ELN interference.

[0047] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of ELN gene and / or ELN protein in regulating skeletal muscle satellite cell proliferation.

2. The application according to claim 1, characterized in that, The regulation is as follows: increasing the expression level of ELN gene and / or ELN protein promotes the proliferation of skeletal muscle satellite cells, while decreasing the expression level of ELN gene and / or ELN protein inhibits the proliferation of skeletal muscle satellite cells.

3. The application according to claim 2, characterized in that, The promotion of skeletal muscle satellite cell proliferation includes enhancing the proliferative capacity of skeletal muscle satellite cells and / or increasing the expression levels of skeletal muscle satellite cell proliferation markers; The inhibition of skeletal muscle satellite cell proliferation includes reducing the proliferative capacity of skeletal muscle satellite cells and / or reducing the expression levels of skeletal muscle satellite cell proliferation markers; The skeletal muscle satellite cell proliferation markers are skeletal muscle satellite cell proliferation marker genes and / or marker proteins.

4. The application according to claim 3, characterized in that, The skeletal muscle satellite cell proliferation marker genes include one or more of the following: CCND1 gene, CDK1 gene, KI67 gene, and PCNA gene; the skeletal muscle satellite cell proliferation marker proteins include one or more of the following: CCND1 protein, CDK1 protein, KI67 protein, and PCNA protein.

5. The application according to any one of claims 1-4, characterized in that, The skeletal muscle satellite cells include bovine skeletal muscle satellite cells.

6. Applications of the ELN gene and / or ELN protein in one or more of the following areas: 1) Regulating meat production traits in meat-producing livestock; 2) Regulating feed conversion efficiency in meat-producing livestock; 3) Improving meat-producing livestock breeds.

7. The application according to claim 6, characterized in that, The improvement of meat livestock breeds includes improving the meat production traits of meat livestock; increasing the expression level of ELN gene and / or ELN protein to improve the meat production traits of meat livestock, and decreasing the expression level of ELN gene and / or ELN protein to decrease the meat production traits of meat livestock.

8. The application according to claim 6, characterized in that, Increasing the expression levels of the ELN gene and / or ELN protein improves feed conversion efficiency in meat livestock, while decreasing the expression levels of the ELN gene and / or ELN protein reduces feed conversion efficiency in meat livestock.

9. The application according to claims 6-8, characterized in that, The livestock used for meat production include beef cattle.

10. A transgenic cattle with improved meat production traits, characterized in that, The transgenic cattle mentioned are beef cattle that overexpress the ELN gene.

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