Application of FGF9 gene in regulation and control of growth and development of chicken skeletal muscle in embryonic period

Abstract

The invention discloses application of an FGF9 gene in regulation and control of growth and development of chicken skeletal muscles, and further discloses application of an overexpression vector of the FGF9 gene or an interference fragment of the overexpression vector in promotion or inhibition of proliferation and differentiation of chicken myoblasts. The invention also discloses a specific primer pair for detecting the expression quantity of the FGF9 influencing the growth and development of the chicken skeletal muscle. The invention also discloses a detection kit and a detection method thereof. The detection kit provided by the invention can be used for detecting the expression quantity of the FGF9 gene in chicken muscle tissues and myoblasts, and the detection method is simple and quick. The FGF9 gene can be used as an important gene for researching a chicken skeletal muscle growth and development regulation theory.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically involving the application of the FGF9 gene in regulating the growth and development of chicken skeletal muscle. Background Technology

[0002] Muscle is a vital component of an animal's body, with skeletal muscle alone accounting for approximately 40% of total body weight. The quantity and quality of skeletal muscle directly affect chicken yield and determine the economic value of broiler chickens. Muscle content is primarily determined by three factors: the number of muscle fibers, the diameter of muscle fibers, and the type of muscle fibers. The proliferation and differentiation of myoblasts are crucial for skeletal muscle development. Skeletal muscle is mainly composed of muscle fibers, which are formed from myoblast precursor cells through proliferation, differentiation, and intercellular fusion. Therefore, studying the genetic regulatory mechanisms of myoblast proliferation and differentiation is of significant scientific importance and guiding value for elucidating the theoretical basis of chicken skeletal muscle growth and development, as well as for the genetic improvement of growth traits in local Chinese chicken breeds.

[0003] The fibroblast growth factor (FGF) family is a crucial regulator of cell migration, proliferation, and differentiation, playing a variety of biological functions. FGF9 is an important member of the fibroblast growth factor family; its gene-encoded protein consists of 208 amino acids and was first isolated from human glioma cells. Studies have found that FGF9 can promote the proliferation of epithelial cells, glial cells, and fibroblasts, and also plays an important role in the development of the embryonic reproductive system and lung tissue.

[0004] However, there are currently no studies or reports on the effects of the FGF9 gene on the proliferation and differentiation of chicken myoblasts. Summary of the Invention

[0005] Purpose of the invention: In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is to provide the application of FGF9 gene in regulating the growth and development of chicken skeletal muscle.

[0006] Another technical problem to be solved by the present invention is to provide an overexpression vector of the FGF9 gene or an interference fragment of the FGF9 gene.

[0007] Another technical problem to be solved by the present invention is to provide an overexpression vector of the FGF9 gene or the interference fragment thereof for the application of promoting or inhibiting the proliferation and differentiation of chicken myoblasts.

[0008] Another technical problem to be solved by this invention is the use of specific primer pairs for detecting the expression level of the FGF9 gene in muscle tissue and / or myoblasts.

[0009] Another technical problem that this invention aims to solve is a fluorescence quantitative detection kit.

[0010] The final technical problem to be solved by this invention is a method for detecting the FGF9 gene.

[0011] Technical solution: In order to achieve the above-mentioned objective, this invention provides the application of the FGF9 gene in regulating the growth and development of chicken skeletal muscle, wherein the GenBank accession number of the FGF9 gene is NC_052532.1.

[0012] The present invention also includes an overexpression vector of the FGF9 gene, wherein the overexpression vector is obtained by introducing the FGF9 gene into the pcDNA3.1 vector.

[0013] The present invention also includes interference fragments of the FGF9 gene, wherein the gene sequence of the interference fragments includes FGF9-si856, FGF9-si1009, FGF9-si1138, or FGF9-si1322, wherein the sense strand of FGF9-si856 is shown in SEQ ID NO.5, the antisense strand of FGF9-si856 is shown in SEQ ID NO.6, the sense strand of FGF9-si1009 is shown in SEQ ID NO.7, the antisense strand of FGF9-si1009 is shown in SEQ ID NO.8, the sense strand of FGF9-si1138 is shown in SEQ ID NO.9, the antisense strand of FGF9-si1138 is shown in SEQ ID NO.10, the sense strand of FGF9-si1322 is shown in SEQ ID NO.11, and the antisense strand of FGF9-si13226 is shown in SEQ ID NO.12.

[0014] The present invention also includes the application of the FGF9 gene overexpression vector or the interference fragment in promoting or inhibiting the proliferation and differentiation of chicken myoblasts.

[0015] The present invention also includes specific primer pairs for detecting the expression level of the FGF9 gene in muscle tissue and / or myoblasts, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0016] The present invention also includes the application of the specific primer pair described herein in the preparation of reagents or kits for detecting the expression level of the FGF9 gene in chicken muscle tissue and / or myoblasts.

[0017] The present invention also includes a fluorescence quantitative detection kit, wherein the kit includes the specific primer pair described above.

[0018] Furthermore, the fluorescence quantitative detection kit also includes 2×ChamQ SYBR qPCR Master Mix reagent and enzyme-free double-distilled water.

[0019] Furthermore, the fluorescence quantitative detection kit also includes a primer pair for the internal reference gene β-actin, the sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0020] The present invention also includes the application of the overexpression vector, the interfering fragment, the specific primer pair, or the fluorescence quantitative detection kit in the regulation of chicken skeletal muscle growth and development.

[0021] The application involves detecting the expression level of the FGF9 gene in chicken muscle tissue and / or myoblasts using quantitative real-time PCR.

[0022] The method uses the specific primer pair or the fluorescence quantitative detection kit to detect the expression level of the FGF9 gene in chicken muscle tissue and / or myoblasts.

[0023] Furthermore, the method includes a real-time quantitative PCR detection method.

[0024] Furthermore, the real-time PCR reaction system comprises: 2×ChamQ SYBR qPCR Master Mix, primers with sequences as shown in SEQ ID NO.1, primers with sequences as shown in SEQ ID NO.2, template cDNA, and enzyme-free double-distilled water.

[0025] Further, the real-time PCR reaction system is as follows: 10 μl of 2×ChamQ SYBR qPCR Master Mix reagent; 0.4 μl of primers with the sequence shown in SEQ ID NO.1; 0.4 μl of primers with the sequence shown in SEQ ID NO.2; 2 μl of template cDNA; and 7.2 μl of enzyme-free double-distilled water.

[0026] Furthermore, the real-time PCR reaction system also includes 0.4 μL of primers with the sequence shown in SEQ ID NO.3 and 0.4 μL of primers with the sequence shown in SEQ ID NO.4.

[0027] This invention involves collecting primary myoblasts from chicken embryo leg muscles, culturing them in vitro, overexpressing or interfering with the FGF9 gene in the myoblasts, extracting total RNA, and performing reverse transcription to obtain cDNA. Quantitative real-time PCR is then performed using specific primers for the FGF9 gene and the internal control β-actin. The PCR reaction system and amplification procedure are the same as conventional quantitative real-time PCR, employing a 2... -△△CTThe expression level of this mRNA was examined. Simultaneously, the expression trends of proliferation and differentiation marker genes were detected. The effects of the FGF9 gene on chicken myoblast proliferation and differentiation were comprehensively analyzed using CCK-8 assay, Edu assay, protein immunoblotting, and indirect immunofluorescence experiments.

[0028] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention is the first to discover that the FGF9 gene can serve as an important gene for studying the regulatory theory of chicken skeletal muscle growth and development. This invention also provides specific primer pairs for detecting FGF9 gene expression, along with a detection kit and detection method. The detection kit provided by this invention can be used to detect the expression level of the FGF9 gene in chicken muscle tissue and myoblasts, and the detection method is simple and rapid. The FGF9 gene provided by this invention can be used to study the molecular mechanisms of chicken skeletal muscle growth and development. Subsequent studies will validate its specific regulatory mechanism in chicken skeletal muscle growth and development by overexpressing and interfering with FGF9. Attached Figure Description

[0029] Figure 1 This is a melting curve diagram for qPCR.

[0030] Figure 2 The image shows the results of FGF9 gene interference and overexpression efficiency detection.

[0031] Figure 3 The figure shows the effect of the FGF9 gene on the expression of proliferation marker genes mRNA.

[0032] Figure 4 Image showing the results of CCK8 assay for cell proliferation;

[0033] Figure 5 Figure showing the results of EDu detection of the effect of FGF9 gene on cell proliferation;

[0034] Figure 6 The figure shows the effect of FGF9 gene on the expression of differentiation marker gene mRNA;

[0035] Figure 7 Figure showing the results of indirect immunofluorescence assay of the effect of FGF9 gene on myotube differentiation. Detailed Implementation

[0036] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0037] Example 1: Effects of FGF9 gene on chicken myoblast proliferation and differentiation

[0038] 1. Obtaining the FGF9 gene

[0039] The fertilized eggs of Bian chickens used in this experiment were provided by the Institute of Animal Husbandry and Veterinary Medicine, Shanxi Academy of Agricultural Sciences, and incubated in the research group's laboratory incubator. Total RNA was extracted from the leg muscle tissue of 12-year-old Bian chickens and reverse transcribed to obtain cDNA. Specific primer pairs for the FGF9 gene (GenBank accession number: NC_052532.1) were designed using the cDNA as templates, and their sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. Primer pairs for the internal reference gene β-actin (GenBank accession number: NC_052545.1) are shown in SEQ ID NO.3 and SEQ ID NO.4. All primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0040] The amplification reaction system consisted of 20 μL, containing 10 μL of 2×ChamQ SYBR qPCR Master Mix reagent; 0.4 μL each of specific primer pairs or internal reference gene primer pairs (specific primers with sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, or internal reference gene primers with sequences as shown in SEQ ID NO.3 and SEQ ID NO.4); 2 μL of template cDNA; and 7.2 μL of enzyme-free double-distilled water.

[0041] The quantitative fluorescence reaction procedure was as follows: pre-denaturation at 95℃ for 30 seconds; 95℃ for 10 seconds, 60℃ for 30 seconds, for 40 cycles; and melting curves were obtained by repeating the process of 95℃ for 15 seconds, 60℃ for 60 seconds, 95℃ for 30 seconds, and 60℃ for 15 seconds.

[0042] The primer sequences are as follows:

[0043] FGF9-F:5'-GTCAGCATCCGAGGAGTAG-3'SEQ ID NO.1

[0044] FGF9-R:5'-CGCCACGTAGTATCGTCTT-3'SEQ ID NO.2

[0045] β-actin-F:5'-CAGCCATCTTTCTTGGGTAT-3'SEQ ID NO.3

[0046] β-actin-R:5'-CTGTGATCTCCTTCTGCATCC-3'SEQ ID NO.4

[0047] Depend on Figure 1 A and Figure 1 As shown in B, the melting curves of the FGF9 gene and the β-actin gene have a single main peak, indicating that the primer design is good.

[0048] 2. The FGF9 gene inhibits the proliferation and differentiation of chicken myoblasts.

[0049] The proliferation and differentiation of myoblasts are a fundamental process in skeletal muscle development; myoblasts must proliferate and differentiate to become mature muscle cells. This invention verifies the effect of the FGF9 gene on the proliferation and differentiation of chicken myoblasts. This invention commissioned Shanghai Gemma Technology Co., Ltd. to construct and synthesize four interference fragment sequences of the FGF9 gene, named FGF9-si856, FGF9-si1009, FGF9-si1138, and FGF9-si1322, respectively. Shanghai Gemma Technology Co., Ltd. also constructed and synthesized an FGF9 overexpression vector. Using BamH1 and EcoRI restriction sites, the CDS sequence of the chicken FGF9 gene was subcloned into pcDNA3.1, named pcDNA3.1-FGF9.

[0050] The sequences of the four interfering segments are as follows:

[0051] FGF9-si856 interference sequence:

[0052] Chain of Justice: GGCUCCCUUAGGUGAAGUUTT SEQ ID NO.5:

[0053] Antonym chain: AACUUCACCUAAGGGAGCCTT SEQ ID NO.6:

[0054] FGF9-si1009 interference sequence:

[0055] Chain of Justice: GGACUUGGACCAUUUAAAGTT SEQ ID NO.7

[0056] Antonym chain: CUUUAAAUGGUCCAAGUCCTT SEQ ID NO.8:

[0057] FGF9-si1138 interference sequence:

[0058] Chain of Justice: GGAGUUCAUCAGUAUAGCATT SEQ ID NO.9;

[0059] Antonym chain: UGCUAUACUGAUGAACUCCTT SEQ ID NO.10;

[0060] FGF9-si1322 interference sequence:

[0061] Chain of Justice: GACACUGGAAGACGAUACUTT SEQ ID NO.11;

[0062] Antonym chain: AGUAUCGUCUUCCAGUGUCTT SEQ ID NO.12.

[0063] Chicken primary myoblasts were extracted from the leg muscle of 12-year-old chicken embryos and seeded in 12-well plates. Transfection was performed when the cell density reached 70%, with three biological replicates per group. The transfection procedure was as follows: Four interfering fragments were diluted to 20 μM with DEPC water, and the overexpression vector concentration was adjusted to 250 μg / μL. The interfering fragments or overexpression vector were mixed with the transfection buffer and vortexed for 10 s. After brief centrifugation, jetPRIME transfection reagent (Catalog No. 101000046) from Polyplus Transfection was added, vortexed again for 1 s, and incubated at room temperature for 20 min. Finally, the mixture was added to cell culture plates to complete the transfection process. The dosages were as follows: 2.75 μL of interfering fragment, 100 μL of transfection buffer, and 3 μL of transfection reagent per replicate; or 3.2 μL of overexpression vector, 75 μL of buffer, and 2.4 μL of transfection reagent per replicate. 24 hours after transfection, cells were collected to detect the FGF9 gene interference efficiency and overexpression efficiency. The results are as follows: Figure 2 The results showed that the interference efficiency of FGF9-si1322 was the highest among the four sequences, reaching a highly significant level (P≤0.01); while the overexpression efficiency reached more than 1600-fold, also reaching a highly significant level (P≤0.01), allowing for subsequent experiments. Ultimately, this invention continued to use the FGF9-si1322 interference fragment and the pcDNA3.1-FGF9 overexpression vector to verify the effect of FGF9 on the proliferation and differentiation of chicken myoblasts.

[0064] Chicken myoblasts were transfected with FGF9-si1322 and pcDNA3.1-FGF9 overexpression vectors during the proliferation and differentiation phases, respectively, with three biological replicates per group. During the proliferation phase, the results of quantitative real-time PCR were as follows: Figure 3 The results showed that the mRNA expression of PCNA, a cell cycle and proliferation marker gene, in FGF9-si1322 transfected cells was not significant, while the mRNA expression of P21 (a negative regulator of the cell cycle) was significantly reduced (P≤0.05 or P≤0.01). Figure 3 A); however, transfection with pcDNA3.1-FGF9 revealed a significant decrease in the mRNA expression of the proliferation marker gene PCNA (P≤0.05). Figure 3 B); CCK8 assay showed that cell viability was significantly higher than that of the control group 24 hours after FGF9-si1322 transfection. Figure 4 A); Transfection with pcDNA3.1-FGF9 revealed that cell viability was significantly lower than that of the control group after 60 hours. Figure 4B); Finally, analysis of the Edu experimental data using ImageJ revealed that the proportion of FGF9-si1322-positive cells was significantly higher than that of the control group (P≤0.05). Figure 5 A, Figure 5 C); The proportion of positive cells after transfection with pcDNA3.1-FGF9 was significantly lower than that in the control group (P≤0.05). Figure 5 B. Figure 5 D).

[0065] Furthermore, during the differentiation phase of chicken myoblasts, quantitative real-time PCR results showed that transfection with FGF9-si1322 significantly increased the mRNA expression of cell differentiation marker genes MYOG, MYOD1, and MYHC. Figure 6 A); The mRNA expression of cell differentiation marker genes MYOG, MYOD1, and MYHC was detected by transfecting pcDNA3.1-FGF9. The mRNA expression of MYOG and MYOD1 was significantly or extremely significantly decreased (P≤0.05 or P≤0.01). Figure 6 B) Indirect immunofluorescence assays showed that FGF9-si1322 transfection significantly increased myotube area (P≤0.05). Figure 7 A, Figure 7 C), while transfection with pcDNA3.1-FGF9 significantly reduced the myotube area (P≤0.05). Figure 7 B. Figure 7 D).

[0066] In summary, the FGF9 gene has the function of inhibiting the proliferation and differentiation of chicken myoblasts. The FGF9 gene provided by this invention can serve as an important gene for studying the theory of regulation of chicken skeletal muscle growth and development. Knocking out the FGF9 gene through gene editing can promote the growth and development of chicken skeletal muscle.

Claims

1. FGF9 The application of genes in regulating the growth and development of chicken skeletal muscle is characterized by, The FGF9 The gene's GenBank accession number is NC_052532.

1.

2. FGF9 Gene overexpression vectors, characterized in that, The overexpression vector includes [a specific vector]. FGF9 The gene was obtained by introducing it into the pcDNA3.1 vector.

3. FGF9 The interference fragment of the gene, characterized in that, The gene sequence of the interfering fragment includes FGF9 -si856、 FGF9 -si1009、 FGF9 -si1138 or FGF9 -si1322, the aforementioned FGF9 The justice chain of -si856 is shown in SEQ ID NO.5, wherein... FGF9 The antisense chain of -si856 is shown in SEQ ID NO.6, which is... FGF9 The justice chain of -si1009 is shown in SEQ ID NO.7, wherein... FGF9 The antisense chain of -si1009 is shown in SEQ ID NO.

8. FGF9 The justice chain of -si1138 is shown in SEQ ID NO. 9, wherein... FGF9 The antisense chain of -si1138 is shown in SEQ ID NO.10, wherein... FGF9 The justice chain of -si1322 is shown in SEQ ID NO.11, wherein... FGF9 The antisense chain of -si13226 is shown in SEQ ID NO.

12.

4. The claim 2 FGF9 The application of gene overexpression vectors or the interference fragments described in claim 3 in promoting or inhibiting the proliferation and differentiation of chicken myoblasts.

5. Used for detection FGF9 A primer pair specific to the expression level of a gene in muscle tissue and / or myoblasts, characterized in that... The specific primer pair sequences are shown in SEQ ID NO.1 and SEQ ID NO.

2.

6. The specific primer pair according to claim 5 in the preparation of chicken muscle tissue and / or myoblasts FGF9 Applications in reagents or kits for gene expression levels.

7. A fluorescence quantitative detection kit, characterized in that, The kit includes the specific primer pair as described in claim 5.

8. The application of the overexpression vector of claim 2, the interfering fragment of claim 3, the specific primer pair of claim 5, or the fluorescence quantitative detection kit of claim 7 in the regulation of chicken skeletal muscle growth and development.

9. The application according to claim 8, characterized in that, The application is to detect chicken muscle tissue and / or myoblasts using quantitative real-time PCR. FGF9 Gene expression levels.

10. A detection method FGF9 The gene-based method is characterized by, The method uses the specific primer pair described in claim 5 or the fluorescence quantitative detection kit described in claim 7 to detect chicken muscle tissue and / or myoblasts. FGF9 Gene expression levels.

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