Circsesn1 gene, overexpression vector thereof, specific primer pair, detection kit and application thereof

Abstract

The application discloses CircSESN1 a gene, an overexpression vector thereof, a specific primer pair, a detection kit and application thereof. CircSESN1 The application also discloses application of the gene, the overexpression vector, the specific primer pair or the fluorescent quantitative detection kit in regulation of chicken skeletal muscle growth and development. CircSESN1 The application further discloses a method for inhibiting proliferation and / or promoting differentiation of chicken myoblasts. The detection kit provided by the application can be used for detecting

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to the CircSESN1 gene, its overexpression vector, specific primer pairs, detection kits, and applications. Background Technology

[0002] Skeletal muscle is a crucial component of an animal's body, accounting for approximately 40% of its body weight. For broiler chickens, the development of skeletal muscle directly impacts chicken yield and determines the economic value of the broiler. Skeletal muscle content is primarily determined by the number, diameter, and type of muscle fibers. Myoblasts, as precursor cells for muscle fibers, determine the number of muscle fibers through their proliferation and differentiation process, ultimately becoming mature muscle fibers after a series of proliferation and differentiation processes. Therefore, studying the genetic regulatory mechanisms of myoblast proliferation and differentiation is of significant scientific importance and provides guidance for elucidating the theoretical basis of skeletal muscle growth and development in chickens and for the genetic improvement of growth traits in local Chinese chicken breeds.

[0003] Circular RNA (circRNA) is a class of non-coding RNA molecules that lack a 5′ cap and a 3′ poly(A) tail, possessing a closed circular structure. They are stably expressed, primarily located in the cytoplasm or stored in exosomes, and have been shown to be widely present in various eukaryotic organisms. Studies have shown that circRNAs play an important role in the regulation of skeletal muscle growth and development in chickens, mainly acting as miRNA sponges to regulate the expression of genes related to the proliferation and differentiation of chicken myoblasts.

[0004] A limited number of studies have shown that the SESN1 gene, from which CircSESN1 originates, can promote the proliferation and differentiation of chicken myoblasts. Another transcription product of the SESN1 gene, CircSESN1, originates from exons three, four, and five of the SESN1 gene (XM_004940327.5), and studies have shown that it can promote the proliferation and differentiation of chicken myoblasts through the CircSESN1 / miR-16-5p / SESN1 pathway.

[0005] However, current research on the structure and function of the CircSESN1 gene is limited and incomplete, especially regarding the structure of the CircSESN1 gene in different chicken breeds, which has not yet been fully and thoroughly studied. Therefore, it is necessary to explore the structure and function of the CircSESN1 gene in different chicken breeds to further investigate the impact of new CircSESN1 genes on chicken skeletal muscle growth and development, reveal new mechanisms of muscle development regulation, and potentially provide new and valuable molecular markers for broiler breed selection and breeding. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a new CircSESN1 gene, which has a circular structure and is distributed in the nucleoplasm of the cell.

[0007] The technical problem that this invention also aims to solve is to provide the application of the CircSESN1 gene in inhibiting the proliferation of chicken myoblasts and / or promoting the differentiation of chicken myoblasts.

[0008] Another technical problem to be solved by the present invention is to provide an overexpression vector and its application in inhibiting cell proliferation and / or promoting chicken myoblast differentiation.

[0009] Another technical problem that this invention aims to solve is to provide specific primer pairs for detecting the expression level of the CircSESN1 gene.

[0010] Another technical problem that this invention aims to solve is to provide a fluorescence quantitative detection kit.

[0011] Another technical problem this invention aims to solve is to provide the application of the CircSESN1 gene, the overexpression vector, the specific primer pair, or the fluorescence quantitative detection kit in the regulation of chicken skeletal muscle growth and development.

[0012] The final technical problem to be solved by this invention is a method for inhibiting the proliferation of chicken myoblasts and / or promoting their differentiation.

[0013] Technical solution: In order to achieve the above-mentioned objective, the present invention provides the CircSESN1 gene, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0014] This invention provides the application of the CircSESN1 gene in inhibiting the proliferation of chicken myoblasts and / or promoting the differentiation of chicken myoblasts.

[0015] The present invention provides an overexpression vector comprising the CircSESN1 gene, preferably comprising pcDNA3.1-CircSESN1.

[0016] This invention provides the application of the overexpression vector in inhibiting cell proliferation and / or promoting chicken myoblast differentiation.

[0017] The present invention provides a specific primer pair for detecting the expression level of the CircSESN1 gene, the sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0018] Furthermore, the present invention also includes specific primer pairs for simultaneously detecting the expression levels of the CircSESN1 gene and the Linear SESN1 gene in chicken muscle tissue and / or myoblasts.

[0019] Furthermore, the specific primer pair sequences of the CircSESN1 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, and the specific primer pair sequences of the Linear SESN1 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0020] This invention provides the application of the specific primer pair for the CircSESN1 gene in the preparation of reagents or kits for detecting the expression level of CircSESN1 in chicken muscle tissue and / or myoblasts.

[0021] The present invention provides a fluorescence quantitative detection kit, the kit comprising the aforementioned specific primer pair.

[0022] Furthermore, the fluorescence quantitative detection kit also includes 2×ChamQ SYBR qPCR Master Mix reagent and enzyme-free double-distilled water.

[0023] Furthermore, the fluorescence quantitative detection kit also includes primer pairs for the internal reference genes β-actin, GAPDH, and U6. The primer pair sequences for the internal reference gene β-actin are shown in SEQ ID NO.5 and SEQ ID NO.6, the primer pair sequences for the internal reference gene GAPDH are shown in SEQ ID NO.7 and SEQ ID NO.8, and the primer pair sequences for the internal reference gene U6 are shown in SEQ ID NO.9 and SEQ ID NO.10.

[0024] This invention provides the application of the CircSESN1 gene, the overexpression vector, the specific primer pair, or the fluorescence quantitative detection kit in the regulation of chicken skeletal muscle growth and development.

[0025] The application involves detecting the expression level of the CircSESN1 gene in chicken muscle tissue and / or myoblasts using quantitative real-time PCR.

[0026] 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.

[0027] 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.

[0028] Furthermore, the real-time PCR reaction system further includes 0.4 μL of primers with the sequence shown in SEQ ID NO.3; 0.4 μL of primers with the sequence shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0029] The present invention provides a method for inhibiting the proliferation of chicken myoblasts and / or promoting their differentiation, the method comprising increasing the expression level of the CircSESN1 gene.

[0030] This invention involves collecting chicken leg muscle tissue and using CircSESN1 and Linear SESN1 specific primer pairs for quantitative real-time PCR of gDNA and cDNA, followed by agarose gel electrophoresis and Sanger sequencing. Primary myoblasts and DF-1 cells from chicken embryo leg muscle are cultured in vitro and subjected to RNase R digestion, actinomycin D assay, and nucleocytoplasmic separation assay. Quantitative real-time PCR is performed using specific primers for CircSESN1, Linear SESN1, and internal controls β-actin, GAPDH, and U6. The PCR reaction system and amplification procedure are the same as conventional quantitative real-time PCR. -△△CT The expression level of this mRNA was examined. Primary myoblasts of chicken embryo leg muscle were cultured in vitro. After overexpression of the novel CircSESN1 in the myoblasts, total RNA was extracted and reverse transcribed to obtain cDNA. Quantitative real-time PCR was performed using the specific primers for CircSESN1 and the internal controls β-actin and GAPDH. The PCR reaction system and amplification procedure were the same as conventional quantitative real-time PCR. -△△CT The expression level of this mRNA was examined. The expression trend of proliferation marker genes was detected, and the effect of CircSESN1 gene on chicken myoblast proliferation and differentiation was comprehensively analyzed by combining EDU detection, protein immunoblotting, and indirect immunofluorescence assay.

[0031] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention, through Sanger sequencing, RNase R digestion, and other experiments, verified the acquisition of a novel CircSESN1 gene with a circular structure distributed in both the cell nucleus and cytoplasm. This invention also designed and synthesized specific primers, a detection kit, and a detection method for detecting the expression level of the novel CircSESN1 gene. The detection kit provided by this invention can be used to detect the expression level of the novel CircSESN1 gene in chicken muscle tissue and myoblasts; the detection method is simple and rapid. The novel CircSESN1 gene provided by this invention can serve as an important non-coding RNA for studying the regulatory theory of chicken skeletal muscle growth and development. The novel CircSESN1 gene provided by this invention can be used to study the molecular mechanisms of chicken skeletal muscle growth and development. In subsequent studies, by overexpressing the novel CircSESN1 gene, the target miRNA and the regulated genes will be verified, and the specific regulatory mechanism of the novel CircSESN1 gene in the process of chicken skeletal muscle growth and development will be determined. Attached Figure Description

[0032] Figure 1 Figure 1 shows the results of the circularization verification of the CircSESN1 gene; (A) Sanger sequencing of CircSESN1; (B) Agarose gel electrophoresis of PCR products of CircSESN1 and Linear SESN1 primers on gDNA and cDNA templates, respectively; (C) RNase R enzyme digestion experiment of CircSESN1 and Linear SESN1 on primary myoblasts (CPMS cells) and DF-1 cells of chicken embryo leg muscle; (D) Actinomycin D test of CircSESN1 and Linear SESN1 on primary myoblasts (CPMS cells) and DF-1 cells of chicken embryo leg muscle.

[0033] Figure 2 This is a diagram showing the expression distribution of the CircSESN1 gene in the cell nucleus and cytoplasm.

[0034] Figure 3 The results show the expression of the CircSESN1 gene in different tissues and at different time points; (A) Expression profile of the CircSESN1 gene in 13 tissues of 1-day-old chicks; (B) Expression pattern of the CircSESN1 gene in primary myoblasts of chicken embryo leg muscle at different time points (GM-50% and GM-100% represent proliferation at 50% and 100% density, respectively; DM1, DM2, DM3, DM4 and DM5 represent induction differentiation from 1 to 5 days).

[0035] Figure 4 The graph shows the efficiency detection results of the CircSESN1 overexpression vector.

[0036] Figure 5 Figure 1 shows the results of qRT-PCR and Western blot analysis of the effect of CircSESN1 gene on cell proliferation; (A) Changes in the expression of proliferation marker genes detected by qRT-PCR; (B) Effect of CircSESN1 gene on PCNA protein expression; (C) Bar chart comparing the gray values ​​of PCNA protein.

[0037] Figure 6 Figure 1 shows the results of EDU detection of the effect of CircSESN1 gene on cell proliferation; (A) Fluorescence pattern after overexpression of CircSESN1 gene; (B) Cell proliferation ratio after overexpression of CircSESN1 gene.

[0038] Figure 7 The figure shows the effect of the CircSESN1 gene on the expression of the differentiation marker gene MYHC.

[0039] Figure 8 The following figures illustrate the effect of the CircSESN1 gene on MYHC protein expression: (A) Effect of the CircSESN1 gene on MYHC protein expression; (B) Bar chart comparing the gray values ​​of MYHC protein.

[0040] Figure 9 Figure 1 shows the results of indirect immunofluorescence detection of the effect of the CircSESN1 gene on myotube differentiation; (A) Fluorescence microscopy results after CircSESN1 gene overexpression; (B) Myotube area statistics. Detailed Implementation

[0041] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0042] Example 1: Primer design for the novel CircSESN1 gene, and verification of CircSESN1 gene circulation and subcellular localization.

[0043] This invention screens for differentially expressed CircSESN1 genes by performing RNA-Seq on leg muscle tissues at different time points during the chicken embryonic stage. The study revealed that this gene is a circular circRNA structure; therefore, the design of specific primers for the CircSESN1 gene needs to span the splice site. The specific primer pairs for the CircSESN1 gene are shown in SEQ ID NO.1 and SEQ ID NO.2. The specific primer pairs for the Linear SESN1 gene are shown in SEQ ID NO.3 and SEQ ID NO.4, and the primer pairs for the internal reference gene β-actin are shown in SEQ ID NO.5 and SEQ ID NO.6. Total RNA and gDNA were extracted from leg muscle tissues of Bian chickens from the Bian Chicken Breeding Farm of Shanxi Agricultural University. The total RNA was reverse transcribed to obtain cDNA. Using cDNA and gDNA as templates, specific primers were used to quantitatively analyze the CircSESN1 and Linear SESN1 genes, respectively. The products were then subjected to agarose gel electrophoresis. Figure 1 (B) The CircSESN1 gene-specific primer pair only amplified bands on cDNA, not on gDNA, while the Linear SESN1 gene-specific primer pair amplified bands on both gDNA and cDNA. The CircSESN1 gene-specific product was subjected to Sanger sequencing. Figure 1 A), Figure 1 A and Figure 1 B confirmed the existence of the circular binding site of the CircSESN1 gene. Total RNA was extracted from CPMs and DF-1 cells, and the total RNA was divided into two parts: one part was treated with RNase R, and the other part was not. The two parts were digested using a PCR instrument, and the digested products were reverse transcribed to obtain cDNA. Quantitative analysis of the cDNA was performed using specific primers for the CircSESN1 gene, Linear SESN1 gene, and β-actin gene. Figure 1 C) The results showed that the Linear SESN1 gene was involved in RNase R + The expression levels in the treatment groups were all significantly lower than those of RNase R. - In the treatment group (P < 0.01), the expression level of CircSESN1 did not change significantly (P > 0.05). CPMs and DF-1 cells were seeded into 6-well plates. After the cell density reached 80%, medium containing actinomycin D was added. Five time periods were set up, with six replicates for each time period. Total RNA was extracted and reverse transcribed into cDNA. Quantitative analysis of the cDNA was performed using CircSESN1 and β-actin-specific primers. Figure 1D), the results showed that with the extension of the incubation time in actinomycin D medium, the decreasing trend of CircSESN1 expression was significantly less than that of Linear SESN1 expression (P < 0.05). These results confirm the actual existence of the circular structure of the CircSESN1 gene. Figure 1 ).

[0044] Chicken primary myoblasts were extracted from the leg muscle of 12-year-old chicken embryos and seeded into 6-well plates. After the cells reached confluence, the nuclei and cytoplasm were separated using a nucleus and cytoplasm extraction kit (Novizan, catalog number E101). RNA was extracted separately and reverse transcribed into cDNA. The cDNA was then quantitatively analyzed using CircSESN1, GAPDH, and U6 specific primers. Figure 2 The specific primer pairs for the CircSESN1 gene are shown in SEQ ID NO.1 and SEQ ID NO.2, the specific primer pairs for the Linear SESN1 gene are shown in SEQ ID NO.3 and SEQ ID NO.4, the primer pairs for the internal reference gene GAPDH are shown in SEQ ID NO:7 and SEQ ID NO:8, and the primer pairs for the internal reference gene U6 are shown in SEQ ID NO:9 and SEQ ID NO:10. All primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0045] The amplification reaction system described above is 20 μL, containing 10 μL of 2×ChamQ SYBR qPCR Master Mix reagent; specific primer pairs or internal reference gene primer pairs (0.4 μL each of the specific primers or 0.4 μL each of the internal reference gene primers); 2 μL of template cDNA; and 7.2 μL of enzyme-free double-distilled water.

[0046] 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.

[0047] The RNase R reaction system consisted of 20 μL of fluid. The RNase R-added group contained 4 μg of RNA sample, 0.6 μL of RNase R enzyme, 2 μL of 10×RNase R Reaction Buffer, and up to 20 μL of DEPC-treated water. The non-RNase R-added group contained 4 μg of RNA sample, 0 μL of RNase R enzyme, 2 μL of 10×RNase R Reaction Buffer, and up to 20 μL of DEPC-treated water.

[0048] The conditions for quantitative fluorescence reaction were: reaction at 37℃ for 15 min, followed by inactivation at 70℃ for 10 min.

[0049] The primer sequences are as follows:

[0050]

[0051]

[0052] Depend on Figure 1 A, Figure 1 B. Figure 1 C Figure 1 As shown in D, the circular structure of the CircSESN1 gene has been experimentally verified, and Figure 1 As shown in B, the specific primer pair for the CircSESN1 gene amplifies a single band, indicating that the primer design is good.

[0053] Depend on Figure 2 It is known that the CircSESN1 gene is expressed in both the cell nucleus and cytoplasm.

[0054] 2. The CircSESN1 gene inhibits the proliferation of chicken myoblasts.

[0055] Myoblast proliferation and differentiation are fundamental processes in skeletal muscle development; myoblasts must proliferate and differentiate to become mature muscle cells. This invention verifies the effect of the CircSESN1 gene on the proliferation and differentiation of chicken myoblasts. This invention commissioned Shanghai Gemma Technology Co., Ltd. to construct a CircSESN1 overexpression vector named pcDNA3.1-CircSESN1. The CircSESN1 gene sequence was provided to Shanghai Gemma Technology Co., Ltd., which cloned the CircSESN1 gene sequence and subcloned it into the pcDNA3.1(+) vector using BamH1 and EcoRI restriction enzyme sites. An empty pcDNA3.1(+) vector was used as a control in the experiment.

[0056] The new CircSESN1 gene sequence is as follows (443 bp): SEQ ID NO.11

[0057]

[0058] Thirteen tissue samples, including heart, liver, and spleen, were collected from one-day-old female border chickens. Four replicates were used to extract RNA, which was reverse transcribed into cDNA. Quantitative real-time PCR was performed on the template cDNA using CircSESN1 and β-actin specific primers to detect the expression level of the CircSESN1 gene in different tissues. Figure 3A) The results showed that the CircSESN1 gene had the highest relative expression level in the pectoral and leg muscles. Primary chicken myoblasts were extracted from the leg muscle of 12-day-old chicken embryos and seeded in 6-well plates. RNA was extracted from the proliferation phase (GM50%, GM100%) and differentiation phases (DM1 induced differentiation day 1, DM2 induced differentiation day 2, DM3 induced differentiation day 3, DM4 induced differentiation day 4, DM5 induced differentiation day 5), with three replicates for each phase. Quantitative analysis of template cDNA was performed using specific primers for CircSESN1 and β-actin to detect the expression level of CircSESN1 at different phases. Figure 3 B) The results showed that the relative expression level of the CircSESN1 gene generally increased, and the expression level was significantly higher in all time periods of the differentiation phase than in the proliferation phase (P < 0.05).

[0059] Chicken primary myoblasts were extracted from the leg muscle of 12-year-old chicken embryos and seeded into 12-well plates. Transfection was performed when the cell density reached 70%, with four biological replicates per group. The transfection procedure was as follows: The overexpression vector concentration was adjusted to 250 μg / μl with DEPC water. The overexpression vector pcDNA3.1-CircSESN1 was mixed with the transfection buffer and vortexed for 10 s. After brief centrifugation, the transfection reagent was added, and the mixture was vortexed again for 1 s. The mixture was then 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: 3.2 μL of overexpression vector, 75 μL of transfection buffer, and 2.4 μL of transfection reagent. Forty-eight hours after transfection, cells were collected to detect the CircSESN1 overexpression efficiency. The results showed an overexpression efficiency of approximately 120,000-fold, reaching a highly significant level (P < 0.001), indicating that subsequent experiments could be performed. Figure 4 ).

[0060] Ultimately, this invention continued to use the pcDNA3.1-CircSESN1 overexpression vector to verify the effect of CircSESN1 on the proliferation and differentiation of chicken myoblasts.

[0061] Chicken myoblasts were seeded in 6-well plates and transfected with pcDNA3.1-CircSESN1 when the cell density reached 70%. Three biological replicates were set up for each group. Quantitative real-time PCR results showed that the mRNA expression of the proliferation marker genes PAX7 and PCNA transfected with pcDNA3.1-CircSESN1 was either extremely significant or significantly decreased (P < 0.001 or P < 0.01). Figure 5 A); Western blotting experiments showed that transfection with pcDNA3.1-CircSESN1 could inhibit PCNA protein expression. Figure 5B, C); Finally, EDU detection revealed that the proportion of cells transfected with pcDNA3.1-CircSESN1 was significantly lower than that in the control group (P < 0.001). Figure 6 A, Figure 6 B).

[0062] In addition, chicken myoblasts were seeded in 6-well plates. When the cell density reached 90%, the medium was replaced with differentiation medium and transfected with pcDNA3.1-CircSESN1. Quantitative real-time PCR results showed that after transfection with pcDNA3.1-CircSESN1, the mRNA expression of the cell differentiation marker gene MYHC significantly increased (P < 0.01). Figure 7 Western blotting experiments revealed that transfection with pcDNA3.1-CircSESN1 significantly increased MYHC protein expression (P < 0.01). Figure 8 A, Figure 8 B); Finally, indirect immunofluorescence assays showed that transfection with pcDNA3.1-CircSESN1 significantly increased the myotube area (P < 0.001). Figure 9 A, Figure 9 B).

[0063] In summary, the newly discovered CircSESN1 gene in this invention has the function of inhibiting chicken myoblast proliferation and promoting differentiation, while another previously reported transcript of the CircSESN1 gene has the function of simultaneously promoting chicken myoblast proliferation and differentiation. During muscle growth and development, inhibiting excessive cell proliferation is a key step in ensuring normal muscle differentiation and functional maturation. Excessive cell proliferation may lead to abnormal muscle hyperplasia (such as fibrosis) and is also accompanied by tumor risk. After cell proliferation stops, energy and raw materials (such as amino acids) can be diverted to differentiation processes such as myotube fusion and myofiber hypertrophy. Therefore, CircSESN1 can ensure that muscle tissue stops dividing and transitions to functional maturation at the correct time by regulating the broiler cell cycle, signaling pathways, and differentiation program, thereby breeding new high-yielding broiler breeds. The novel CircSESN1 provided by this invention can serve as an important non-coding RNA for studying the theory of regulation of chicken skeletal muscle growth and development.

Claims

1. CircSESN1 Genes, characterized by, The CircSESN1 The nucleotide sequence of the gene is shown as SEQ ID NO.

11.

2. The claim 1 CircSESN1 Application of genes in the preparation of products that inhibit the proliferation of chicken myoblasts and / or promote the differentiation of chicken myoblasts.

3. An overexpression vector, characterized in that, The overexpression vector includes the one described in claim 1. CircSESN1 Gene.

4. The overexpression vector according to claim 3, characterized in that, The overexpression vector includes pcDNA3.1-CircSESN1.

5. The use of the overexpression vector according to claim 3 in the preparation of products that inhibit the proliferation of chicken myoblasts and / or promote the differentiation of chicken myoblasts.

6. The claim 1 CircSESN1 The application of the gene and the overexpression vector of claim 3 in the preparation of products that promote the growth and development of chicken skeletal muscle.

7. A method for inhibiting the proliferation of chicken myoblasts and / or promoting their differentiation, characterized in that, The method is a non-disease treatment method, and the method includes improving the method described in claim 1. CircSESN1 Gene expression levels.

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