Application of TAGLN gene in regulating the growth and development of skeletal muscle in chicken embryos

Abstract

The present invention discloses TAGLN The invention discloses a method for detecting genes affecting the growth and development of skeletal muscle in the chicken embryo stage. TAGLN The invention also discloses a detection kit and a detection method thereof. The detection kit provided by the invention can be used to detect TAGLN The gene expression in chicken muscle tissue and myoblasts is detected in a simple and quick manner. This gene can be used as an important gene for studying the regulatory theory of chicken skeletal muscle growth and development during the embryonic period.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and in particular relates to the application of TAGLN gene in regulating the growth and development of skeletal muscle in chicken embryonic period. Background Art

[0002] Muscle is an important part of the animal body, and skeletal muscle alone accounts for about 40% of the total body weight. The quantity and quality of skeletal muscle directly affect chicken production and determine the economic value of broilers. Muscle content is mainly determined by three factors: the number of muscle fibers, the diameter of muscle fibers, and the type of muscle fibers. Among them, the higher the content of oxidative muscle fibers and the lower the content of glycolytic muscle fibers in the muscle, the better the main quality indicators of the muscle. Myoblasts, as precursor cells of skeletal muscle fibers, undergo a series of proliferation and differentiation and eventually become mature muscle fibers. The proliferation of myoblasts and their differentiation into myotubes largely determine the number of muscle fibers after birth. Therefore, studying the genetic regulatory mechanism of myoblast proliferation and differentiation has important scientific significance and guiding role in clarifying the theoretical basis of chicken skeletal muscle growth and development and the genetic improvement of growth traits of local chickens in my country.

[0003] As a nucleic acid demethylase, FTO can remove methyl groups from single-stranded DNA and RNA, thereby regulating the level of N6-methyladenosine (m6A) in RNA in cells. Therefore, lowering the mRNAm6A methylation level and enhancing its stability is one of the important mechanisms for FTO to exert its biological function. The applicant previously found that FTO was significantly upregulated during chicken embryonic development and proliferation and differentiation of myoblasts, and its expression level was significantly negatively correlated with the m6A methylation modification level, indicating that the FTO gene may mediate RNAm6A demethylation and affect the proliferation and differentiation of chicken myoblasts. TAGLN is an actin-binding protein and a member of the calcium-regulated protein family. Its N-terminal calcium-regulated protein homology region and C-terminal calcium-regulated protein module region are necessary for actin binding and are expressed in smooth muscle, cardiac muscle and skeletal muscle lineages in early embryonic development.

[0004] However, there are currently no relevant studies or reports on the correlation between the FTO gene and the TAGLN gene, and the effect of the TAGLN gene on the proliferation and differentiation of chicken myoblasts. Summary of the invention

[0005] Purpose of the invention: The present invention uses RNA m6A methylation sequencing (meRIP-seq) and transcriptome sequencing technology (RNA-seq) to detect the effects of interfering with FTO on RNA m6A methylation levels and gene expression in chicken myoblasts, and finally targets the TAGLN gene. The technical problem to be solved by the present invention is to provide the application of the TAGLN gene in regulating the growth and development of skeletal muscle in the chicken embryonic period.

[0006] The technical problem that the present invention needs to solve is to provide an overexpression vector of TAGLN or an interfering sequence thereof.

[0007] The technical problem that the present invention is to solve is to provide the application of the TAGLN overexpression vector or its interference sequence in promoting or inhibiting the proliferation and differentiation of chicken myoblasts.

[0008] The technical problem that the present invention needs to solve is to provide a specific primer pair for detection.

[0009] The technical problem that the present invention needs to solve is to provide a detection kit and a detection method thereof.

[0010] The final technical problem to be solved by the present invention is to provide the application of the TAGLN overexpression vector or its interference sequence, the specific primer pair, and the detection kit in studying the regulation of skeletal muscle growth and development in chicken embryos.

[0011] Technical solution: In order to solve the above technical problems, the present invention provides the application of TAGLN gene in regulating the growth and development of skeletal muscle in chicken embryos.

[0012] The present invention also includes an overexpression vector of TAGLN or its interfering sequence, wherein the TAGLN overexpression vector includes pcDNA3.1-TAGLN, the TAGLN interfering sequence includes TAGLN-si104, TAGLN-si392, TAGLN-si486 or TAGLN-si518, the sense chain of the TAGLN-si104 is shown in SEQ ID NO:5, the antisense chain of the TAGLN-si104 is shown in SEQ ID NO:6, the sense chain of the TAGLN-si392 is shown in SEQ ID NO:7, the antisense chain of the TAGLN-si392 is shown in SEQ ID NO:8, the sense chain of the TAGLN-si486 is shown in SEQ ID NO:9, the antisense chain of the TAGLN-si486 is shown in SEQ ID NO:10, the sense chain of the TAGLN-si486 is shown in SEQ ID NO:11, the antisense chain of the TAGLN-si486 is shown in SEQ ID NO:12.

[0013] As shown in SEQ ID NO:12.

[0014] The present invention also includes the use of the TAGLN overexpression vector or its interference sequence in promoting or inhibiting the differentiation of chicken myoblasts.

[0015] The present invention also includes a specific primer pair for detecting the expression of TAGLN in chicken embryonic muscle tissue and / or myoblasts. The sequences of the specific primer pair are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0016] The present invention also includes the use of the specific primer pair in preparing a reagent or a kit for detecting the expression level of TAGLN in embryonic muscle tissue and / or myoblasts.

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

[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 of the internal reference gene β-actin, and the primer pair sequence of the internal reference gene β-actin is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0020] The present invention also includes the use of the TAGLN overexpression vector or its interfering sequence, the specific primer pair or the fluorescent quantitative detection kit in studying or regulating the growth and development speed of skeletal muscle in chicken embryos.

[0021] Furthermore, the application is to detect the expression level of TAGLN in chicken embryonic muscle tissue and / or myoblasts by fluorescent quantitative PCR.

[0022] The present invention also includes a detection method of the detection kit, which specifically involves detecting the expression of TAGLN in chicken embryonic muscle tissue and / or myoblasts by fluorescent quantitative PCR.

[0023] Furthermore, the fluorescent quantitative PCR reaction system comprises: 2×ChamQ SYBR qPCR Master Mix, a primer having a sequence as shown in SEQ ID NO: 1, a primer having a sequence as shown in SEQ ID NO: 2, a template cDNA, and enzyme-free double-distilled water.

[0024] Furthermore, the fluorescent quantitative PCR reaction system is: 10 μl of 2×ChamQ SYBR qPCR Master Mix reagent; 0.4 μl of primers with a sequence as shown in SEQ ID NO: 1; 0.4 μl of primers with a sequence as shown in SEQ ID NO: 2; 2 μl of template cDNA; and 7.2 μl of enzyme-free double-distilled water.

[0025] Furthermore, the fluorescent quantitative PCR reaction system also includes 0.4 μl of a primer having a sequence as shown in SEQ ID NO:3; and 0.4 μl of a primer having a sequence as shown in SEQ ID NO:4.

[0026] The present invention collects primary myoblasts of chicken embryo leg muscle for in vitro culture, overexpresses and interferes with TAGLN in the myoblasts, extracts total RNA of the cells for reverse transcription, and obtains cDNA. Specific primers of TAGLN and internal reference β-actin in the present invention are used for fluorescence quantitative PCR. The PCR reaction system and amplification procedure are the same as conventional fluorescence quantitative PCR. 2 -△△CT Methods The expression level of the mRNA was tested. The expression trend of proliferation and differentiation marker genes was also detected. The effects of TAGLN on the proliferation and differentiation of chicken myoblasts were comprehensively analyzed by combining CCK-8, Edu detection, protein immunoblotting and indirect immunofluorescence assay.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention obtains specific primers for detecting the expression of the TAGLN gene, a detection kit and a detection method. The detection kit provided by the present invention can be used to detect the expression of TAGLN in muscle tissue and myoblasts in chicken embryos, and the detection method is simple and quick. The TAGLN provided by the present invention can be used as an important gene for studying the theory of regulation of skeletal muscle growth and development in embryonic chickens. The detection method is simple and quick. The TAGLN provided by the present invention can be used for studying the molecular mechanism of skeletal muscle growth and development in chicken embryos. In subsequent studies, TAGLN is verified by overexpression and interference treatment to determine its specific regulatory mechanism in the growth and development of skeletal muscle in chicken embryos. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 qPCR melting curve and RT-qPCR verification of differentially expressed genes; (A) TAGLN melting curve;

[0029] (B) β-actin dissolution curve; (C) RT-qPCR verification of differentially expressed genes.

[0030] Figure 2 TAGLN interference and overexpression efficiency detection;

[0031] Figure 3 Effect of TAGLN on the mRNA expression of proliferation marker genes;

[0032] Figure 4 Effect of TAGLN on P21 protein expression;

[0033] Figure 5 CCK8 detects cell proliferation;

[0034] Figure 6 EDu detected the effect of TAGLN on cell proliferation;

[0035] Figure 7 Effect of TAGLN on the mRNA expression of differentiation marker genes;

[0036] Figure 8 Effect of TAGLN on MYHC protein expression

[0037] Fig. 9 Indirect immunofluorescence was used to detect the effect of TAGLN on myotube differentiation. DETAILED DESCRIPTION

[0038] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0039] Example 1: Mining process of TAGLN gene and its effect on the proliferation and differentiation of chicken myoblasts

[0040] 1. TAGLN gene mining process and RT-qPCR verification of TAGLN gene primer pairs

[0041] Previous experiments found that the expression of the FTO gene was significantly upregulated during the embryonic development of "Heying Black Chicken" and the proliferation and differentiation of myoblasts, and its expression level was significantly negatively correlated with the m6A methylation modification level, indicating that FTO may regulate RNAm6A demethylation and affect the proliferation and differentiation of chicken myoblasts. Further experiments have proved that FTO has a significant promoting effect on the proliferation and differentiation of chicken myoblasts. In order to screen the downstream target genes of FTO, we interfered with FTO in primary chicken myoblasts and used RNA m6A methylation sequencing (meRIP-seq) and transcriptome sequencing technology (RNA-seq) were used to detect the changes in RNAm6A methylation levels and gene expression levels in cells after interfering with FTO. The results showed that differential methylation sites were generated after interfering with FTO, which also led to significant changes in gene expression levels. Studies have shown that FTO upregulates mRNA expression mainly by enhancing its stability. Therefore, the nine-image diagram analysis was used to further screen the genes whose m6A methylation levels were significantly upregulated and whose mRNA expression levels were significantly downregulated after interfering with FTO as candidate target genes of FTO. According to the above principles and sequencing results, candidate target genes such as TAGLN, TMEM132A, TBXA2R, SOX18, NOG2, NPB, NR4A3, and PTCRA were screened out. Then, RT-qPCR technology was used to verify the candidate target genes. Finally, the key candidate target gene TAGLN with significant differential expression was determined, that is, after interfering with FTO, the methylation level of the TAGLN gene was significantly upregulated, while its mRNA expression level was significantly downregulated.

[0042] Taking the TAGLN gene as an example, RT-qPCR was performed to verify the differentially expressed genes shown by sequencing. In addition, the verification methods of seven other genes, including TMEM132A, TBXA2R, SOX18, NOG2, NPB, NR4A3, and PTCRA, were consistent with that of the TAGLN gene.

[0043] Total RNA was extracted from leg muscle tissue of 12-embryonic-age Bian chicken and reverse transcribed to obtain cDNA. Using cDNA as a template, a specific primer pair of TAGLN gene (GenBank accession number: NC_052555.1) was designed, and its sequence is shown in SEQ ID NO.1 and SEQ ID NO.2. The primer pair of internal reference gene β-actin (GenBank accession number: NC_052545.1) is shown in SEQ ID NO:3 and SEQ ID NO:4. The primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0044] The amplification reaction system is 20 μl, including 10 μl of 2×ChamQ SYBR qPCR Master Mix reagent; 0.4 μl of each specific primer pair or internal reference gene primer pair (0.4 μl of each specific primer with a sequence as shown in SEQ ID NO: 1 and SEQ ID NO: 2, or 0.4 μl of each primer with an internal reference gene primer sequence 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.

[0045] Fluorescence quantitative reaction program: pre-denaturation at 95°C for 30 seconds; 95°C for 10 seconds, 60°C for 30 seconds, 40 cycles; 95°C for 15 seconds, 60°C for 60 seconds, 95°C for 30 seconds, 60°C for 15 seconds to obtain the melting curve.

[0046] The primer sequences are as follows:

[0047] TAGLN-F:5'-TGTTCCAGACCGTTGACCTC-3'SEQ ID NO:1

[0048] TAGLN-R:5'-TGATGTCTTGCCCTCCTTC-3'SEQ ID NO:2

[0049] β-actin-F:5'-CAGCCATCTTTCTTGGGTAT-3'SEQ ID NO:3

[0050] β-actin-R:5'-CTGTGATCTCCTTCTGCATCC-3'SEQ ID NO:4

[0051] Depend on Figure 1 As shown in A and B, the melting curves of TAGLN and β-actin are smooth and have a unique main peak, indicating that the primers are well designed.

[0052] Depend on Figure 1 C shows that interfering with FTO reduced the expression levels of candidate target genes such as TMEM132A, TBXA2R, SOX18, NOG2, NPB, NR4A3, and PTCRA, but did not reach a significant level. However, interfering with FTO significantly reduced the expression of TAGLN mRNA, indicating that TAGLN may be a target gene of FTO and may affect the proliferation and differentiation of chicken myoblasts.

[0053] 2. TAGLN promotes the proliferation and differentiation of chicken myoblasts

[0054] The proliferation and differentiation of myoblasts is a basic process of skeletal muscle development. Myoblasts need to undergo proliferation and differentiation to become mature muscle cells. The present invention verifies the effect of TAGLN on the proliferation and differentiation of chicken myoblasts. The present invention commissioned Shanghai Jima Company to construct four interference sequences of TAGLN genes, named TAGLN-si104, TAGLN-si392, TAGLN-si486 and TAGLN-si518, and commissioned Shanghai Jima Company to construct a TAGLN overexpression vector named pcDNA3.1-TAGLN.

[0055] The TAGLN gene sequence is as follows:

[0056]

[0057] The four interference sequences are as follows:

[0058] TAGLN-si104 interference sequence:

[0059] Sense strand: GCAUGAGCAGGGAUGUCCATT SEQ ID NO: 5:

[0060] Antisense strand: UGGACAUCCCUGCUCAUGCTT SEQ ID NO: 6:

[0061] TAGLN-si392 interference sequence:

[0062] Sense strand: GCGUGGUCAAGACAGACAUTT SEQ ID NO: 7

[0063] Antisense strand: AUGUCUGUCUUGACCACGCTT SEQ ID NO: 8:

[0064] TAGLN-si486 interference sequence:

[0065] Sense strand: GGCGGUGACCAAGAAUGAUTT SEQ ID NO: 9;

[0066] Antisense strand: AUCAUUCUUGGUCACCGCCTT SEQ ID NO: 10;

[0067] TAGLN-si518 interference sequence:

[0068] Sense strand: GGGAUCCCAACUGGUUCAUTT SEQ ID NO: 11;

[0069] Antisense strand: AUGAACCAGUUGGGAUCCCTT SEQ ID NO:12.

[0070] Chicken primary myoblasts were extracted from leg muscles of 12-year-old chicken embryos and inoculated in 12-well plates. Transfection was performed when the cell density reached 70%. Four biological replicates were set for each group. The transfection steps were as follows: the interference sequence concentration was diluted to 20 μM with DEPC water, and the overexpression vector concentration was adjusted to 250 μg / μl. The interference sequence or overexpression vector was mixed with transfection buffer and vortexed for 10 seconds. After brief centrifugation, the transfection reagent was added, vortexed again for 1 second, and incubated at room temperature for 20 minutes. Finally, it was added to the cell culture plate to complete the transfection process. The added dosage was as follows: 2.75 μl of interference sequence, 100 μl of buffer, 3 μl of transfection reagent or 3.2 μl of overexpression vector, 75 μl of buffer, and 2.4 μl of transfection reagent were added to each replicate. After 48 hours of transfection, cells were collected to detect the TAGLN interference efficiency and overexpression efficiency. The results showed that the interference efficiency of TAGLN-si392 was the highest among the four sequences, reaching more than 2 times; reaching a significant level (P≤0.05); and the overexpression efficiency was close to 20 times, reaching an extremely significant level (P≤0.01), and subsequent experiments can be carried out ( Figure 2 ).

[0071] Finally, the present invention continues to select TAGLN-si392 interference sequence and pcDNA3.1-TAGLN overexpression vector to verify the effect of TAGLN on the proliferation and differentiation of chicken myoblasts.

[0072] Chicken myoblasts were transfected with TAGLN-si392 and pcDNA3.1-TAGLN overexpression vectors during the proliferation and differentiation stages, respectively. Four biological replicates were set in each group. During the proliferation stage, the results of fluorescence quantitative PCR showed that the mRNA expression of cell cycle and proliferation marker genes PCNA, CDK1 and CCND1 in transfected TAGLN-si392 cells was significantly or extremely significantly reduced, and the mRNA expression of P21 (a negative regulator of the cell cycle) was extremely significantly increased (P≤0.05 or P≤0.01) ( Figure 3 A); however, the mRNA expression of proliferation marker genes PCNA, CCND1 and CCNA2 was significantly or extremely significantly increased after transfection with pcDNA3.1-TAGLN, and the negative regulatory gene P21 was significantly decreased (P≤0.05 or P≤0.01); Western blotting experiments showed that transfection with TAGLN-si392 could promote the expression of P21 (cell cycle negative regulatory factor) protein ( Figure 4 A, B), transfection with pcDNA3.1-TAGLN can inhibit the expression of P21 protein ( Figure 4 A, C); CCK8 detection showed that the cell activity was significantly lower than that of the control group 24 hours after transfection with TAGLN-si392 ( Figure 5A); after transfection with pcDNA3.1-TAGLN, the cell activity was significantly higher than that of the control group ( Figure 5 B); Finally, EDu detection showed that the proportion of TAGLN-si392-transfected positive cells was significantly lower than that of the control group (P≤0.05) ( Figure 6 A); The proportion of positive cells after transfection with pcDNA3.1-TAGLN was significantly higher than that in the control group (P≤0.05) ( Figure 6 B).

[0073] In addition, during the differentiation period of chicken myoblasts, the results of fluorescence quantitative PCR showed that the mRNA expressions of cell differentiation marker genes MYOG, MYOD1 and MYHC detected by transfection with TAGLN-si392 were significantly or extremely significantly reduced (P≤0.05 or P≤0.01) ( Figure 7 A); After transfection with pcDNA3.1-TAGLN, the mRNA expressions of cell differentiation marker genes MYOG, MYOD1 and MYHC were significantly or extremely significantly increased (P≤0.05 or P≤0.01) ( Figure 7 B), Western blotting experiments revealed that transfection of TAGLN-si392 inhibited MYHC protein expression ( Figure 8 A, B); Transfection of pcDNA3.1-TAGLN promotes MYHC protein expression ( Figure 8 A, C), and finally the results of indirect immunofluorescence experiments showed that transfection of TAGLN-si392 significantly reduced the myotube area (P≤0.01) ( Fig. 9 A, C), while transfection with pcDNA3.1-TAGLN significantly increased the myotube area (P≤0.05) ( Fig. 9 B, D).

[0074] In summary, the TAGLN gene has the function of promoting the proliferation and differentiation of chicken myoblasts. The TAGLN provided by the present invention can be used as an important gene for studying the growth and development regulation theory of chicken skeletal muscle.

Claims

1. Overexpression TAGLN Application of gene in positively regulating skeletal muscle growth and development in chicken embryos, the TAGLN 2. Application of an overexpression vector of the TAGLN gene in promoting the proliferation and differentiation of chicken myoblasts, wherein the overexpression vector of the TAGLN gene is pcDNA3.1-TAGLN, TAGLN 3. TAGLN Application of a gene overexpression vector in positively regulating the growth and development of skeletal muscle in chicken embryos, wherein the TAGLN gene overexpression vector is pcDNA3.1-TAGLN, and the TAGLN

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