CN112779262B

CN112779262B - Application of pig RagC gene

Info

Publication number: CN112779262B
Application number: CN202110034472.9A
Authority: CN
Inventors: 王修启; 金成龙; 秦颖超; 朱敏; 高春起; 严会超
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-01-11
Filing date: 2021-01-11
Publication date: 2023-04-18
Anticipated expiration: 2041-01-11
Also published as: CN112779262A

Abstract

The invention provides application of a pig RagC gene. The invention designs a specific primer according to the homology of a human RagC gene sequence and a mouse RagC gene sequence, clones pig RagC gene ORF for the first time, and applies and explores the function of the pig RagC gene participating in induction of different amino acids in different histiocytes of a pig. The invention discovers that the over-expression RagC gene participates in the expression level of the lysine activated porcine skeletal muscle satellite cell mTORC1 signal channel protein, and can obviously improve the protein synthesis capacity of the porcine skeletal muscle satellite cell; the RagC gene is suggested to be involved in the process that mTORC1 senses the change of lysine concentration to regulate and control the synthesis of skeletal muscle satellite cell protein and promote the growth of pig muscle.

Description

Application of pig RagC gene

Technical Field

The invention belongs to the technical field of molecular biology. More particularly, it relates to the application of pig RagC gene.

Background

The mTORC1 can sense the concentration change of intracellular and extracellular amino acids, and further mediate the amino acids to regulate and control protein synthesis. RagC is an important target protein for recruitment of mTORC1 to lysosomal activation. However, the pathways of mTORC1 activation by different amino acids are different, and the pathways of mTORC1 activation by amino acids are not completely consistent among different cells. RagC is one of the members of Rag GTPase, and the other 3 members in mammals are RagA, ragB and RagD respectively, and all belong to the Ras small G protein family. The RagA and RagB are highly homologous, and the RagC and RagD are highly homologous, and the RagA or RagB can be combined with the RagC or RagD to form a heterodimer. Existing studies demonstrate that Rag GTPase is able to mediate amino acid activation of the mTORC1 signaling pathway, particularly leucine and arginine.

Patent CN111394370A discloses a pig RagA gene and application thereof, and particularly discloses that overexpression of the RagA gene can remarkably increase the activity of intestinal epithelial cells and simultaneously activate a mTORC1 signal pathway. However, at present, research on RagC genes at home and abroad is concentrated on human, mice and yeast, and research on pig RagC genes is not reported, and particularly, application of RagC participating in activation of mTORC1 signal channels in different histiocytes of pigs by different amino acids is unknown. In the process of resolving amino acid sensing, functional studies of RagC are crucial, however, there are studies that suggest that the involvement of RagC is not required for glutamine activation of mTORC 1. Therefore, it is necessary to carry out systematic research on the function of amino acid induction of the RagC gene in porcine histiocytes, and an important basis is provided for the subsequent application of the porcine RagC gene in improving the growth quality of pigs.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects of the pig RagC gene in the research of the functions of pig cells in the prior art and provides the application of the pig RagC gene.

The first purpose of the invention is to provide the application of pig RagC gene in improving the protein synthesis capacity of animal cells.

The second purpose of the invention is to provide the application of the pig RagC gene and/or the protein coded by the pig RagC gene as mTORC1 pathway activator for improving the protein synthesis capability of animal cells.

The third purpose of the invention is to provide the application of the pig RagC gene and/or the protein coded by the pig RagC gene in promoting the growth of animal muscle.

The fourth purpose of the invention is to provide a method for improving the protein synthesis capability of the pig cells.

The above object of the present invention is achieved by the following technical solutions:

the invention designs specific primers according to the homology of a human RagC gene sequence and a mouse RagC gene sequence, clones a pig RagC gene for the first time, the nucleotide sequence of the pig RagC gene is shown as SEQ ID NO.5, and the application and the exploration are carried out on the function of the pig RagC gene participating in the induction of different amino acids in different histiocytes of pigs. The invention discovers that the over-expression of the RagC gene is involved in the expression level of the protein of the mTORC1 signal pathway of the pig skeletal muscle satellite cell activated by lysine, but the over-expression of the RagC gene is not involved in the mTORC1 signal pathway of the pig intestinal epithelial cell activated by glutamic acid, which indicates that the functions of the RagC in different cells involved in different amino acid sensing are different.

The invention therefore claims the following applications:

the application of pig RagC gene in improving the protein synthesis capacity of animal cells.

The application of the pig RagC gene and/or the protein coded by the pig RagC gene in the improvement of the protein synthesis capability of animal cells as mTORC1 pathway activators.

Preferably, the animal cell is a porcine tissue cell.

More preferably, the porcine tissue cells are porcine skeletal muscle satellite cells.

The nucleotide sequence of the pig RagC gene is shown as SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 6.

Likewise, the application of the pig RagC gene and/or the protein coded by the pig RagC gene in promoting the muscle growth of animals is also within the protection scope of the invention.

Preferably, the animal is a pig.

Preferably, the above-described application is the overexpression of the porcine RagC gene in porcine skeletal muscle satellite cells.

A method for increasing protein synthesis ability of a pig cell, which comprises over-expressing a pig RagC gene in the pig cell, comprising the steps of:

s1, amplifying the pig RagC gene ORF by PCR;

s2, constructing a lentivirus over-expression vector containing a pig RagC gene ORF;

s3, infecting the pig cells with the lentivirus overexpression vector obtained in the step S2 to obtain a pig cell strain which expresses a pig RagC gene.

Preferably, the porcine cells are porcine skeletal muscle satellite cells.

Preferably, the nucleotide sequence of the primer amplified by PCR in step S1 is shown in SEQ ID NO. 1-2.

Preferably, the plasmid used for constructing the lentiviral over-expression vector containing the porcine RagC gene ORF in the step S2 is pLVX-AcGFP1-N1.

Preferably, real-time PCR is adopted to identify whether an over-expression cell strain of the RagC gene is obtained or not, and the sequence of a primer used for identifying the over-expression of the RagC gene by PCR is shown in SEQ ID NO. 7-8.

The invention has the following beneficial effects:

the invention provides a new application of pig RagC gene. According to the invention, a specific primer is designed according to homology of a human RagC gene sequence and a mouse RagC gene sequence, the pig RagC gene ORF is cloned for the first time, and the application and exploration are carried out on the function of the pig RagC gene participating in induction of different amino acids in different histiocytes of a pig. The invention discovers that the over-expression RagC gene participates in the expression level of the lysine activated porcine skeletal muscle satellite cell mTORC1 signal channel protein, and can obviously improve the protein synthesis capacity of the porcine skeletal muscle satellite cell; the result suggests that the gene of RagC participates in the process that mTORC1 senses the change of lysine concentration to regulate and control the synthesis of skeletal muscle satellite cell protein and promote the growth of pig muscle, and can be used for the subsequent functional research of the RagC gene of pigs on pig animals and the development of amino acid sensing.

Drawings

FIG. 1 is a diagram showing the result of electrophoresis of a PCR amplification product of the RagC gene; in the figure, M: DNA Marker DL2000,1 and 2: the target strand of RagC (1200 bp).

FIG. 2 is a diagram showing the results of cutting gel and recovering the RagC gene after agarose gel electrophoresis; in the figure, M: DNA Marker DL2000,1: the target strand of RagC (1200 bp).

FIG. 3 is a diagram showing the result of electrophoresis of a PCR amplification product of the ragC gene having a restriction enzyme cleavage site and a Flag tag; in the figure, M: DNA Marker DL2000,1: contains a RagC gene band (1247 bp).

FIG. 4 is a diagram showing the results of recovering the RagC gene with the cleavage site and Flag tag by cutting; in the figure, M: DNA Marker DL2000,1: contains the RagC gene band (1247 bp).

FIG. 5 is a graph showing the results of the purified and recovered RagC gene after Xho I and EcoR I double digestion; in the figure, M: DNA Marker DL2000,1: contains the RagC gene band (1241 bp).

FIG. 6 is a diagram showing the results of the purification and recovery of pLVX-AcGFP1-N1 fragment by Xho I and EcoR I; in the figure, M1: DNA Marker DL15000,1: pLVX-AcGFP1-N1 fragment (8771 bp).

FIG. 7 is a result diagram of the recombinant vector obtained by enzyme digestion identification; in the figure, M2: DNA Marker DL10000,1 and 2 are respectively used for identifying the recombinant vector (10012 bp) by single enzyme digestion of Xho I and EcoR I restriction enzymes, and 3 is used for identifying the recombinant vector (8771 bp) by double enzyme digestion of Xho I and EcoR I restriction enzymes.

FIG. 8 is a graph of the sequencing peaks of RagC in the recombinant overexpression vector (CMV-F one-way sequencing).

Fig. 9 shows GFP positive cells obtained using the MoFlo XDP ultra-flow cytometric sorting system, negative control: negative Control group (wild type porcine intestinal epithelial cells), control: control group (empty plasmid group, expression GFP tag), ragC overlay: ragC overexpression panel (expression GFP tag).

FIG. 10 is a graph showing the intensity of GFP fluorescence signals observed after sorting in the control group and the RagC overexpression group.

Fig. 11 is the identification of stable transgenic porcine intestinal epithelial cell line overexpressing RagC, a: ragC fluorescence signal intensity, B: mRNA abundance and C: protein expression level (n = 3); wherein, control: control group (empty plasmid group), ragC overlay expression: ragC overexpression group.

FIG. 12 shows that immunofluorescence identifies that the Flag tag is simultaneously over-expressed in the RagC stably transfected porcine intestinal epithelial cell line.

Figure 13 is a graph showing that glutamate activates mTORC1 signaling in porcine intestinal epithelial cells and down-regulates the level of RagC total protein expression.

Figure 14 is that RagC is not involved in glutamate activation of mTORC1 signaling events in porcine intestinal epithelial cells.

Fig. 15 is a graph showing that lysine up-regulates porcine skeletal muscle satellite cell RagC protein expression levels and activates the mTORC1 signaling pathway.

FIG. 16 shows the GFP fluorescence signal intensity of control and RagC-overexpressing groups after stable transformation of porcine skeletal muscle satellite cells by overexpression of RagC.

FIG. 17 shows that overexpression of RagC significantly increases the expression level of mTORC1 signal pathway protein of porcine skeletal muscle satellite cells.

FIG. 18 shows that overexpression of RagC significantly improves the protein synthesis capacity of porcine skeletal muscle satellite cells.

Detailed Description

The invention is further described with reference to the following detailed description and accompanying drawings, but the invention is not limited thereto in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 PCR amplification of porcine RagC Gene ORF

1. Primer design

Based on the homology between the human ragC gene sequence (accession No. NM-022157.4) and the mouse ragC gene sequence (accession No. NM-017475.2), specific primers F1/R1 were designed using Primer Premier 5 software, and the nucleotide sequences of the primers are shown in Table 1.

TABLE 1 nucleotide sequences of primers for cloning

2. Extraction of total RNA of porcine intestinal epithelial cells

The culture medium in the 6-well plate was aspirated and the cells rinsed twice with PBS. Adding 1mL Trizol reagent into each hole, standing for 5min at room temperature, centrifuging for 10min at 12000r/min at 4 ℃, and taking supernatant to a new centrifuge tube. Adding 0.2mL of chloroform, shaking vigorously and mixing uniformly, standing at room temperature for 5min, centrifuging at 4 ℃ for 15min at 12000r/min (the speed reduction is zero), and taking the upper-layer water phase to a new centrifuge tube. Adding isopropanol according to the proportion of 1. The RNA precipitate was rinsed 2 times with 75% ethanol, centrifuged at 12000r/min at 4 ℃ for 5min, and the supernatant was discarded. Standing at room temperature, drying the precipitate, and adding a proper amount of DEPC water for dissolving. The RNA concentration was measured by a nucleic acid protein analyzer, and the quality of the RNA was checked by ordinary agarose gel electrophoresis.

3. Digestion of DNA in total RNA of porcine intestinal epithelial cells

The ingredients were added and mixed uniformly in the digestion reaction system shown in Table 2, reacted at 37 ℃ for 30min and at 65 ℃ for 5min (inactivating DNase) to digest DNA in total RNA of porcine small intestine epithelial cells.

TABLE 2 digestion reaction System

4. cDNA Synthesis

Reverse transcription was performed according to M-MLV reagent instructions, taking 2. Mu.g of RNA, adding 3. Mu.L of random primer, adding DEPC water to 15. Mu.L, denaturing at 70 ℃ for 5min, cooling on ice, and centrifuging briefly. Then adding other components according to a reverse transcription reaction system shown in the table 3, oscillating, uniformly mixing, centrifuging for a short time, and standing for 60min at 37 ℃; inactivating at 80 deg.C for 5min, standing at 12 deg.C for 10min, subpackaging, and storing at-20 deg.C.

TABLE 3 reverse transcription reaction System

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5. PCR amplification of porcine RagC gene ORF

(1) Experimental method

And (3) taking the cDNA obtained in the step (4) as a template, and amplifying the pig RagC gene by using the specific primer designed in the step (1) through a PCR amplification system shown in a table 4.

TABLE 4 CDS region system amplification

(2) Recovery of PCR amplification products

Preparing 1.2% agarose gel, carrying out agarose electrophoresis for 30min at 110V after spotting the PCR amplification product, and taking a picture by a gel imaging system after the electrophoresis is finished. Then, the agarose gel with the target fragment was excised, and the target fragment was recovered using an agarose gel recovery kit (TIANGEN DP 209) according to the kit instructions.

(3) Results of the experiment

FIGS. 1 and 2 show that the PCR amplification product of the RagC gene and the size of the RagC gene recovered by cutting gel after agarose gel electrophoresis are 1200bp.

6. PCR amplification of pig RagC gene ORF containing enzyme cutting site

(1) Design of pig RagC gene amplification primer containing enzyme cutting site

A pair of primers F2/R2 is designed at two ends of the ORF of the RagC gene, the nucleotide sequence of the primers is shown in Table 5, an upstream primer has an Xho I enzyme cutting site, and a downstream primer has an EcoR I enzyme cutting site. For later functional verification, a Kozak sequence (for enhancing the expression of a target gene) and a Flag tag are added into an upstream primer, 3 bases of CTA (CTA) are removed from a downstream primer, so that amplified RagC gene ORF and a connected vector GFP are subjected to fusion expression, and G bases are added at the same time, so that no frameshift mutation is generated.

TABLE 5 nucleotide sequences of primers for cloning

(2) Results of the experiment

As shown in FIGS. 3 and 4, the PCR amplification product of the ragC gene with the restriction enzyme site and Flag tag and the size of the ragC gene with the restriction enzyme site and Flag tag recovered from the gel cutting were all 1247bp.

7. Amplification extraction of pLVX-AcGFP1-N1 plasmid

Inoculating a bacterial liquid containing a lentivirus overexpression vector pLVX-AcGFP1-N1 on a LA plate, culturing overnight at 37 ℃, picking a single colony from the LA plate by using a sterilized toothpick after the colony grows out, inoculating the single colony in 5mL LA liquid culture medium, and shaking overnight at 37 ℃. Plasmids were extracted by the method of reference plasmid miniprep kit (TIANGEN DP 103).

8. Recovering double enzyme digestion target fragment sequence and slow virus over-expression vector

(1) Experimental methods

Referring to the recovery method of the DNA product in the step 5, the target fragment is recovered, double enzyme digestion of the target fragment and the vector is performed according to the reaction system shown in Table 6, and after enzyme digestion, the Universal DNA purification recovery kit (TIANGEN DP 214) is used for recovery, and the recovery is performed according to the method of the kit specification.

TABLE 6 double digestion reaction System

(2) Results of the experiment

The experimental results are shown in FIG. 5 and FIG. 6, the size of the purified and recovered RagC gene after Xho I and EcoR I double digestion is 1241bp, and the size of the purified and recovered pLVX-AcGFP1-N1 fragment after Xho I and EcoR I double digestion is 8771bp.

9. Ligation reaction

The pLVX-AcGFP1-N1 and the RagC fragment double enzyme digestion products are uniformly mixed according to a reaction system shown in the table 7, the mixture is placed in an environment at 16 ℃ for overnight connection, the result of the recombinant vector obtained by enzyme digestion identification is shown in the figure 7, and the size of the recombinant vector is 10012bp.

TABLE 7 ligation reaction System of pLVX-AcGFP1-N1 and RagC Gene fragments

10. Transformation of Escherichia coli stbl3 competent cells by ligation product

And (3) uniformly mixing 10 mu L of the ligation product with 50 mu L of stbl3 competent cells, carrying out ice bath for 30min, carrying out heat shock for 45s at 42 ℃, standing on ice for 3-5 min, adding 600 mu L of antibiotic-free LB culture medium, and shaking for 1h at 37 ℃ at 250 r/min. Spreading on LA plate surface with 200 μ L sterile glass spreader, standing at 37 deg.C for 20min, and culturing for 12h.

11. Sequencing

(1) Experimental method

And after transformation and single bacterium selection of the ligation product, carrying out amplification and extraction on the operation of the plasmid, carrying out PCR identification on the extracted recombinant plasmid, and selecting a bacterial solution with positive enzyme digestion identification and sending the bacterial solution to Guangzhou Scout Biotechnology Limited company for sequencing. After the result is determined, the positive bacteria liquid is taken and extracted with a Kangji corporation endotoxin-free Plasmid Maxi Kit (CWBIO CW 2104) to obtain plasmids, and the plasmids are stored at-20 ℃ for later use after the concentration is measured.

(2) Results of the experiment

Sequencing the obtained recombinant plasmid pLVX-Flag-RagC-AcGFP1-N1, wherein a sequencing peak diagram is shown in figure 8, a nucleotide sequence of the porcine RagC gene obtained after sequencing is shown in SEQ ID No.5, and an amino acid sequence of the protein coded by the ORF is shown in SEQ ID No. 6.

Example 2 construction of cell line overexpressing pig RagC Gene

1. Construction of porcine RagC overexpression vector

The successfully sequenced recombinant plasmid pLVX-Flag-RagC-AcGFP1-N1 in the example 1 is used as a pig RagC gene overexpression vector, and the amplification and plasmid extraction are the same as those in the example 1.

2. The transformation, amplification and extraction methods of the lentiviral vector helper plasmids pMD2.G and psPAX2 are the same as those in example 1, except that the cells used for transformation are Escherichia coli DH5 alpha competent cells.

3. Culture and passage of 293T cell of lentivirus packaging cell

Resuspending the cells in complete medium (high-glucose DMEM medium containing 10% FBS and 1% penicillin/streptomycin), inoculating into a flask, incubating at 37 deg.C, saturation humidity, 5% CO ₂ Cultured in an incubator.

When the fusion rate of the 293T cell reaches 90%, the passage is carried out by the following specific method: the medium was aspirated, rinsed 1 time with PBS, and digested with 0.25% trypsin solution for 4min. The trypsin solution was aspirated off, an equal volume of complete medium was added to stop digestion, and the cell suspension was transferred to a 10mL centrifuge tube and centrifuged at 1000r/min at 4 ℃ for 3min. The supernatant was aspirated off, the cells were resuspended in complete medium, inoculated into a new flask and cultured as 1.

4. Lentiviral packaging

The recombinant plasmid pLVX-Flag-RagC-AcGFP1-N1 and the empty plasmid pLVX-AcGFP1-N1 are co-transfected into 293T cells together with a lentivirus packaging helper plasmid pMD2.G and psPAX2 respectively to carry out lentivirus packaging (the mass ratio of the recombinant plasmid to the lentivirus packaging helper plasmid is pLVX-RagC-AcGFP1-N1: psPAX2: pMD2.G = 3).

5. Obtaining an overexpression porcine RagC stable transgenic cell strain

(1) Experimental methods

Infecting pig intestinal epithelial cells and pig skeletal muscle satellite cells with the harvested lentivirus particles, screening by a flow cytometry and a puromycin drug (2.5 mu g/mL and 3 mu g/mL respectively) to obtain pig intestinal epithelial cells and pig skeletal muscle satellite cells of stably-transformed pig RagC, and identifying RagC overexpression by applying a cellular immunofluorescence technique, real-time PCR and Western blotting to show whether the stably-transformed pig intestinal epithelial cells and pig skeletal muscle satellite cells over-expressing the RagC gene are obtained; wherein, the primer F3/R3 and the sequence thereof used for identifying the overexpression of the RagC gene by Real-time PCR are shown in the table 8.

TABLE 8 primers and sequences thereof for Real-time PCR identification of overexpression of RagC gene

(2) Results of the experiment

The results are shown in FIGS. 9 to 12, which indicate that stable porcine intestinal epithelial cells and porcine skeletal muscle satellite cells overexpressing RagC have been obtained. From the area size of the R3 region in FIG. 9 and the detectable GFP green fluorescence signal in FIG. 10, ragC overexpression can be successfully found; FIG. 11 shows that a stably transformed porcine intestinal epithelial cell line with over-expressed RagC has been obtained; fig. 12 suggests successful RagC overexpression in porcine skeletal muscle satellite cells.

Example 3 functional verification of the porcine RagC Gene

1. Function of RagC gene after over-expression in pig intestinal epithelial cell

(1) Experimental methods

Uses pig intestine epithelial cell line as material, 5 x 10 ⁴ 6-well cell culture plates are inoculated on each cell/well, after 24h of adherence, 15h of serum starvation and 4h of amino acid starvation, the cells are divided into a control group (0 mmol/L) and a glutamic acid group (5 mmol/L) for treatment, and cell protein samples are collected. Detecting the expression level of key protein of mTORC1 signal channel and the expression level of RagC by western blotting; secondly, wild type, empty plasmid and RagC overexpressing cells were used as test materials, 5X 10 ⁴ And inoculating 6-well cell culture plates into each cell/well, treating the cells by dividing the cells into a control group (0 mmol/L) and a glutamic acid group (5 mmol/L), collecting cell protein samples, detecting the expression level of key proteins of the mTORC1 signal pathway through western blotting, and observing the influence of the RagC gene on the mTORC1 signal pathway after the RagC gene is over-expressed in porcine intestinal epithelial cells.

(2) Results of the experiment

The results are shown in fig. 13 and 14, which indicate that glutamate treatment activates mTORC1 signaling in porcine intestinal epithelial cells (fig. 13A-B) and down-regulates the level of RagC total protein expression (fig. 13C-D); the results in fig. 14 show that the over-expression of the RagC gene is not involved in the mTORC1 signaling event in glutamate-activated porcine intestinal epithelial cells.

2. Function of RagC gene after over-expression in pig skeletal muscle satellite cell

(1) Experimental method

Using pig skeletal muscle satellite cell as material, and using 5X 10 ⁴ Inoculating 6-well cell culture plates to each cell/well, and detecting the influence of lysine (0, 0.25, 0.5 and 1 mmol/L) treatment with different concentrations on the key protein expression and RagC expression level of the mTORC1 signal pathway after 24-hour adherence, 15-hour serum starvation and 4-hour amino acid starvation; secondly, the influence of RagC gene overexpression on the expression of key proteins of mTORC1 signal pathway and the expression level of RagC in pig skeletal muscle satellite cells is detected.

(2) Results of the experiment

The results are shown in fig. 15-17, and the results in fig. 15 show that 0.50mmol/L lysine significantly improves the expression level of the total protein of the porcine skeletal muscle satellite cell RagC and activates the mTORC1 signal pathway; FIG. 16 shows that RagC gene was successfully overexpressed in porcine skeletal muscle satellite cells; the results of FIG. 17 show that the overexpression of the RagC gene remarkably improves the protein expression level of the mTORC1 signal pathway of porcine skeletal muscle satellite cells.

3. Effect of overexpression of RagC on protein synthesis ability of skeletal muscle satellite cells of pigs

(1) Experimental methods

Takes empty plasmid and RagC over-expression pig skeletal muscle satellite cell as materials, and takes 5 multiplied by 10 ⁴ 6-well cell culture plates were inoculated per cell/well, and after 48 hours of culture, 1.00. Mu.g/ml puromycin was added for 30min of treatment. Cell samples were collected from RIPA lysates and puromycin levels were detected by Western blotting to determine protein synthesis in porcine skeletal muscle satellite cells.

(2) Results of the experiment

The result is shown in figure 18, and the over-expression of RagC remarkably improves the protein synthesis capacity of porcine skeletal muscle satellite cells. The over-expression of the RagC gene is involved in the lysine-activated pig skeletal muscle satellite cell mTORC1 event, and the fact that the RagC gene is involved in the process that the mTORC1 senses the change of lysine concentration to regulate the synthesis of skeletal muscle satellite cell protein and promote the growth of pig muscles is suggested.

The invention proves that the functions of RagC participating in different amino acid induction in different cells are different, and the gene of RagC can participate in mTORC1 induced lysine concentration change so as to regulate and control the synthesis of pig skeletal muscle satellite cell protein, and can be used for the subsequent functional research of pig RagC gene in pig animals and the development of amino acid induction.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> southern China university of agriculture

Application of <120> pig RagC gene

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Glu Lys Ala Phe Leu Phe Asp Val Val Ser Lys Ile Tyr Ile Ala Thr

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Asp Tyr Asn Phe His Cys Phe Arg Lys Ala Ile His Glu Val Phe Glu

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385 390 395

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acagagggat attcatcaaa ggg 23

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggttggcagt tgtggaataa gt 22

Claims

1. The application of pig RagC gene in improving the protein synthesis capacity of animal cells is characterized in that the animal cells are pig skeletal muscle satellite cells; the application is that pig RagC genes are over-expressed in pig skeletal muscle satellite cells; the nucleotide sequence of the RagC gene is shown as SEQ ID NO. 5.

2. Use of a porcine RagC gene and/or its encoded protein for promoting muscle growth in an animal, wherein the animal is a pig; the application is that the pig RagC gene is over-expressed in pig skeletal muscle satellite cells; the nucleotide sequence of the RagC gene is shown as SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 6.

3. A method for improving the protein synthesis capability of a pig cell, which is characterized in that the pig RagC gene is over-expressed in the pig cell, and the method comprises the following steps:

s1, PCR amplification of pig RagC gene ORF;

s3, infecting the pig cells with the lentivirus overexpression vector obtained in the step S2 to obtain a pig cell strain which expresses a pig RagC gene;

the pig cell is a pig skeletal muscle satellite cell; the nucleotide sequence of the RagC gene is shown as SEQ ID NO. 5; the nucleotide sequence of the primer amplified by the PCR in the step S1 is shown as SEQ ID NO. 1-2.