CN111690682B - Methods and uses for modulating skeletal muscle development - Google Patents

Abstract

The invention discloses a method for regulating skeletal muscle development, which comprises the overexpression of RASGRP1 gene at an embryo level. Therefore, a key target gene RASGRP1 which is modified by H3K27me3 in the pig skeletal muscle development process is found, the enrichment level of the gene is firstly up-regulated and then down-regulated under the modification effect of H3K27me3 in the pig skeletal muscle fiber forming process, and the proliferation function of pig skeletal muscle satellite cells can be promoted so as to promote the development of pig skeletal muscle; the development of skeletal muscle of the pig can be promoted by over-expressing RASGRP1 gene, thereby providing a new method and target spot for improving the lean meat percentage of the pig and a new research direction; moreover, the application of the over-expression RASGRP1 gene in promoting skeletal muscle development can provide a new thought and target spot for the breeding of lean pigs; in addition, the RASGRP1 gene is overexpressed in the embryonic period of the pig, the effect of promoting the development of skeletal muscle can be achieved by various methods, and the application range is wide.

Description

Methods and uses for modulating skeletal muscle development

Technical Field

The invention relates to the field of biotechnology, in particular to a method for regulating skeletal muscle development and application.

Background

With the continuous improvement of the living standard of people, high-quality meat is more and more favored by consumers, and the demand of people for pork is also continuously increased.

In nature, the growth, muscle mass and growth development of skeletal muscle are related to the individual growth and development of mammals. The growth and development of skeletal muscle are one of the important factors influencing the pork yield, which accounts for more than 40% of the animal body, is an important component of the animal body and accounts for a higher proportion in the animal body in the growth period. The research on the growth and development mechanism of the skeletal muscle of the pig is beneficial to improving the occupation ratio of the skeletal muscle of the pig, increasing the pork yield and promoting the production level of the pig industry in China.

Skeletal muscle is composed primarily of muscle fibers embedded in the paratendineus connective tissue, each muscle fiber containing a number of muscle nuclei. The number of muscle fibers determines the number and size of skeletal muscles, while muscle mass is also determined by the number and cross-sectional area of muscle fibers, an increase in the number of muscle fibers is manifested as muscle hyperplasia, and an increase in the cross-sectional area of muscle fibers is manifested as muscle hypertrophy. In animals, the bottom layer of mature muscle fibers contains muscle stem cells, also known as Satellite cells (myoblasts), which can be activated to generate new myoblasts (myoblasts) that exit the cell cycle and differentiate to form myotubes that fuse with muscle-specific proteins to generate muscle fibers that promote the growth and regeneration of skeletal muscle.

Skeletal muscle formation occurs mainly during gestation, and muscle fiber development in pig skeletal muscle forms primary fiber around 35 days after gestation, and the process is continued until 60 days of gestation; secondary fibers are formed at 50-60 days of gestation, a process that reaches peak proliferation at about 75 days; the number of muscle fibers thereafter did not change by the time of day 90 of gestation.

Disclosure of Invention

The invention analyzes the modification level of H3K27me3 in the process of forming pig skeletal muscle fibers by a western blot technology, and screens out a target gene RASGRP1 regulated and controlled by H3K27me3 in the process of forming pig skeletal muscle fibers by combined analysis of a high-throughput sequencing technology ChIP-seq and an RNA-seq. Further experimentally, it was demonstrated that the level of enrichment of RASGRP1 gene was continuously down-regulated during pig skeletal muscle fibrogenesis with up-regulation of the level of H3K27me3 modification. Meanwhile, the overexpression of the RASGRP1 gene in C2C12 myoblasts is preliminarily verified to promote the proliferation function of the cells and finally promote the development of skeletal muscles of pigs. Therefore, the RASGRP1 gene can be overexpressed at the embryo level to promote the development of skeletal muscle, further improve the muscle content of animals and breed lean animal strains.

The invention aims to provide a method and application for regulating skeletal muscle development, which are used for promoting skeletal muscle development, further improving the muscle content of animals and breeding lean animal strains.

According to one aspect of the invention, there is provided a method of modulating skeletal muscle development, the method comprising overexpressing the RASGRP1 gene at the embryonic level to promote skeletal muscle development.

In certain embodiments, the method comprises preparing a transgenic animal that overexpresses the RASGRP1 gene to promote skeletal muscle development in the animal.

In certain embodiments, the method comprises microinjecting the RASGRP1 gene overexpression vector into fertilized eggs, transplanting the fertilized eggs into the uterus of a surrogate pregnant animal, and obtaining an animal with an overexpressed RASGRP1 gene to promote the skeletal muscle development of the animal.

In certain embodiments, the method comprises microinjecting the RASGRP1 gene overexpression vector into blastocysts, implanting into the uterus of a surrogate pregnant animal, and obtaining an animal overexpressing RASGRP1 gene to promote skeletal muscle development in the animal.

In certain embodiments, the method comprises artificial insemination of an animal overexpressing RASGRP1 gene after incubating the RASGRP1 gene overexpression vector with semen to promote skeletal muscle development in the animal.

In certain embodiments, the above methods are applicable to swine.

In certain embodiments, the nucleotide sequence of the RASGRP1 gene overexpression vector comprises the nucleotide sequence set forth in SEQ ID NO. 1.

According to another aspect of the invention, there is provided the use of a method of promoting skeletal muscle development in lean animal breeding.

In certain embodiments, the use of a method of promoting skeletal muscle development in lean animal breeding is accomplished by any one of the following methods: overexpression of the RASGRP1 gene at the embryonic level; preparing a transgenic animal over-expressing RASGRP1 gene; microinjecting RASGRP1 gene overexpression vectors into fertilized eggs, and transplanting the fertilized eggs into the uterus of a surrogate pregnant animal to obtain an overexpression RASGRP1 gene animal; microinjecting RASGRP1 gene overexpression vector to blastocyst, and transplanting into uterus of surrogate pregnant animal to obtain overexpression RASGRP1 gene animal; after the RASGRP1 gene overexpression vector and semen are incubated together, artificial insemination is carried out to obtain the animal with the RASGRP1 gene overexpression.

In certain embodiments, the lean animal comprises a lean pig.

The invention has the beneficial effects that:

1. the method finds a key target gene RASGRP1 which is modified by H3K27me3 in the pig skeletal muscle development process, the enrichment level of the gene is firstly up-regulated and then down-regulated under the modification effect of H3K27me3 in the pig skeletal muscle fiber forming process, and the proliferation function of pig skeletal muscle satellite cells can be promoted so as to promote the development of pig skeletal muscle;

2. by over-expressing RASGRP1 gene, the development of pig skeletal muscle can be promoted, thereby providing a new method and target spot for improving the lean meat percentage of pigs and a new research direction;

3. the application of the over-expression RASGRP1 gene in promoting skeletal muscle development can provide a new thought and target spot for the selection of lean type pigs.

4. The RASGRP1 gene is overexpressed in the embryonic period of the pig, the effect of promoting the development of skeletal muscle can be achieved by various methods, and the application is wide.

Drawings

FIG. 1 shows the expression level of embryonic skeletal muscle H3K27me 3: FIG. 1A shows that the H3K27me3 modification is present in porcine embryonic skeletal muscle at different stages, and FIG. 1B shows that the level of H3K27me3 modification in porcine skeletal muscle increases with the development of the embryo;

FIG. 2 shows the results of RNA-seq and ChIP combined analysis: wherein FIG. 2A is a differentially expressed gene common to ChIP-seq and RNA-seq, FIG. 2B is a gene with reduced level of enrichment but upregulated expression modified by H3K27me3, and FIG. 2C is a gene with upregulated level of enrichment but decreased expression modified by H3K27me 3;

FIG. 3 is the level of recruitment of the embryonic skeletal muscle RASGRP1 gene at different stages under modification with H3K27me 3;

FIG. 4 is a graph showing the relative expression amounts of RASGRP1 gene in embryonic skeletal muscle at different stages based on the average Ct value of RASGRP1 gene in skeletal muscle at 65 days of age;

FIG. 5 shows the results of the double restriction enzyme identification of the overexpression vector pcDNA3.1-RASGRP 1: lane 1 is pcDNA3.1-RASGRP1 recombinant plasmid, Lane 2 is pcDNA3.1-RASGRP1 fragment double digested by BamHI and EcoRI restriction enzyme, M is Marker KBLAder;

FIG. 6 is a graph showing the effect of promoting proliferation of C2C12 myoblasts after overexpression of RASGRP1 gene, on a scale of 100. mu.m.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings.

Example 3 detection of the expression level of H3K27me3 in porcine skeletal muscle samples at different time periods

Taking 6 tissue samples of 33-day, 65-day and 90-day dorsoventral longissimus samples of Duroc pig embryos for detection, wherein the specific experimental process is as follows:

(1) extraction of Total protein

Preparing protein lysate.

Using clean scissors and tweezers to cut about 0.1g of tissue sample, quickly putting the tissue sample into a pre-cooled homogenizer, and mixing the tissue sample with the water according to the ratio of 1: protein lysate was added at a ratio of 10.

③ homogenize the slurry on ice until the tissue is digested and transfer the sample to a 1.5mL centrifuge tube.

Fourthly, centrifuging for 10 minutes at 4 ℃ by 12,000 Xg, and taking the supernatant into a new 1.5mL centrifuge tube.

(2) Protein quantification

The quantification was performed according to the BCA protein quantification kit, as follows:

first, 40. mu.l of protein standard (25mg/mL) was diluted with 1960. mu.l of PBS to prepare 0.5mg/mL of protein standard.

② adding PBS to dilute the BSA standard product according to the instruction.

③ add the protein standard to the standard well of 96-well plate in an amount of 0. mu.l, 1. mu.l, 2. mu.l, 4. mu.l, 8. mu.l, 12. mu.l, 16. mu.l, 20. mu.l, add PBS to make up to 20. mu.l, and repeat three times for each concentration.

And fourthly, adding 20 mu l of protein solution to be detected (diluted by 1: 10) into the sample hole of the 96-well plate, and repeating the steps.

Adding 20 mul BCA working solution into each hole, and standing at 60 ℃ for 60 minutes (the specific time is determined according to the color change).

Sixthly, after the incubation is finished, cooling to room temperature, reading an absorbance value at 562nm, deducting the absorbance value of a blank control group, calculating the average absorbance of each concentration protein standard, drawing a standard curve, and calculating a regression equation.

(3) Expression of Western blotting detection protein

Firstly, assembling and fixing a cleaned glass plate and a comb, selecting and preparing 12% separation glue according to the molecular weight of experimental detection indexes, adding TEMED into the prepared separation glue, immediately shaking up, and pouring glue (reserving the height of about 1.5cm as concentrated glue).

② adding 400 mul of ultrapure water to make the surface of the separation glue smooth and straight.

And thirdly, after about 20 minutes, a remarkable gel line is seen between the water and the glue, which indicates that the separation glue is fully solidified, the water on the upper layer of the glue is poured off, and the water between the glass slides is sucked to be dry by using filter paper.

Fourthly, 5% of concentrated glue is prepared, TEMED is added and then the mixture is immediately shaken up, the rest space is filled with the concentrated glue, and then a comb is obliquely inserted into the concentrated glue and is smoothed out, so that bubbles are avoided.

Fifthly, after the concentrated glue is solidified, taking out the finished film and the glass plate integrally, putting the film and the glass plate into an electrophoresis tank for clamping, taking out a comb, and adding enough electrophoresis buffer (the liquid level of the inner tank is higher than the glue).

Sixthly, sucking 10 mu l of sample by using a micropipette and slowly adding the sample into the sample hole.

Seventhly, adjusting the voltage to 80V for performing concentrated gel electrophoresis, adjusting the voltage to 90V after the indicator runs out of the separation gel in about 30 minutes, and stopping electrophoresis after the indicator runs out of the separation gel in 60 minutes.

Cutting a PVDF membrane and 4 pieces of filter paper which have the same size as the gel sample, soaking the PVDF membrane in methanol for 5min, and then soaking the PVDF membrane, the filter paper and the sponge in a transfer buffer solution.

Ninthly, sequentially stacking the sponge, the two layers of filter paper, the glue, the film, the two layers of filter paper and the sponge at the negative electrode end (black), and placing the stacked sponges on a transfer device; and (5) rotating the film for 70min at 350 mA.

The membrane is immersed in the sealing liquid for sealing at 37 ℃ for 1-2 h or at 4 ℃ overnight.

The membrane was taken out and directly placed in primary antibody, incubated at 37 ℃ for 1h, and washed 5 times with TBST, 5 minutes each.

Transferring the membrane into a second antibody, and incubating for 40 minutes at 37 ℃; washing the membrane for 5 times by TBST, 5 minutes each time; and developing by a chemiluminescence method (ECL).

As shown in fig. 1, the H3K27me3 modification exists in the muscle tissue, and the level of the H3K27me3 modification is increased along with the increase of the embryonic development time, and the expression level of the H3K27me3 in the embryonic day 90 skeletal muscle tissue is higher than that in the embryonic day 65 skeletal muscle tissue, and the difference is not significant; the expression level of H3K27me3 in the embryonic day 65 skeletal muscle tissue is higher than that in the embryonic day 33 skeletal muscle tissue, and the difference is not significant; the expression level of H3K27me3 in the embryonic day 90 skeletal muscle tissue is higher than that in the embryonic day 33 skeletal muscle tissue, and the difference is obvious.

Example two ChIP-seq and RNA-seq Joint analysis and screening of differentially expressed genes

1. ChIP-seq experiment

(1) Preparation of working reagent

The working reagent preparation system used in the ChIP-seq experimental process is as follows:

dounching Buffer (1mL) was added with 10. mu.l Tris-HCl (pH 7.5), 4. mu.l MgCl2, 2. mu.l CaCl2, 10. mu.l PIC, 974. mu.l ddH2O, respectively;

(1mL) 10. mu.l of Tris-HCl (pH 8.0), 10. mu.l of Triton X-100, 10. mu.l of Deoxycholate (DOC), 10. mu.l of SDS, 18. mu.l of NaCl, 4. mu.l of EDTA, 10. mu.l of PIC, and 928. mu.l of ddH2O were added;

(1mL) 0.4. mu.l of EDTA (pH 8.0), 0.1. mu.l of Benzamidine, 1. mu.l of PMSF, 3. mu.l of DTT, 10. mu.l of PIC, and 985.5. mu.l of ddH2O were added;

(1mL) 20. mu.l of Tris-HCl (pH 8.0), 10. mu.l of SDS, 10. mu.l of Triton X-100, 4. mu.l of EDTA, 30. mu.l of NaCl, 10. mu.l of PIC, and 916. mu.l of ddH 2O;

respectively adding 20. mu.l Tris-HCl (pH 8.0), 10. mu.l SDS, 10. mu.l Triton X-100, 4. mu.l EDTA, 100. mu.l NaCl, 10. mu.l PIC and 846. mu.l ddH2O into ChiP Final Wash Buffer (1 mL);

sixthly, Elution Buffer (1mL) is added with 100 mu l NaHCO3, 100 mu l SDS and 800 mu l ddH2O respectively.

(2) Preparation of magnetic beads

The protein A and protein G coupled magnetic bead stock solution is turned upside down for several times, and the protein agarose magnetic beads are resuspended to be uniformly mixed.

60 mul of magnetic beads are taken out from each tube of magnetic bead storage liquid, the two kinds of magnetic beads are added into a 1.5mL siliconized centrifuge tube, and the two kinds of magnetic beads are blown and beaten up and down by a 1mL tip with the tip cut off for several times and are mixed evenly.

③ add 1mL of IP Buffer into the centrifuge tube, and turn upside down for several times to clean the magnetic beads.

Fourthly, freezing and centrifuging for 2min at 4,000rpm and 4 ℃ to ensure that the beads are primarily precipitated into blocks.

Fifthly, placing the centrifuge tube on a magnetic frame for 15S until all the magnetic beads are precipitated, and sucking out and removing the supernatant by using a 200 mu l gun head.

Sixthly, repeating the step four once.

Seventhly, 1mL of IP Buffer, 30. mu.l of 10mg/mL single-stranded salmon sperm DNA, and 75. mu.l of 10mg/mL BSA were added to the magnetic beads. Rotating at 4 deg.C, mixing uniformly and sealing for 3 h. 4,000rpm, refrigerated centrifugation at 4 ℃ for 2min, the centrifuge tube was placed on a magnetic rack for 15S until all the magnetic beads were precipitated, and the supernatant was removed by aspiration with a 200. mu.l pipette tip.

Sixthly, repeating the step four, and repeating the step four once.

Ninthly resuspend the beads with 300. mu.l of IP Buffer and the suspension stored at 4 ℃ until needed.

(3) Micrococcus nuclease digestion

Adding prepared Douncing Buffer into a 1.5mL centrifuge tube, and placing on ice for precooling.

② weighing 20-30mg of muscle tissue sample, transferring the muscle sample into a mortar containing liquid nitrogen by using clean scissors and tweezers, and grinding the tissue into powder by using a grinding rod.

③ blowing and beating the mixture evenly by using a 1mL blue gun head, then sucking all the reagents by using an injector with the specification of 255/8 gauge L, beating the reagents out, and repeatedly sucking and beating the reagents out for 20 times.

Adding 50U/mL micrococcus nuclease to incubate for 7min in a water bath at 37 ℃.

Fifthly, 5 mul of 0.5M EDTA is added and incubated for 5min on ice.

Sixthly, adding 1mL of Hypotonic lysine Buffer, mixing evenly, and incubating for 60min on ice.

Seventhly, centrifuging at 3,000 Xg and 4 ℃ for 5min, and transferring the supernatant into a 1.5mL centrifuge tube.

(4) Phenol extraction for detecting DNA size

Adding 100 ul of supernatant obtained in the previous step and 100 ul of ddH2O, 200 ul of 25: 24:1 phenol-chloroform-isoamyl alcohol.

② mixing the mixture by reversing for several times, then centrifuging for 5min at the maximum rotating speed, and transferring the upper liquid into a new 1.5mL tube.

③ adding 500. mu.l of ice absolute ethyl alcohol and 20. mu.l of 3M NaOAc, shaking and mixing uniformly, and standing and incubating for 30min at-20 ℃.

Fourthly, centrifuging for 20min at the maximum rotating speed of 4 ℃, and abandoning the supernatant.

Fifthly, cleaning with 70 percent alcohol, centrifuging for 5min at the maximum rotation speed of 4 ℃, and discarding the supernatant.

Sixthly, the EP tube cover is opened and put in the air for air drying, and 50 mu l of ddH2O is added for dissolution.

Seventhly, taking 5 mu l of solution to detect the size of the DNA after enzyme digestion on 1.5 percent of glue.

(5) Immunoprecipitation

First, 100. mu.l of the blocked magnetic bead suspension was added to the Micrococcus nuclease digested portion of the solution and then rotated at 4 ℃ for 2 h.

② 4,000rpm 4 ℃ refrigerated centrifugation for 2min, and supernatant is taken to be put into a 1.5mL centrifuge tube.

Thirdly, 10 percent of the supernatant in the second step is divided into 1.5mL centrifuge tubes to be used as Input, and the centrifuge tubes are frozen and stored at the temperature of minus 20 ℃. The remaining supernatant was divided into 1.5mL centrifuge tubes as IP tubes, 0.2-0.25mL per tube. Input was used to calculate the amount of DNA that was extracted with each antibody.

And fourthly, adding the IP Buffer until the volume of each IP tube reaches 325 mu l.

Fifthly, adding the antibody H3K27me3 and the control IgG respectively, then rotating and mixing uniformly for 1H at 4 ℃, and sealing the pipe orifice with a sealing film.

Sixthly, adding 20 mu l of magnetic bead suspension into each IP tube, uniformly mixing at 4 ℃ for one night in a rotating way, and sealing the tube opening by a membrane.

(6) Cleaning of

The mixture was subjected to refrigerated centrifugation at 4,000rpm in an IP tube at 4 ℃ for 2min, and then placed on a magnetic stand for 15 seconds to sufficiently precipitate magnetic beads, and the supernatant was removed from a 200. mu.l pipette tip.

② adding 400 mul ChIP Wash buffer, then lightly shaking for 10s, rotating and sealing at 4 ℃ for 3min, then freezing and centrifuging at 4,000rpm and 4 ℃ for 2min, placing on a magnetic frame for 15s to fully precipitate magnetic beads, removing supernatant with 200 mul gun head.

③ the same method uses ChIP Wash buffer to Wash again, then uses ChIP Final Wash buffer to Wash again.

Fourthly, taking out the Input, and adding the Elution buffer until the volume reaches 200 mu l.

Fifthly, adding 100 mul of Elution buffer into the IP pipe.

Sixthly, 0.5 mu l of RNase with the concentration of 10 mu l/mu l is added into each of the Input and the IP, and the mixture is incubated for 2 hours in a water bath at 68 ℃.

Seventhly, freezing and centrifuging the IP tube at 4000rpm and 4 ℃ for 2min, and taking the supernatant into a 1.5mL centrifuge tube.

Eighty percent (eight) percent (100 μ l) of Elution buffer is added into the IP tube, the mixture is incubated for 5min in water bath at 68 ℃, refrigerated and centrifuged for 2min at 4,000rpm and 4 ℃, and the supernatant is taken out to the same centrifuge tube of 1.5 mL.

(7) Kit purification of DNA

Using OMEGA

DNA was purified using a DNA Clean-Up Kit (model: D6296). The specific operation refers to the specification.

(8) Selecting DNA fragments which are obtained from the chromatin co-immunoprecipitation and have enough enrichment capacity to interact with the H3K27me3 antibody, and sending the selected DNA fragments to a Novista genesis science and technology company for DNA sequencing.

2. RNA-seq experiment

(1) Tissue RNA extraction

RNA extraction was carried out according to the OMEGA's Total RNA Kit II Kit (model: R6934-02) operating manual, as follows:

weighing 20-30mg of animal tissue sample into a mortar containing liquid nitrogen, and grinding the tissue into powder by using a grinding rod.

② adding lmL RNA-Solv Reagent and 20 mul 2-mercaptoethanol into a 1.5mL centrifuge tube, transferring the powder in the step (I) into the centrifuge tube, and standing for 5 minutes at room temperature.

③ adding 200 mul of chloroform into the centrifuge tube respectively, whirling for 20 seconds, and standing for 2-3 minutes at room temperature.

Fourthly, centrifuging for 15min at 4 ℃ by 12,000 Xg.

Fifthly, transferring the supernatant to a new 1.5mL centrifuge tube, adding equal volume of 70% ethanol, and vortexing to mix the supernatant sufficiently.

Sixthly, the

The RNA collecting column is sleeved into a 2mL collecting tube, and 700 mul of the mixed solution is transferred to the step of the fifth step

RNA collection column. The mixture was centrifuged at 10,000 Xg for 30s at room temperature, and the filtrate was discarded.

Seventhly, repeating the step sixthly until all the mixed liquid passes through the collecting column to be filtered.

Era (u)

RNA collection column in the recovery header, adding 500 u l RNA Wash buffer I to the collection column. Centrifuge at 10,000 Xg for 1 minute at room temperature, discard filtrate and collection tube.

Ninthly handle

The RNA collection column was fitted into a new 2mL collection tube, 500. mu.L of RNA Wash Buffer II was added to Wash the column, the column was centrifuged at 10,000 Xg for 1 minute at room temperature, and the filtrate was discarded. Before use, RNA Wash Buffer II was diluted with absolute ethanol as required according to the instructions.

In the front part of the car

The RNA collection column was pulled back into the collection tube, 500. mu.l of RNase Buffer II was added to wash the column, and the column was centrifuged at 10,000 Xg for 1 minute at room temperature, and the filtrate was discarded.

Handle

The RNA collection column is sleeved in the collection tube, and the empty tube is centrifuged at the maximum rotating speed for 2min at room temperature to ensure that

The RNA collection column was dried.

Handle

The RNA collection column was inserted into a new 1.5mL centrifuge tube, 45-70. mu.l DEPC water was added to the center of the collection column, allowed to stand at room temperature for 3-5 minutes, and centrifuged at 10,000 Xg for 2min at room temperature. The DEPC water needs to be preheated to 70 ℃.

(2) RNA reverse transcription to synthesize cDNA

PrimeScript Using TaKaRa^TMThe RT reagent Kit with gDNA Eraser (Perfect Real Time) Kit instructions for reverse transcription of RNA into cDNA.

(3) The RNA samples were sent to Nuo He Sourcez for sequencing.

3. Data processing of ChIP-seq and RNA-seq

(1) RNA-seq data processing

Firstly, in order to ensure the quality and reliability of data analysis, the original data obtained by sequencing is filtered. The filtering content is as follows: reads with a linker (adapter) are removed, reads containing N (N indicates that base information cannot be determined) are removed, and low-quality reads (reads whose number of bases with a quality value Qphred of 20 or less accounts for 50% or more of the length of the whole read) are removed. And then, respectively carrying out sequencing error rate check and GC content distribution check to obtain clean reads used for subsequent analysis. The pretreated clean reads were subjected to pig genome mapping analysis on HISAT software.

Secondly, differential analysis is carried out, and the gene differential expression analysis is mainly divided into three parts:

a. firstly, standardizing read data;

b. then calculating a hypothesis testing probability (P-value) according to the model;

c. finally, multiple hypothesis testing correction was performed to obtain the FDR value (false discovery rate).

And thirdly, in order to research the significance correlation between the differential genes and the biological functions or the pathways, performing GO function enrichment analysis and KEGG pathway enrichment analysis on the differential gene set by using clusterProfiler software.

(2) ChIP-seq data processing

Data quality control

Quality control is carried out on raw data (raw reads) generated by an Illumina sequencing platform, and the quality of the raw data is subjected to primary statistics by using fast QC v0.11.5 software. In order to ensure the data quality and more efficiently utilize sequencing data to facilitate subsequent analysis, Skewer v0.1.126 software is used for filtering the original data, and the data filtering steps are as follows:

a. cutting off low quality reads, wherein reads with the average quality value lower than 20 are cut off;

b. cutting off the reads with the base N, wherein the ratio of N to all the bases of the whole reads exceeds 15%;

c. removing reads with a connector (adapter);

d. reads shorter than 18nt after pruning are discarded.

② sequence comparison

For filtered data (clean reads), BWA v0.7.12 software is used to compare clean data with pig genome, the comparison result is formed into a bam file, and the bam file is visually analyzed by an IGV (Integrated genetics viewer) browser.

(iii) prediction of fragment size

Clear reads can be significantly enriched at specific binding sites, the fragment size of an IP sample is predicted by using MACS2 software, and the enrichment degree of the clear reads is counted.

(iv) analysis of Strand Cross Correlation (SCC)

Clear reads obtained by sequencing are approximately and evenly distributed on positive and negative chains, and the chain cross-correlation detection of IP and Input data can not only obtain the correlation coefficient between the positive and negative chains, but also reflect the chromatin co-immunoprecipitation effect.

Analysis and annotation of peak value

And (3) utilizing MACS2 software to complete peak detection analysis (peak calling), setting a threshold value to be that P-value is less than or equal to 0.005, counting the number, width, distribution and the like of peaks, and screening out related genes of the peaks and the like. Peak-related genes were confirmed using peakanostat software, followed by GO functional enrichment analysis using GOseq software and KEGG pathway enrichment analysis using KOBAS software.

The results of the joint analysis of RNA-seq and ChIP are shown in FIG. 2. According to the results, genes of which the enrichment level of H3K27me3 is reduced along with the increase of the embryonic development period and the gene expression level is improved along with the increase of the embryonic development period are analyzed, genes of which the expression difference multiples are more than 2 between 90 days of an embryo and 65 days of the embryo, between 90 days of the embryo and 33 days of the embryo and between 65 days of the embryo and 33 days of the embryo are screened, and finally, RASGRP1 gene which can play a key role in regulating and controlling the growth and development of skeletal muscle is screened, and the expression trend and the channel prediction result are shown in Table 1.

TABLE 1 expression trends and pathway predictions for RASGRP1 Gene

Example three ChIP-qPCR validation of differentially expressed Gene RASGRP1

The primer design software Oligo 7.56 was used to design quantitative PCR primers for pig genes as shown in Table 2. IP samples which are obtained in the chromatin co-immunoprecipitation process and incubated with antibodies are diluted to the same concentration, the Input of 9 samples are respectively diluted to 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL and 0.625ng/mL, the Input of different concentrations is used for drawing a standard curve, and all samples are prepared into an amplification system according to the table 3. Absolute quantitative PCR was performed using the EcoRM Real-time PCR instrument from illumina, with 3 biological replicates for each IP sample and different concentrations of Input, under the following reaction conditions: 95 ℃ for 5 min; at 95 ℃, 10s, 60 ℃, 15s, 72 ℃, 20s, 40-50 cycles; 95 ℃ for 15 s; 60 ℃ for 15 s; 95 ℃ for 15 s.

TABLE 2 ChIP-qPCR primers

TABLE 3 ChIP-qPCR reaction System

The result is shown in figure 3, ChIP-qPCR result shows that under the condition of histone H3K27me3 modification, the enrichment amount of RASGRP1 in embryo day 65 is 55.1 percent lower than that of embryo day 33, the difference is obvious (p is less than or equal to 0.05), the enrichment amount of RASGRP1 in embryo day 90 is 91.4 percent lower than that of embryo day 33, the difference is extremely obvious (p is less than or equal to 0.01), the enrichment amount of RASGRP1 in embryo day 90 is 81.2 percent lower than that of embryo day 65, and the difference is obvious (p is less than or equal to 0.05); .

Example four RT-PCR validation of differentially expressed Gene RASGRP1

1. The quantitative PCR primers for the designed gene and the reference gene β -actin on the NCBI (national Center for Biotechnology information) website are shown in Table 4. The cDNA obtained by reverse transcription was diluted to the same concentration, an amplification system was prepared according to Table 5, 3 biological replicates per sample, and then Real-time quantitative fluorescent PCR was performed using an EcoTM Real-time PCR instrument of illumina, under the reaction conditions: 95 ℃ for 5 min; at 95 ℃, 10s, 60 ℃, 15s, 72 ℃, 20s, 40-50 cycles; 95 ℃ for 15 s; 60 ℃ for 15 s; 95 ℃ for 15 s.

TABLE 4 RT-qPCR primers

TABLE 5 RT-qPCR reaction System

2. Beta-actin is used as an internal reference, the relative expression quantity of RASGRP1 genes in skeletal muscle of pigs at different periods (embryo day 33, embryo day 65 and embryo day 90) is detected by real-time PCR, and the average Ct value of RASGRP1 genes in the embryo day 65 is used as a reference and is 2^-△△CtThe method calculates the relative expression of the genes and plots the results.

As shown in FIG. 4, RT-qPCR results show that the RASGRP1 gene expression level is 155.4% higher than that of 33 days of embryos at 65 days of embryos, the difference is very obvious (p is less than or equal to 0.01), the RASGRP1 gene expression level is 430.4% higher than that of 33 days of embryos at 90 days of embryos, the difference is very obvious (p is less than or equal to 0.01), and the RASGRP1 gene expression level is 107.7% higher than that of 65 days of embryos at 90 days of embryos, and the difference is very obvious (p is less than or equal to 0.01).

EXAMPLE cloning of the five porcine RASGRP1 Gene

Total RNA, TaKaRa PrimeScript, was extracted from the longissimus dorsi tissue of Duroc pigs using the Total RNA kit II from OMEGA^TMThe RT reagent Kit with gDNA Eraser Kit carries out reverse transcription (the specific operation method refers to the Kit instruction) to obtain the cDNA of the pig RASGRP1 gene.

(1) Primer design

The CDS region sequence of the porcine RASGRP1 Gene (Gene ID: 100522721) was searched at the NCBI website of national institute of Biotechnology (http:// www.ncbi.nlm.nih.gov /), and homologous recombination primers were designed using the In-Fusion Cloning primer design tool of TaKaRa (primer sequences are shown In Table 6).

TABLE 6 amplification primers for CDS of porcine RASGRP1 gene

Note: f represents the upstream primer, R represents the downstream primer, and the underlined parts represent the homologous recombinase ligation sites.

(2) PCR amplification reaction

cDNA qualified in quality detection is used as a template, CDS of the RASGRP1 gene of the pig is specifically amplified by high-fidelity enzyme, and a specific PCR reaction system is shown in a table 7; the reaction conditions are as follows: 95 ℃ for 4 min; at 95 ℃, 30s, 58 ℃, 30s, 72 ℃, 1min, 35 cycles; 72 ℃ for 5 min; 4 ℃ for 10 min.

TABLE 7 PCR reaction System

The PCR amplification product was detected by 1.5% agarose Gel electrophoresis, purified and recovered using Gel extraction kit from Omega, and stored in a refrigerator at-20 ℃ for further use.

EXAMPLE construction of six overexpression vectors pcDNA3.1-RASGRP1

(1) Carrying out double enzyme digestion on the pcDNA3.1(+) eukaryotic expression vector by using BamHI and EcoRI, wherein a specific reaction system is shown in Table 8;

TABLE 8 pcDNA3.1(+) double enzyme digestion reaction System

The fragment was digested at 37 ℃ for 2 hours, followed by electrophoresis on a 1.5% agarose gel to detect the cleavage and recover the desired fragment.

(2) The recovered fragments (including the target fragment and the digested pcDNA3.1(+) vector) were prepared according to the reaction system of Table 9, and reacted at 50 ℃ for 15min (the procedure of this step was performed according to Takara In-Fusion HD Cloning kits);

TABLE 9 In-Fusion Cloning reaction System

(3) The recombinant ligation product is transformed by adopting Trans5 alpha competent cells of Beijing Quanyujin biology company, and the specific operation steps are as follows:

taking 50 mu L of competent cells melted on ice bath, adding the target DNA, gently mixing uniformly, and placing in the ice bath for 30 min;

② heat shock is carried out for 45s in 42 ℃ water bath, then the heat shock is rapidly transferred into an ice bath for 2min, and the process does not need to shake a centrifuge tube;

③ adding 500 mu L of sterile LB culture medium (without antibiotic) into the centrifuge tube, mixing uniformly, putting into a shaker at 37 ℃ and 200rpm for resuscitation for 1 h;

fourthly, according to the experimental requirements, sucking a proper volume of transformed competent cells, adding the competent cells to an LB agar culture medium containing the aminobenzyl resistance, and uniformly spreading the cells;

fifthly, placing the flat plate in an incubator at 37 ℃ for positive culture for 30min, inverting the flat plate and culturing overnight. Then, monoclonal colonies are picked from the resistant plate in a clean bench by using a sterilizing gun head to be placed into 500 mu L of LB liquid culture medium containing ampicillin and are placed in a constant-temperature shaking table at 37 ℃ for amplification culture for 4-6h, 1 mu L of cultured bacterial liquid is taken as a template, PCR amplification is carried out by using specific primers (shown in table 6), agarose gel electrophoresis detection is carried out, and sequencing is carried out after positive clones are determined. And comparing the sequencing result by using BLASTn in the NCBI website to identify whether the sequence is correct. The endotoxin-removed Plasmid was extracted according to the instruction of the Endo-free Plasmid Mini Kit II Kit of OMGEA, and the extracted Plasmid was identified by double digestion with BamH I and EcoRI, the digestion system is shown in Table 10, the digestion result is shown in FIG. 6, the construction of the overexpression vector pcDNA3.1-RASGRP1 (nucleotide sequence shown in SEQ ID NO: 1) was completed, and the vector was placed in a refrigerator at-20 ℃ for further use.

TABLE 10 cloning vector double digestion

Example effects of seven pig RASGRP1 Gene overexpression vector pcDNA3.1-RASGRP1 in C2C12 myoblast proliferation

(1) Cell transfection of recombinant plasmids

One day before transfection, well-formed and vigorously growing C2C12 cells were inoculated into a cell culture dish at 37 ℃ with 5% CO₂Culturing in an incubator, and transfecting when the density of the cells reaches 60-70%;

② diluting the Lipofectamine TM 3000 reagent by using Opti-DEETM culture medium, fully mixing uniformly, standing for 5min at room temperature;

③ using Opti-DEMETM culture medium to dilute the recombinant plasmid to prepare a premixed solution, and then adding P3000^TMMixing the reagents, and standing at room temperature for 5 min;

fourthly, mixing the liquid 1 in the third step: 1, uniformly mixing, and incubating for 15min at room temperature;

fifthly, adding the mixed liquid in the fourth step into the cell culture hole, shaking up gently, placing at 37 ℃ and 5% CO₂Culturing in an incubator;

sixthly, after culturing for 4-6h, removing the transfection mixed solution by suction, and adding a complete culture medium for continuous culture.

(2) EdU cell proliferation assay

Before the experiment, the C2C12 cells are inoculated into a 96-well plate at the number of 5 multiplied by 103 per well and cultured in an incubator at 37 ℃ and 5% CO2 for 24 hours, then the recombinant plasmids are transfected, and the detection of cell proliferation imaging is carried out by utilizing an EdU kit of Guangzhou Ruibo biology company 24 hours after the transfection, and the specific operation steps are as follows:

the method comprises the following steps of using a cell complete culture medium according to the volume ratio of 1000: 1 (reagent A) to prepare an appropriate amount of 50. mu.M EdU medium;

adding 100 mu L of 50 mu M EdU into each hole, incubating for 2h in an incubator based on 5% CO2 at 37 ℃, and removing the culture medium;

③ the cells are cleaned by DPBS for 1 to 2 times, 5min each time;

adding 50 mu L of 4% paraformaldehyde stationary liquid into each hole, incubating for 30min at room temperature, and removing the stationary liquid;

fifthly, adding 50 mu L of 2mg/mL glycine into each hole, incubating for 5min by a decoloring shaker, and discarding the glycine solution;

sixthly, 100 mu of LDPBS is added into each hole, the decoloration shaking table is cleaned for 5min, and the DPBS is removed;

seventhly, adding 100 mu L of penetrating agent (DPBS containing 0.5 percent of TritonX-100) into each hole, decoloring and shaking the holes for incubation for 10min, and washing the DPBS for 5 min;

adding 100 mu L of 1 XApollo dyeing reaction solution into each hole, carrying out shaking table incubation for 30min in a dark room temperature, and discarding the dyeing reaction solution;

ninthly, adding 100 mu L of penetrant to decolor and shake the table for cleaning for 2-3 times, and removing the penetrant after 10min each time;

ddH for R₂O is as follows 100: 1, preparing a proper amount of 1 × Hoechst33342 reaction solution, and storing in a dark place;

adding 100 μ L of 1 × Hoechst33342 reaction solution into each well, decolorizing and shaking table for 30min in dark at room temperature, and discarding the staining reaction solution;

and adding 100 mu L of DPBS into each hole, washing for 1-3 times, and performing imaging detection by using a fluorescence microscope.

The results are shown in FIG. 6, the positive rate of EdU of the cells in the pcDNA3.1-RASGRP1 transfected group is higher than that in the control group, which indicates that the proliferation of C2C12 myoblasts is promoted by up-regulating the expression of RASGRP1 gene.

Example eight overexpression of the RASGRP1 Gene to promote skeletal muscle development

1) The development and yield of skeletal muscle of pigs are promoted by preparing transgenic pigs over-expressing RASGRP1 gene

Transfecting a pig fibroblast with the over-expression vector pcDNA3.1-RASGRP1, screening a positive cell line of the over-expression RASGRP1 gene, and taking the positive cell line as a donor cell for nuclear transplantation to perform somatic cloning.

Comparing the longissimus dorsi of 33-day, 65-day and 90-day transgenic cloned embryos with non-transgenic cloned embryos of the same age, the RASGRP1 gene expression of the transgenic cloned embryos is remarkably increased, and the mature muscle fiber number of the transgenic cloned embryos is remarkably increased.

Meanwhile, after birth, when the obtained transgenic pig over-expressing RASGRP1 gene is slaughtered, compared with non-transgenic pig of the same age, skeletal muscle development is better and muscle mass is higher.

Therefore, the over-expression RASGRP1 gene can improve the development of skeletal muscle of pigs, improve the muscle content of the pigs, is beneficial to culturing lean type pig strains and can improve the pork quality through breeding.

2) The pig skeletal muscle development and yield are improved by microinjecting the overexpression vector pcDNA3.1-RASGRP1 into the fertilized ovum/blastocyst

Injecting 10pL of pcDNA3.1-RASGRP1 with the concentration of 20nM into fertilized ovum/blastocyst fertilized in vitro of pig, and then transplanting the treated embryo into the uterus of surrogate pregnant sow.

Comparing the treated embryos of 33 days, 65 days and 90 days with untreated embryos of the same age, the RASGRP1 gene expression of the embryos developed from the treated fertilized eggs/blastocysts is remarkably increased, and the number of mature muscle fibers of the embryos developed from the treated fertilized eggs/blastocysts is remarkably increased.

Meanwhile, the fertilized eggs/blastocysts treated by the method are transplanted to the uterus of a surrogate sow until the piglet is born, and the skeletal muscle development and the muscle mass are better and higher when the piglet is slaughtered compared with the untreated pig.

Therefore, the over-expression RASGRP1 gene can improve the development of skeletal muscle of pigs, improve the muscle content of the pigs, is beneficial to culturing lean type pig strains and can improve the pork quality through breeding.

3) The overexpression vector pcDNA3.1-RASGRP1 is used for treating the boar semen to improve the development and the yield of the boar skeletal muscle.

20nM of the overexpression vector pcDNA3.1-RASGRP1 was added to the semen and incubated for half an hour and the treated semen was subjected to in vitro fertilization of pigs.

Comparing the 33-day, 65-day and 90-day dorsominal longissimus of the processed embryo with the untreated embryo at the same age, the RASGRP1 gene expression of the processed embryo is obviously increased, and the mature muscle fiber number of the processed embryo is obviously increased.

Meanwhile, after the treated piglets are born, the skeletal muscle development is better and the muscle mass is higher when the piglets are slaughtered compared with untreated pigs.

Therefore, semen is treated by the overexpression vector pcDNA3.1-RASGRP1 to promote RASGRP1 gene expression in embryo, so that the development of skeletal muscle of the pig can be improved, the muscle content of the pig can be improved, the lean type pig strain can be cultured, and the pork quality can be improved by breeding.

In other embodiments, animals overexpressing the RASGRP1 gene to increase skeletal muscle development and yield include, but are not limited to, swine, but may also be other animals such as cattle, sheep, mice, and the like.

What has been described above are merely some embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made without departing from the inventive concept herein, and it is intended to cover all such modifications and variations as fall within the scope of the invention.

Sequence listing

<110> southern China university of agriculture

<120> methods and uses for modulating skeletal muscle development

<130> 20200727

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7805

<212> DNA

<213> Sus scrofa

<400> 1

gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta ccgagctcgg atccatgggc accctgggaa agacaaggga 960

ggccccaaga aagccaagcc acggctccag ggctgctcct aagaccagac tggaggctaa 1020

gccagctagc tccccatgcc cttctcaccc tagcctggct cagatcacac agtttaggat 1080

gatggtgtcc ctgggacacc tggctaaggg agcttctctg gacgatctga tcgatagctg 1140

cgtgcagtcc ttcgacgccg atggaaacct gtgcaggagc aaccagctgc tgcaggtcat 1200

gctgaccatg cacagaatcc tgatctctag cgccgagctg ctgcagaaag tgatcaccct 1260

gtacaaggat gccctggcta agaactcccc aggcctgtgc ctgaagatct gttactttgt 1320

gcggtactgg atcaccgagt tttggatcat gttcaagatg gacaccagcc tggcccacac 1380

aatggaggag ttccaggagc tggtgaaggc taacggagag gagctgcact cccgcctgat 1440

cgataccaca cagatcaact ctagggactg gagcaggaag ctgacccagc ggatcaagtc 1500

caacacatct aagaagcgca aggtgagcct gctgtttgat cacctggagc cagaggagct 1560

gtctgagcac ctgacctacc tggagtttaa gagcttcagg agaatcagct tctccgacta 1620

ccagaactac ctggtgaact cctgcgtgaa ggagaacccc accatggaga gatctatcgc 1680

cctgtgcaac ggcatcagcc agtgggtgca gctgatggtg ctgtctaggc caacaccaca 1740

gctgagggct gaggtgttta tcaagttcat ccaggtggct cagaagctgc accagctgca 1800

gaactttaac accctgatgg ctgtgatcgg cggactgtgc cactcctcta tcagcaggct 1860

gaaggagacc agctcccacg tgccacacga gatcaacaag gtgctgggcg agatgacaga 1920

gctgctgtct agctgcagaa actacgacaa ctaccggcgc gcctacggcg agtgtacaca 1980

cttcaagatc cccatcctgg gagtgcacct gaaggatctg atctccctgt acgaggccat 2040

gcctgactac ctggaggagg gaaaggtgaa cgtgcacaag ctgctggctc tgtacaacca 2100

catcaacgag ctggtgcagc tgcaggaggt ggctccacct ctggaggcta acaaggatct 2160

ggtgcacctg ctgaccctga gcctggatct gtactacaca gaggacgaga tctacgagct 2220

gtcctacgct agggagccaa ggaaccacaa ggctccacca ctgaccccaa gcaagcctcc 2280

agtggtggtg gattgggcta gcggcgtgtc ccctaagcca gaccccaaga caatcagcaa 2340

gcacgtgcag cggatggtgg actccgtgtt taagaactac gaccacgatc aggacggcta 2400

catctcccag gaggagttcg agaagatcgc cgcttccttc cccttttctt tctgcgtgat 2460

ggataaggac agggagggac tgatctctag agatgagatc accgcctact tcatgcgcgc 2520

ttcctctatc tacagcaagc tgggcctggg atttcctcac aacttccagg agaccacata 2580

cctgaagcca acattttgcg acaactgtgc cggcttcctg tggggagtga tcaagcaggg 2640

ctaccggtgc aaggattgtg gaatgaactg ccacaagcag tgtaaggacc tggtggtgtt 2700

cgagtgcaag aagcgcgcca agaactccac cgctcctaca gagaactcta ccagcgtggg 2760

ccctacacca aacctgtgct ctctgggagc caaggatctg ctgcacgctc ctgaggaggg 2820

ccccttcacc ttccccaacg gagaggccgt ggagcactct gaggagagca aggacaggac 2880

aatcatgctg atgggcgtga gctcccagaa gatctctgtg aggctgaaga gaaccgtggc 2940

ccacaaggct acccagacag agtccctgtc ttggctgtct agcgagggcc ccagcggaca 3000

ctttgtgctg tcctctccta gaaagaccgc ccaggacaca ctgtacgtgc tgccctcccc 3060

tacatctcca tgcccctccc ctgtgctggt gaggaagaga gctttcgtga agtgggagaa 3120

caaggagagc ctgatcaagt ccaaggagga gctgaggcac ctgaggctgc caacctacca 3180

ggagctggag caggagatca acacactgaa ggccgataac aacgctctga agatccagct 3240

gaagtacgcc cagaagaaga tcgagaccct gcagctggct cggtccaacc acgtgctggc 3300

tcagatggag cacggcgact gttcttgaga attctgcaga tatccagcac agtggcggcc 3360

gctcgagtct agagggcccg tttaaacccg ctgatcagcc tcgactgtgc cttctagttg 3420

ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 3480

cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 3540

tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 3600

gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc 3660

tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 3720

gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 3780

ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 3840

agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 3900

ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 3960

gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 4020

ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 4080

ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag 4140

tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 4200

aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 4260

tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 4320

tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 4380

gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 4440

tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga 4500

ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 4560

gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 4620

ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 4680

ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 4740

tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 4800

gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 4860

gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 4920

gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 4980

gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 5040

cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 5100

atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 5160

cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 5220

gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 5280

tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag 5340

cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg 5400

gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc 5460

tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca 5520

atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 5580

ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg 5640

cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 5700

acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 5760

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 5820

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 5880

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 5940

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 6000

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 6060

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 6120

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 6180

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 6240

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 6300

gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 6360

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 6420

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 6480

gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 6540

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt 6600

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 6660

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 6720

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 6780

gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 6840

cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 6900

cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 6960

caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7020

gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7080

gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7140

cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7200

catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7260

gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7320

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7380

gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7440

cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7500

tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7560

gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 7620

atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 7680

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 7740

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 7800

acgtc 7805

Claims (7)

1. A method of modulating skeletal muscle development in a pig, wherein the method comprises overexpressing the RASGRP1 gene at the embryonic level to promote skeletal muscle development in a pig, the method not involving diagnosis or treatment of disease.

2. The method of claim 1, wherein the method comprises preparing a transgenic pig overexpressing the RASGRP1 gene to promote skeletal muscle development in pigs.

3. The method according to claim 1, wherein the method comprises microinjecting RASGRP1 gene overexpression vector into fertilized eggs, transplanting the fertilized eggs into the uterus of surrogate pigs, and obtaining the RASGRP1 gene-overexpressed pigs to promote the development of skeletal muscles of the pigs.

4. The method according to claim 1, wherein the method comprises microinjecting RASGRP1 gene overexpression vector into blastocyst, transplanting the vector into the uterus of a surrogate pig, and obtaining the RASGRP1 gene overexpression pig for promoting the development of skeletal muscle of the pig.

5. The method according to claim 1, wherein the method comprises artificial insemination of pigs overexpressing the RASGRP1 gene after co-incubation of the RASGRP1 gene overexpression vector with pig semen to promote skeletal muscle development in pigs.

6. The method according to any one of claims 3-5, wherein the nucleotide sequence of the RASGRP1 gene overexpression vector is the nucleotide sequence shown in SEQ ID NO. 1.

7. Use of a method for modulating skeletal muscle development in a lean pig, wherein said use is effected by the method of any one of claims 1-5.

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