Methods and uses for modulating skeletal muscle development
阅读说明：本技术 调节骨骼肌发育的方法和应用 (Methods and uses for modulating skeletal muscle development ) 是由 吴珍芳 顾婷 谭宝华 周健 甘炎民 邢萍萍 乔佳鑫 严冠豪 蔡更元 李紫聪 洪林 于 2020-07-28 设计创作，主要内容包括：本发明公开了一种调节骨骼肌发育的方法,该方法包括在胚胎水平超表达RASGRP1基因。由此,不仅发现了一个在猪骨骼肌发育过程中受H3K27me3修饰作用的一个关键靶基因RASGRP1,该基因的富集水平在猪骨骼肌肌纤维形成过程中在H3K27me3的修饰作用下先上调后下调,并能够促进猪骨骼肌卫星细胞的增殖功能进而促进猪骨骼肌的发育；还可以通过超表达RASGRP1基因,来促进猪骨骼肌的发育,从而为提高猪的瘦肉率提供了一种新的方法和靶点,以及研究的新方向；再者,超表达RASGRP1基因在促进骨骼肌发育上的应用,可为瘦肉型猪的选育提供新的思路和靶点；另外,在猪的胚胎时期通过超表达RASGRP1基因,通过多种不同的方法均能达到促进骨骼肌发育的效果,具有广泛的适用性。(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.)
1. A method of modulating skeletal muscle development, wherein the method comprises overexpressing the RASGRP1 gene at the embryonic level to promote skeletal muscle development.
2. The method of claim 1, wherein the method comprises preparing a transgenic animal that overexpresses the RASGRP1 gene to promote skeletal muscle development in the animal.
3. The method as claimed in claim 1, wherein the method comprises microinjecting RASGRP1 gene overexpression vector into fertilized egg, transplanting into uterus of surrogate pregnant animal, and obtaining animal with RASGRP1 gene overexpression to promote the development of skeletal muscle of the animal.
4. The method of claim 1, wherein the method comprises microinjecting the RASGRP1 gene overexpression vector into blastocyst, transplanting into uterus of surrogate pregnant animal, and obtaining RASGRP1 gene overexpression animal to promote skeletal muscle development of the animal.
5. The method according to claim 1, wherein the method comprises artificial insemination of the animal overexpressing the RASGRP1 gene after incubating the RASGRP1 gene overexpression vector with semen to promote skeletal muscle development in the animal.
6. The method of any one of claims 2-5, wherein the animal comprises a pig.
7. The method according to any one of claims 3-5, wherein the nucleotide sequence of the RASGRP1 gene overexpression vector comprises the nucleotide sequence shown as SEQ ID NO. 1.
8. Use of a method of modulating skeletal muscle development in lean animal breeding.
9. The use according to claim 8, wherein said use is achieved by a method according to any one of claims 1-5.
10. The use of claim 8, wherein the lean animal comprises a lean pig.
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:
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.
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