CN116179561B - Application of Braket black cattle skeletal muscle differentiation regulation gene LOC112441863 - Google Patents

Abstract

The invention belongs to the technical field of biological breeding, and particularly relates to application of a skeletal muscle differentiation regulating gene LOC112441863 of Bulkhead black cattle. The Brachite black cattle gene LOC112441863 has the functions of regulating proliferation, differentiation and expression of bovine skeletal muscle cells and/or promoting generation of type II muscle fibers of cattle. Can be used for regulating proliferation and differentiation expression of bovine skeletal muscle cells and promoting generation of bovine type II myofibers. Experiments prove that LOC112441863 can regulate proliferation and growth of skeletal muscle of Bulkhead black cattle and can also regulate type II myofiber generation. LOC112441863 can influence proliferation and growth of muscle cells of the Brakeat black cattle, thereby influencing growth of meat quality, and individuals with high LOC112441863 content should be selected as beef cattle with high meat yield in breeding, so that a new direction is provided for breeding of high-quality Brakeat beef cattle.

Description

Application of Braket black cattle skeletal muscle differentiation regulation gene LOC112441863

Technical Field

The invention belongs to the technical field of biological breeding, and particularly relates to application of a skeletal muscle differentiation regulating gene LOC112441863 of Bulkhead black cattle.

Background

In livestock production, the meat producing performance of animals is an important economic indicator, and activation, proliferation, migration, arrangement, fusion and differentiation of skeletal muscle satellite cells to form contractile and beating multinuclear myotubes is critical for myogenesis. Therefore, research on the muscle growth and development regulation network from different layers plays a very important guiding role in understanding and improving the meat production performance of animals.

The Bulkhead black cattle is a new germplasm of high-quality beef cattle, and is a good germplasm resource obtained by improving local Ruxi yellow cattle and Bohai sea black cattle through combining modern biotechnology such as somatic cell cloning and the like with a conventional breeding hybridization method. The hybrid germplasm fully shows hybrid vigor, maintains beef quality characteristics and partial appearance characteristics, and obtains physiological characteristics of Luxi cattle and Bohai black cattle adapting to local environment and geographic conditions. The beef has the characteristics of good meat quality, typical marble patterns, also called snowflake meat, tenderness and succulent, contains rich proteins, has amino acid composition which is more similar to the needs of human bodies than pork, can improve the disease resistance of organisms, has the functions of nourishing cardiac muscles and enhancing resistance, contains rich iron, has very low content of saturated fatty acid in intramuscular fat, has high content of unsaturated fatty acid, has unique flavor, fresh and tender meat quality and good taste, contains rich trace elements and CoQ10, and is a new high-quality beef germplasm which is not available in beef germplasm resources in China.

Long non-coding RNA, abbreviated as lncRNA, refers to non-coding RNA that is greater than 200 nucleotides in length. LncRNA has very important regulatory functions, and almost participates in various biological processes and pathways, and is closely related to the occurrence and development of various diseases, so that the LncRNA becomes a research hot spot and an important point in the past few years and in the future. Several muscle-specific lncRNAs that control muscle gene expression have been reported to date, including muscle-specific linc-MD1. Recently, a LncRNA-FKBP1C has been reported to regulate muscle growth by inhibiting DNMT3B during myogenic differentiation and muscle regeneration. In bovine myoblasts LncYYW positively regulate the expression of the growth hormone 1 (GH 1) gene and its downstream genes AKT1 and PIK3 CD. Linc-MD1 proved to be competitive endogenous RNA for miR-133 and miR-135 targets during myoblast differentiation, and these transcription factors activate muscle-specific gene expression, thereby controlling the time of muscle differentiation.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides an application of LOC112441863 in regulating proliferation and differentiation expression of skeletal muscle cells of Bullet black cattle, and LOC112441863 can regulate proliferation and growth of skeletal muscle of Bullet black cattle and can regulate type II myofiber generation.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

An application of LOC112441863 in the breeding of Brakeet black cattle.

A briKate black bovine gene LOC112441863, said briKate black bovine gene LOC112441863 having a function of regulating proliferation and differentiation expression of bovine skeletal muscle cells and/or a function of promoting bovine type ii myofiber production.

The application of the Brachiensis gene LOC112441863 in regulating proliferation and differentiation expression of bovine skeletal muscle cells; the Braket black bovine gene LOC112441863 can promote proliferation of bovine skeletal muscle cells and inhibit differentiation of bovine skeletal muscle cells.

The Brachiensis gene LOC112441863 is applied to promoting the generation of type II muscle fibers of cattle.

Further, the method of operation of the application is to transfect a vector containing the LOC112441863 mimetic or inhibitor into a target cell.

Further, the vectors are lentiviruses, adenoviruses, adeno-associated viruses, liposomes and plasmids for constructing viruses.

Further, the target cell is selected from somatic cells, fertilized eggs, cell lines of Bullebrand black cattle.

The application is to screen individuals with high LOC112441863 content as beef cattle with high meat yield.

An application of LOC112441863 in regulating proliferation and differentiation of skeletal muscle cells of Bullet black cow is provided.

Advantageous effects

Experiments prove that LOC112441863 can regulate proliferation and growth of skeletal muscle of Bulkhead black cattle and can also regulate type II myofiber generation. LOC112441863 can influence proliferation and growth of muscle cells of the Brakeat black cattle, thereby influencing growth of meat quality, and individuals with high LOC112441863 content should be selected as beef cattle with high meat yield in breeding, so that a new direction is provided for breeding of high-quality Brakeat beef cattle.

Drawings

Fig. 1 is a graph of the histological properties of the muscle fibers of brisket black cattle and ruxi yellow cattle, wherein fig. 1A and 1B are a graph of the fluorescence staining of the transverse and longitudinal cut fast and slow muscles of the muscle fibers (brisket black cattle on top 2 and ruxiyellow cattle on bottom 2) and statistical analysis of the area of the fast/slow muscle fibers, the total area of the fast/muscle fibers, and the total area of the slow/muscle fibers. Fig. 1C and 1D are graphs of the transverse and longitudinal HE staining of muscle fibers (left 2 is briKate black cattle, right 2 is Ruxi yellow cattle) and statistical analysis of the area, diameter, length, density and number of muscle fibers.

FIG. 2 is a LOC112441863 screen, FIG. 2A is a Wen diagram of the final 5810 lncRNAs dataset using four analysis methods (CNCI, CPC, PFAM, CPAT) to determine non-coding transcripts; FIG. 2B is a diagram showing schematically the distribution of differential lncRNA and differential gene Circos; FIGS. 2C and 2D are LOC112441863 and meat quality trait association analysis;

FIG. 3 is an analysis of LOC112441863 expression in different tissues of Bulkhead black cattle and Ruxi yellow cattle;

FIG. 4 is a lentiviral vector;

FIG. 5 shows the efficiency of LOC112441863 adenovirus in infecting cells;

FIG. 6 shows the induction of differentiation of Bullechet black cow myoblasts;

FIG. 7 is an EDU cell proliferation assay;

FIG. 8 shows the detection of a myoblast proliferation differentiation marker gene;

FIG. 9 is a graph showing the effect of LOC112441863 on type II muscle fiber of Bulkhead black cattle, and FIGS. 9A and 9B are type II muscle fiber marker gene assays; FIGS. 9C and 9D are LOC112441863 expression level and marker gene correlation analysis.

Detailed Description

The present invention will be further described with reference to examples and drawings, but the present invention is not limited to the examples.

EXAMPLE 1 measurement of myofiber histological Properties

As a result of performing fast/slow myofluorescence staining on muscle fibers of brisket black cattle and ruxol cattle, as shown in fig. 1A, B, it was found that the ratio of the number of muscle fibers of the fast muscle (red representing fast muscle fiber (type 2))/slow muscle (green fluorescence is slow muscle fiber (type 1)) of brisket black cattle was extremely lower than the ratio data (P < 0.01) of ruxol cattle, and it can be seen from the picture: the Braikite black cattle have more slow muscle fibers and the Ruxi yellow cattle have more fast muscle fibers. There was no significant difference (P > 0.05) in the index of the fast/total muscle fiber area ratio and slow/total muscle fiber area ratio.

According to the results of the dyeing of the muscle fibers HE of the brisket black cattle and the luxi yellow cattle in fig. 1C and 1D, we find that the shape of the muscle fibers of the brisket black cattle is an irregular polygon and the shape of the muscle fibers of the luxiyellow cattle is an irregular ellipse when the muscle fibers are transected; when the muscle fibers are slit, the intramuscular fat of the Braiki black cattle is more than that of the Ruxi cattle. When the HE staining chart is analyzed by Image J software, the average area of the muscle fiber of the Brachikungunya cattle is found to be significantly higher than that of the Brachikungunya cattle (P < 0.05), the average area of the muscle fiber of the Brachikungunya cattle reaches 5490.22 mu m ², and the average area of the muscle fiber of the Brachikungunya cattle reaches 4869.00 mu m ². The muscle fiber density of the muscle fibers of briKate black cattle is significantly higher than that of Ruxi yellow cattle (P < 0.01). There were no significant differences in the indices of myofiber diameter, length and number (P > 0.05).

Example 2LOC112441863 screening

To find lncRNAs modulated by meat quality traits, these lncRNAs have a potential causal contribution to certain specific benefits of skeletal muscle growth and development adaptation, a dataset of lncRNAs differentially expressed by the longissimus brainstorming and Lu Xihuang bovine dorsum was established according to the small RNA sequencing protocol. For the analysis of published datasets (GSM 4904154, GSM4904155, GSM4904156, GSM4904157, GSM4904158, GSM 4904159), four analysis methods (CNCI, CPC, PFAM, CPAT) were used to discriminate the non-coding transcripts as the final 5810 lncRNAs dataset (fig. 2A). Finally 480 differentially expressed lncRNAs were determined. We visually showed the distribution of the differential lncRNA and the differential gene using Circos panels (FIG. 2B), and found that the differentially expressed lncRNA was most distributed on chromosome 9. Wherein the expression level estimation and differential expression analysis employ the following methods:

(1) Expression level estimation

Gene expression levels are generally measured by how much mRNA the gene is transcribed from. The amount of RNA transcribed from each gene is regulated by various factors such as space time, and the amount of mRNA transcribed from the gene varies in individuals at different stages of growth or in different tissues. FPKM is a very effective tool for quantitatively estimating gene expression values using the RNA-Seq technique. FPKM is an abbreviation for FRAGMENTS PER Kilobase per Million MAPPED FRAGMENTS, which is calculated by:

If FPKM (A) is used as the expression quantity of the gene A, F is the number of Fragments which are uniquely compared with the gene A, N is the total number of Fragments which are uniquely compared with the reference gene, L is the length of the exon region of the gene A, the FPKM method can eliminate the influence of the difference of the gene length and the sequencing quantity on the calculated gene expression, and the calculated gene expression quantity can be directly used for comparing the gene expression difference among different samples.

(2) Differential expression analysis

For experiments with biological replicates we used DEseq for differential expression analysis and DEGseq for samples without biological replicates. Comparing the treatment group with the reference group, and selecting genes with |log ₂ ratio|not less than 1 and q <0.05 as obvious differential expression screening conditions to obtain 480 differential expression lncRNAs.

With the identification of a panel of lncRNA, we sought to elucidate the potential biological functions of lncRNA in muscle tissue. We identified co-expression modules from transcriptome data. A total of 16 co-expression modules were obtained and 16 colors were assigned for visual differentiation (fig. 2C). In order to evaluate the correlation between each module and 12 meat quality traits (Type I muscle fiber,TypeⅡmuscle fiber,skeletal muscle area(SMA),muscle fiber diameters(MFD),fascicle length,fiber density,muscle fibre number(MFN),Body weight,Body height,dressing percentage,Ribeye weight,ribeye area(RA)), we performed a correlation analysis between the module and the meat quality traits, the correlation analysis adopts a weighted gene co-expression analysis flow, which specifically includes the steps of:

(1) Input data preparation

Reading fpkm the expression matrix, and performing log ₂ (x+1) transformation; and finally, transposing the matrix to form a behavior sample and a column in the form of genes.

(2) Judging the data quality, and drawing a systematic clustering tree of samples

Checking the data quality of the sample and the gene, and removing low-quality data; drawing a systematic cluster tree of the sample, and removing if a significant outlier exists; and a PCA diagram can be also made to check the distribution condition of the sample.

(3) Selecting an optimal threshold Power

Selection criteria: r ² >0.8, slope≡1, select the power value at the inflection point of the R ² vs. power graph.

(4) Construction of weighted co-expression network (one-step method and fractional method) and identification of gene modules

(5) Association of gene modules and phenotypes: module and phenotype association heatmaps, module and phenotype association boxplot, gene and module, phenotype association scattergrams.

(6) WGCNA map, module correlation display

WGCNA's map TOMplot/Network heapmap plot, which depicts a Topological Overlap Matrix (TOM) between all genes in the analysis, with deeper colors indicating greater correlation between genes;

Eigengene-adjacency-heatmap shows the correlation between gene modules. The "black", "ping", "royalblue" modules with higher correlation to phenotype were chosen for GO analysis.

We found that modules 5 and 16 correlated significantly positively (correlation >0.90, p < 0.001) with SMA, FASCICLE LENGTH and MFN, respectively (fig. 2D). These results indicate that the modules exhibit specific expression of the meat quality trait. When we rank these lncRNAs according to their reactivity (magnitude of effect and statistical significance) to muscle, the lncRNAs that are most reactive to muscle tissue are identified as LOC112441863. Next, the expression level of LOC112441863 was verified on different tissues of Bullet black cattle and Russell yellow cattle, and the PCR reaction system was 25. Mu.L, and 12.5. Mu.LPremix Ex Taq II, 1. Mu.L of upper primer, 1. Mu.L of lower primer, 2. Mu.L of cDNA and 8.5. Mu.L of ddH ₂ O. The reaction procedure is: 95; total 304s0ec cycles: 95℃for 5sec and 62℃for 30sec. Three replicates were performed for each sample. As shown in FIG. 3, the result of the one-way analysis of variance using SPSS 20.0 software shows that LOC112441863 is significantly different in the dorsi longus and heart tissues of Bullet black cattle and Russell yellow cattle (P > 0.01). Table 1 shows LOC112441863 and marker gene primer information.

Table 1 qPCR primers for genes

Example 3LOC112441863 lentiviral construction

The pAdEasy-EF1-MCS-CMV-EGFP (EcoRI MCS) vector map is shown in FIG. 4, the target fragment LOC112441863 is connected with the vector after PCR amplification of the target fragment, the connection reaction solution is reacted for 30min at 50 ℃, and the reaction solution is placed on ice for 5min and immediately converted. Sequencing after bacterial liquid PCR identification, and sequencing results show that: the sequencing result was consistent with the target sequence, as shown in Table 2, and the construction of the target plasmid was successful. Cells were transfected after adenovirus vector recombination, and LOC112441863 sequence information and vector sequencing result sequence information are shown in table 2.

TABLE 2LOC112441863 sequence information and sequencing results

Gene name	Sequence information
		LOC112441863	SED ID NO：1
Vector sequencing	SED ID NO：2

Adenovirus packaging and purification: the day before transfection, 293 cells were inoculated in 60mm dishes in DMEM+10% Hyclon foetal calf serum and incubated overnight at 37℃in an incubator containing 5% CO ₂. When the cells grow to 70-80% of the basal area, taking adenovirus vector linearization plasmid which is successfully recombined, carrying out transfection by Lipofiter ^TM transfection reagent, and replacing fresh cell culture solution after 6 hours of transfection. Cells were observed daily for signs of toxicity. The toxic phenomenon is that cells become larger and round, are grape-shaped, and begin to appear as obvious plaques. And (5) taking the virus out after most of the cells are diseased and fall off from the bottom. All cells and culture medium in 60mm dishes were collected in 15ml centrifuge tubes.

The thermostat water bath was opened to 37℃and the 15ml centrifuge tube was repeatedly freeze-thawed three times in a liquid nitrogen and 37℃water bath. Centrifugation at 3000rpm for 5 min, collecting the virus-containing supernatant, and discarding the pellet. The supernatant was the first virus seed (P1) of the control virus and was used as the seed for subsequent amplification of a large number of viruses.

2Ml of cells (cell density of 90% or more) from a10 cm cell culture dish were collected from the P1 virus supernatant (about 3 ml). The remaining virus supernatant is put into a frozen tube with external rotation and reserved at-80 ℃ and is used as the virus seed.

After two days of virus amplification, all cells fall off the bottom surface to be subjected to virus collection (P2), the cells and the culture solution are taken into a 15ml centrifuge tube together, and the cells are subjected to freeze thawing for three times according to the freeze thawing method, and the supernatant is taken for next generation amplification or stored at the temperature of minus 80 ℃. The virus amplification and virus collection are repeated in each generation.

Inoculating 4×10 ⁶ 293 cells into each 75cm ² square bottle, inoculating 6 75cm ² culture bottles, culturing overnight, and when the cells grow to 90%, inoculating the P2 generation virus (except for taking a small amount of virus retaining seeds) into the culture bottles completely, observing under a microscope after 24 hours to find 60% cytopathy, and completely treating the cells after 46 hours. After harvesting the diseased cell suspension, centrifuging at 2000rpm for 5min, discarding the supernatant, adding 6ml of ST buffer (culture solution+10% serum+2.5% glycerol), mixing votex, freezing and thawing three times between liquid nitrogen and 37 ℃, centrifuging at 3000rpm for 5min, and taking the supernatant as the third generation virus (P3).

The adenovirus was purified and then quality tested, and the titer results are shown in Table 3.

TABLE 3 titer results

HBAD-EGFP overexpression control	3.16*1010PFU/mL
		HBAD-Adeasy-LOC112441863-Null-EGFP	1.99*1010PFU/mL

Example 4 effect of LOC112441863 on skeletal muscle cell differentiation and proliferation of Bullechet black cattle

And (3) taking hind limb muscles of the 5-month-old Bulleyan black cow fetus as a source to obtain cow primary cells through isolated culture. When the cells were grown to 80% confluence, in vitro myogenic induction differentiation was performed by adding DMEM medium containing 2% horse serum, the cells were collected, the cell suspension concentration was adjusted with complete medium, and the cells were divided into 6-well plates, 2×10 ⁵ cells/well, 2mL per well, and cultured in a 5% CO ₂ incubator at 37 ℃ for 24 hours, and infection blank (blank), LOC112441863 over-expression lentivirus (mimic) and LOC112441863 over-expression lentivirus negative control (NC inhibitor), and the infection efficiency was as shown in fig. 5. After 48 hours of further culture, the medium was replaced with a high-sugar DMEM medium containing 2% horse serum, and the culture was continued. After transfection, cell morphology was observed at 2d,3d,4d,5d, and cell proliferation rates were measured at 0h, 24h, 48h, 72 hEdU.

The morphology of the cells treated differently 2-5 days after transfection is shown in FIG. 6: cell fusion phenomenon starts to appear after 2 days of induced differentiation by using 2% horse serum, and compared with the blanc group, the LOC112441863mimic group has more cell fusion quantity; at 3 days, a large number of myotubes are formed by the LOC112441863mimic group and the blanc group, so that the cell length is obviously increased; at 4 days and 5 days, the LOC112441863mimic group myotubes had increased diameter and no significant difference in number compared to the blank group. LOC112441863 can be seen to promote skeletal muscle cell differentiation in briKate black cattle.

The result of Edu method for detecting the cell proliferation rate is shown in FIG. 7: LOC112441863mimic significantly reduced the number of Edu positive cells compared to the blank group (P < 0.01), and in summary, both CCK-8 and EdU results indicate that LOC112441863 inhibits the proliferation of briketjen skeletal muscle cells.

QRT-PCR was performed on the expression levels of proliferation marker genes (CDK 2, PCNA) and differentiation marker genes (MYOD 1, MYH 1) by the method of example 1, and the results are shown in FIG. 8. The results show that LOC112441863mimic significantly increases the expression level of differentiation marker gene (P < 0.01), significantly inhibits the expression level of proliferation marker gene (P < 0.01), and that LOC112441863 promotes skeletal muscle cell differentiation and inhibits proliferation.

Example 5 influence of LOC112441863 on type II muscle fiber of Bullet black cattle

Re-analysis of Hematoxylin and Eosin (HE) stained sections showed that the dorsum longus muscle of Lu Xiniu consisted mainly of type II muscle fibers. The analysis result of LOC112441863 tissue expression shows that compared with a Blacket black cow sample, LOC112441863 has higher expression amount in the longus muscle of the back of the Rumex flavus, and the Rumex flavus tissue mainly consists of type II muscle fibers, which indicates that LOC112441863 can participate in regulating type II muscle fibers.

The expression level of type II myofiber marker genes (MYL 1, MYH 4) was examined by referring to the method in example 1, as shown in FIGS. 9A, 9B. It can be seen that LOC112441863mimic increases the expression level of the marker gene very significantly (P < 0.001), and LOC112441863 is presumed to promote type ii myofibril formation. Next, correlation analysis is performed on the expression level of LOC112441863 and the expression levels of MYH4 and MYL1, as shown in fig. 9C and 9D, and the result shows that LOC112441863 is positively correlated with the expression levels of MYH4 and MYL1, and further illustrates that LOC112441863 promotes type ii myofiber generation.

Sequence listing

SED ID NO: the sequence 1 is:

CTACCACGTGTGGTCAGCTGGAGAGGGCAGTGCCCAGGGAGCTCAGAGCTGCATGCACAGTGTCCCGAGTGCCAGATCAGAAGGGTGAGGAGACGTGGATCAGGCTTCCCCGGCCATCAGACACTGGGGGTGTTGGGGGTCCCAGGAGCACCCCATACCCCAGGAAGTGCTGCTTTCTGAGGGAAAGAGCCCTTGCTGAGGAGTCCAAGGGGCTCTGGTCCCACTCAGCAGCCTGGGACCCCGGGGCCTCAGGTGTCTCTGCCGCTATCAGATCCCTCCTCCTTGGAGTCAACAAGAACCAGCGTGCAGCCAGCTCTGGGCTTCGCAGGGGTTCAGTGGTAAAGAATCTGCCTGCAATGCAGGAGACGTGGGTTCGATCCCTGGGTCGGGAAGATTCCCTGGAGAAGGAAATGGCAACCCACTGCAGTATTCTTGCCTGGAGAATCCCATGGACAGAGGAGTCCAGCGGGCTACAGTCCATGCGGTTGCAAAAGAGTTGAATGTGACTTAGCAACTGAGCACCAACAACAGCCAGTTCTGCACAGACTGTATGACCTCGGGGAAGATGCTGGGCTGTTCCCCACTGGCCCTTCCAGCTCTGTGCCCCGGAAGCTGGCCTAAAGCTGCACTGGTGGGCTCCCTCACCGTCTGGCTTCAGCTGGACGAGAGGAGAATGTGGCCTGGATGCCCTCCCTGCTGGACTGGGGCTTGGCAGTGGCAGATGTCTCTCCCAGAGGCCCTTGGCTCCTCGTGGGCAGCTGTCTCCTCCGGTTACGGCACATACCAGATCCCAGAGGCCTAGAGCGGCAACAGCCCCCTGCTTTGCAGGCTCTGGGTGCCCACCGGCCCTTGTGGGTCCCCCAACTCTGCTCACACGTCTGTACATTGTCTTTTCATCAAGCCCTTCAGCCCCTGTTCTGCATGTGTCTTCTGCCTCTGAACTGCCTGCAATATCACTGAACCCTCTTAGCTTGTTTCCCCATCCTTCTGCTGGGGACCATGATGACATCTGCCTCTAGAAGCTGGAAGAGACAAGGAAACGGATTCTTTCCTAGAGTGATGCAAAGGAACGCTGCTTTGTAGACACCTTGGTTTAGCCCAGACACTGATTTTGGAGATCTGACCTCTGGGCTGTAAGAAGATAAACTTTCGTTGTTTTAAGCCACAACGTTTGTGGCCATTTGTTACAGCGGCCATAGGATACTGCTACACGGGTCCAGGCCCCAGATAAGCAGTGACTGTAATAAATTCTGGGCTCTTCCCCAGCTCCAGCTGAACATTCCAGGAGCAGCTGCTCAGGGGCAACCTGACCGTGACCTCCCAGAACCTCAGTGTCTTCATTTTGGCCTAGGAGGCACATGTGTTTTTTTTTTTTTTCCCCTGCCAGGGTGAAATGCTGACAACAAATTATGTCTCAAGGGTTTCAGACTCCAGTGTTATCAGGAGCCTGAGTAACATGGCAAGTCGCCTGAGTGAGTGGGATGGTGTGAAGCAGTGGGGAGTGGTGGGCCTGTGGCAAACTGGAGAACTCACACCTTGCTTAAAGACATCAACTTTACGACTTCCCCAGCTTCCCTGGTGGCTCAGATGGTAAAGTGTCTGCCTGCAATGCGAGAGACCTGGGTTCGATCCCTGGGTGGAGAAGATCCCCTGGAGAAGGAAATGGAAACCCACTCCAGTACTCTTGCCTGGAAAATTCCATGGACGGAGGAGCCTGGTAGGCTACAGTCCATGGGGTTGCAAAGAGTCAGACAAGACAAAGCGAGTTTGCTTTCACTTTAGGACTTCCCTGGTGGTCCAGTGGTTGAGAATCCACCTGCCAATGGAGGGGACATGGGTTCGATCCCTGGTCAGGGAAGACCCGTGAGGCAGCTGGCCTGCGCACCACCACCACTGAGCCCACGCTCTGGAGCCTGAGCGTTTCAACAGCTGAGGTCTGCTTGCTCGAGAGCCCACACACCAAAATTAGAGAAAGCCCAGGCACAACAACAAACACCCAGCACAGCCAGATAAATAATTTTAAAAAAAGATATCCACTTTAAGTGACAGGAAGAGCAATAAATCCAGCCAAACAAAGTCAACATCTTATCTGTCCACGTGGCTTACGGACTAGGACTTTGGGATCCCATATTCACCTTCTAGTTTCACAGTGACTCCAAGGGGAGGTGCTCCACAGGGTAGGGAGAGGCCCTGAATATGATGAGGGTGAGCCTTGGCTGCCCCTTAGAGTCACCCTTCCCCTACCCCAGACCTCTACCACATCTAGCCTCTGTTTCCTAAAAGGGGATTTTGATACAGAATCTGGTGAGAGCCATTGGGCCCGCTGGCTCACTGGCATCGCCTCACCTCCATCATCCTTTCCCACCCACACAAAACTCTCACAGTGCCCCCTGGAGAAGTCGGTCCATTAGCCCTGGGGCACCACCTCTCACCTTCCTCTCCAACATGGGAGGAACTTGTGTGGAATAACCAAAAGTCTACTACTGTTGGGTCAAGCTCTAACCAATCCAAACCACAGGTTGGGGAGTAGGAGACACAGAAAAGAGGGGCACTGTCCCCCCAAAATGATGAGAACAAAGATGTCCTGGTAGAGACCATGAGTTTTGGAGTCAGACAGACCTGGGTGAGTCCCCACTTCACCACATACTAGCTGTGTGGCTTCAGGAAACATATTTGACCACTCTGAGCCTCAGTTGTCACATCTTTAAAATTGGAATAACATTAATAATACCTCTTTGCATTGTTGCTGTGAGAACTAAATCAAATAATGTATGTGAGGGGTGGTCCCTGGCCTACAGGAGGGACTCCGCTCAGACTCTGGACCCAGACAACTTGGGTTAGAGATCCCTTTAGTGGTTGCGTGACCTTGGCAAGTTACCTCATGTGGCGTTCTCATCTCACATGTGGGTTCACTGGTGTCCTCACAGGGCTGTCGGGAGGATGAGGTCAAGGGTGTGAGACCTTGCCTGGACTTCGTAAGGACTCCGTGAGCCCACGAATTATTTACTAGGTGTGTTCTGACATTGGTGGGTGTGTGTCTCCCGTACCCGGTGGGCCCTGGCATGTGTTTCTGGGATGTGAAGTCAGAGCCTGTTTCCTGGTTGTCGGGAGAGGCTGACCACATTCTACCTATACCTGCCTGTCTAGTTCCTTCTCCCGTTCCTCACCCCACCTCCAAATCTTCCACCCTCTTCATCTCCTGTCCCCCAGCTGCTGTCTGTGACTTTCCCCAGACACCTCTCTGGTAGCCCCCAAGCCTGGGGTTGCAGGGCTACTGGGCTCCGTGGTGTGCCTGGGAGGGGCCTGGGGCTGCCCCCTGAGTAGCCCCGATGTCACCGGAGAAGGCAAGCATTGCGCTGCCCTCGCTCCAACGCGTGTGCACATGCCAGGCAGTCTCGGATGAATCATGTCCACACCGTGCTATTACTAGGCTTGTAACAGAGGCCCTGTGTGCTGGCTTGGGGTATAGTGTTTCTTTCCATTCCATGATTCAGAAAACATCAGTCACTCAGCCATGTGATTCCCTCATCTTCCGCCAAGGAGCTGAGCACAGAGGTTTGCTGGGAAACCCTCAAAGGACAGGGGAGGGCAGCTGTGTGTGGTTGCCAGAAAGGGGATTGAGCTGAGAGAACTGGAAACAGGAACTGGAGAGACTGTCATCAGGAAAAGAGCATCAGGGGACTCAGGTGTGATGAGGCCAGAGCCCTGCCCACGAGAGCAGCTGCTGGCAGATGAAGGGAGCTCACTCAGCTATACACTACCCCAGCCCCAGAAAAAACAATGATTCTTGGGCCTTATTATAAATGTAAAGTTTGCTTATGCAAAATTCTGATCAGCACAGAAAGTTGTAAAAAAGAAAGGACATACCTCTCACATGTTGCTACCAGACACAGTTTTAAATTGTTGGCACACATCTCTGTGCTTGGAACTGCCAGGTTCTGGCCCTCCAAACTACTGACCAGCCGGGGTCTGATCCGGGCTCCTGCCCCAGTCCTCAGGGGACTGTCCTGCCTCAGGGGACACTGAGCTCCAAATGCCCTTGGAGATGGGCCAGGTCTGGGCTCTGGCATCTGGGTTCTAGTCCCAATTTGGTTGTGTGCCCTTAGGCAAATCACTCTGAGTCCTATTTGTGCGGTTACAAATGGAATAATAGGGACTCCCCAGATTGCCCACTGGTTAAGACTCCATGCTCCCAAGGCAGGGGGGCCTGGTTTCAATCCCCGGTCAGGGCACTGAGACCTCACATGCCAGAACTAAGACCCAGGAAAGTGAAAAAGTGAAAGTCGCTCAGTTGTGTCTGACTCTTTGTGATCCCATGGACTCTACTGTCCATGGAATTCTCCAGACCAGAACACTGGAGTGGGTAGCCTTTTCCTTCTCCAGGGGATCTTCCCAACCCTGGGATTGAACCCAGGTCTCCTGCATTGCAGGCAGAGTCTAGACCCAGGGTAGCCAATAAATAAATAAAACAAATATTTTTTAAAAATAGGATAAGAATTGGGCTGATTTGTCCAGAGGATGAAGCATATGAGGGGATATGAAAACTGTAAACCTGCAGAATCTCTTCAAACAGAAGGGGAGGGCCTGCATGATGCCCATTCTCTTGGGGAGGCTCCTTTATCTAGGGGCAGCCTTGAGCCAGCTCTCTTGCTCCACAGCAGAAACTGGTCTGCCCATGTTGACCAAAGGCCTTCCCCCAACCCCGGACTGGCTCAGAGCAGGGAAGCCTCAGTTTGGGATCCCCAGTCATGCCAGTACCACCTCTGAACACCAGTTGAGCACCAGAGTGGGCAGGCACCTGGTTCAGAAAGCTTCAAAACCTTGTTGTGTCGCCAGCTTTGCACCCAGGTAACTGTCTTCCTTTATCTCCCTCACAGATACTGTCACCCCAGCGGGGCCCGTTCTCAGAGCCATCACTGCTCAGAAGCCTCGTTTGATTTCAGGGACCTTTGCGGATGCCCCAAGCGTTGTATCAGTTTACACTGCAGTGATTTGTAGTAAATCCTTGTGTTCTTGTGGAGGGACGGTAATGACAATATAAGACCATTAGATCGGCTATTTCCAAA

SED ID NO:2 sequences are:

CGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACTCTGGTACCCTACCACGTGTGGTCAGCTGGAGAGGGCAGTGCCCAGGGAGCTCAGAGCTGCATGCACAGTGTCCCGAGTGCCAGATCAGAAGGGTGAGGAGACGTGGATCAGGCTTCCCCGGCCATCAGACACTGGGGGTGTTGGGGGTCCCAGGAGCACCCCATACCCCAGGAAGTGCTGCTTTCTGAGGGAAAGAGCCCTTGCTGAGGAGTCCAAGGGGCTCTGGTCCCACTCAGCAGCCTGGGACCCCGGGGCCTCAGGTGTCTCTGCCGCTATCAGATCCCTCCTCCTTGGAGTCAACAAGAACCAGCGTGCAGCCAGCTCTGGGCTTCGCAGGGGTTCAGTGGTAAAGAATCTGCCTGCAATGCAGGAGACGTGGGTTCGATCCCTGGGTCGGGAAGATTCCCTGGAGAAGGAAATGGCAACCCACTGCAGTATTCTTGCCTGGAGAATCCCATGGACAGAGGAGTCCAGCGGGCTACAGTCCATGCGGTTGCAAAAGAGTTGAATGTGACTTAGCAACTGAGCACCAACAACAGCCAGTTCTGCACAGACTGTATGACCTCGGGGAAGATGCTGGGCTGTTCCCCACTGGCCCTTCCAGCTCTGTGCCCCGGAAGCTGGCCTAAAGCTGCACTGGTGGGCTCCCTCACCGTCTGGCTTCAGCTGGACGAGAGGAGAATGTGGCCTGGATGCCCTCCCTGCTGGACTGGGGCTTGGCAGTGGCAGATGTCTCTCCCAGAGGCCCTTGGCTCCTCGTGGGCAGCTGTCTCCTCCGGTTACGGCACATACCAGATCCCAGAGGCCTAGAGCGGCAACAGCCCCCTGCTTTGCAGGCTCTGGGTGCCCACCGGCCCTTGTGGGTCCCCCAACTCTGCTCACACGTCTGTACATTGTCTTTTCATCAAGCCCTTCAGCCCCTGTTCTGCATGTGTCTTCTGCCTCTGAACTGCCTGCAATATCACTGAACCCTCTTAGCTTGTTTCCCCATCCTTCTGCTGGGGACCATGATGACATCTGCCTCTAGAAGCTGGAAGAGACAAGGAAACGGATTCTTTCCTAGAGTGATGCAAAGGAACGCTGCTTTGTAGACACCTTGGTTTAGCCCAGACACTGATTTTGGAGATCTGACCTCTGGGCTGTAAGAAGATAAACTTTCGTTGTTTTAAGCCACAACGTTTGTGGCCATTTGTTACAGCGGCCATAGGATACTGCTACACGGGTCCAGGCCCCAGATAAGCAGTGACTGTAATAAATTCTGGGCTCTTCCCCAGCTCCAGCTGAACATTCCAGGAGCAGCTGCTCAGGGGCAACCTGACCGTGACCTCCCAGAACCTCAGTGTCTTCATTTTGGCCTAGGAGGCACATGTGTTTTTTTTTTTTTTCCCCTGCCAGGGTGAAATGCTGACAACAAATTATGTCTCAAGGGTTTCAGACTCCAGTGTTATCAGGAGCCTGAGTAACATGGCAAGTCGCCTGAGTGAGTGGGATGGTGTGAAGCAGTGGGGAGTGGTGGGCCTGTGGCAAACTGGAGAACTCACACCTTGCTTAAAGACATCAACTTTACGACTTCCCCAGCTTCCCTGGTGGCTCAGATGGTAAAGTGTCTGCCTGCAATGCGAGAGACCTGGGTTCGATCCCTGGGTGGAGAAGATCCCCTGGAGAAGGAAATGGAAACCCACTCCAGTACTCTTGCCTGGAAAATTCCATGGACGGAGGAGCCTGGTAGGCTACAGTCCATGGGGTTGCAAAGAGTCAGACAAGACAAAGCGAGTTTGCTTTCACTTTAGGACTTCCCTGGTGGTCCAGTGGTTGAGAATCCACCTGCCAATGGAGGGGACATGGGTTCGATCCCTGGTCAGGGAAGACCCGTGAGGCAGCTGGCCTGCGCACCACCACCACTGAGCCCACGCTCTGGAGCCTGAGCGTTTCAACAGCTGAGGTCTGCTTGCTCGAGAGCCCACACACCAAAATTAGAGAAAGCCCAGGCACAACAACAAACACCCAGCACAGCCAGATAAATAATTTTAAAAAAAGATATCCACTTTAAGTGACAGGAAGAGCAATAAATCCAGCCAAACAAAGTCAACATCTTATCTGTCCACGTGGCTTACGGACTAGGACTTTGGGATCCCATATTCACCTTCTAGTTTCACAGTGACTCCAAGGGGAGGTGCTCCACAGGGTAGGGAGAGGCCCTGAATATGATGAGGGTGAGCCTTGGCTGCCCCTTAGAGTCACCCTTCCCCTACCCCAGACCTCTACCACATCTAGCCTCTGTTTCCTAAAAGGGGATTTTGATACAGAATCTGGTGAGAGCCATTGGGCCCGCTGGCTCACTGGCATCGCCTCACCTCCATCATCCTTTCCCACCCACACAAAACTCTCACAGTGCCCCCTGGAGAAGTCGGTCCATTAGCCCTGGGGCACCACCTCTCACCTTCCTCTCCAACATGGGAGGAACTTGTGTGGAATAACCAAAAGTCTACTACTGTTGGGTCAAGCTCTAACCAATCCAAACCACAGGTTGGGGAGTAGGAGACACAGAAAAGAGGGGCACTGTCCCCCCAAAATGATGAGAACAAAGATGTCCTGGTAGAGACCATGAGTTTTGGAGTCAGACAGACCTGGGTGAGTCCCCACTTCACCACATACTAGCTGTGTGGCTTCAGGAAACATATTTGACCACTCTGAGCCTCAGTTGTCACATCTTTAAAATTGGAATAACATTAATAATACCTCTTTGCATTGTTGCTGTGAGAACTAAATCAAATAATGTATGTGAGGGGTGGTCCCTGGCCTACAGGAGGGACTCCGCTCAGACTCTGGACCCAGACAACTTGGGTTAGAGATCCCTTTAGTGGTTGCGTGACCTTGGCAAGTTACCTCATGTGGCGTTCTCATCTCACATGTGGGTTCACTGGTGTCCTCACAGGGCTGTCGGGAGGATGAGGTCAAGGGTGTGAGACCTTGCCTGGACTTCGTAAGGACTCCGTGAGCCCACGAATTATTTACTAGGTGTGTTCTGACATTGGTGGGTGTGTGTCTCCCGTACCCGGTGGGCCCTGGCATGTGTTTCTGGGATGTGAAGTCAGAGCCTGTTTCCTGGTTGTCGGGAGAGGCTGACCACATTCTACCTATACCTGCCTGTCTAGTTCCTTCTCCCGTTCCTCACCCCACCTCCAAATCTTCCACCCTCTTCATCTCCTGTCCCCCAGCTGCTGTCTGTGACTTTCCCCAGACACCTCTCTGGTAGCCCCCAAGCCTGGGGTTGCAGGGCTACTGGGCTCCGTGGTGTGCCTGGGAGGGGCCTGGGGCTGCCCCCTGAGTAGCCCCGATGTCACCGGAGAAGGCAAGCATTGCGCTGCCCTCGCTCCAACGCGTGTGCACATGCCAGGCAGTCTCGGATGAATCATGTCCACACCGTGCTATTACTAGGCTTGTAACAGAGGCCCTGTGTGCTGGCTTGGGGTATAGTGTTTCTTTCCATTCCATGATTCAGAAAACATCAGTCACTCAGCCATGTGATTCCCTCATCTTCCGCCAAGGAGCTGAGCACAGAGGTTTGCTGGGAAACCCTCAAAGGACAGGGGAGGGCAGCTGTGTGTGGTTGCCAGAAAGGGGATTGAGCTGAGAGAACTGGAAACAGGAACTGGAGAGACTGTCATCAGGAAAAGAGCATCAGGGGACTCAGGTGTGATGAGGCCAGAGCCCTGCCCACGAGAGCAGCTGCTGGCAGATGAAGGGAGCTCACTCAGCTATACACTACCCCAGCCCCAGAAAAAACAATGATTCTTGGGCCTTATTATAAATGTAAAGTTTGCTTATGCAAAATTCTGATCAGCACAGAAAGTTGTAAAAAAGAAAGGACATACCTCTCACATGTTGCTACCAGACACAGTTTTAAATTGTTGGCACACATCTCTGTGCTTGGAACTGCCAGGTTCTGGCCCTCCAAACTACTGACCAGCCGGGGTCTGATCCGGGCTCCTGCCCCAGTCCTCAGGGGACTGTCCTGCCTCAGGGGACACTGAGCTCCAAATGCCCTTGGAGATGGGCCAGGTCTGGGCTCTGGCATCTGGGTTCTAGTCCCAATTTGGTTGTGTGCCCTTAGGCAAATCACTCTGAGTCCTATTTGTGCGGTTACAAATGGAATAATAGGGACTCCCCAGATTGCCCACTGGTTAAGACTCCATGCTCCCAAGGCAGGGGGGCCTGGTTTCAATCCCCGGTCAGGGCACTGAGACCTCACATGCCAGAACTAAGACCCAGGAAAGTGAAAAAGTGAAAGTCGCTCAGTTGTGTCTGACTCTTTGTGATCCCATGGACTCTACTGTCCATGGAATTCTCCAGACCAGAACACTGGAGTGGGTAGCCTTTTCCTTCTCCAGGGGATCTTCCCAACCCTGGGATTGAACCCAGGTCTCCTGCATTGCAGGCAGAGTCTAGACCCAGGGTAGCCAATAAATAAATAAAACAAATATTTTTTAAAAATAGGATAAGAATTGGGCTGATTTGTCCAGAGGATGAAGCATATGAGGGGATATGAAAACTGTAAACCTGCAGAATCTCTTCAAACAGAAGGGGAGGGCCTGCATGATGCCCATTCTCTTGGGGAGGCTCCTTTATCTAGGGGCAGCCTTGAGCCAGCTCTCTTGCTCCACAGCAGAAACTGGTCTGCCCATGTTGACCAAAGGCCTTCCCCCAACCCCGGACTGGCTCAGAGCAGGGAAGCCTCAGTTTGGGATCCCCAGTCATGCCAGTACCACCTCTGAACACCAGTTGAGCACCAGAGTGGGCAGGCACCTGGTTCAGAAAGCTTCAAAACCTTGTTGTGTCGCCAGCTTTGCACCCAGGTAACTGTCTTCCTTTATCTCCCTCACAGATACTGTCACCCCAGCGGGGCCCGTTCTCAGAGCCATCACTGCTCAGAAGCCTCGTTTGATTTCAGGGACCTTTGCGGATGCCCCAAGCGTTGTATCAGTTTACACTGCAGTGATTTGTAGTAAATCCTTGTGTTCTTGTGGAGGGACGGTAATGACAATATAAGACCATTAGATCGGCTATTTCCAAACTCGAGCCTAAGCTTCTAGATAAGATATCCGATCCACCGGATCTAGATAACTGATCATAATCAGCCATACCACATTTGTA.

Claims (2)

1. Use of the briKate black bovine gene LOC112441863 for promoting bovine type ii myofibrilia, characterized in that the use is operated by transfecting a vector comprising a LOC112441863 mimetic or inhibitor into a target cell;

the vector is lentivirus, adenovirus, adeno-associated virus, liposome or plasmid for constructing virus.

2. The use according to claim 1, wherein the target cell is a somatic cell, fertilized egg or cell line of a brisket black cow.