Chicken skeletal muscle fast and slow muscle typing marker and application thereof

Abstract

The invention provides a chicken skeletal muscle fast and slow muscle typing marker and application thereof, the marker is MYH1E and / or MYH7B, the MYH1E is associated with a chicken skeletal muscle fast muscle feature indicator gene, and the MYH7B is associated with a chicken skeletal muscle slow muscle feature indicator gene. By analyzing an expression map of an MYH gene family, symbolic genes of chicken skeletal muscle fiber typing are deeply studied, and two genes MYH1E and MYH7B are explored and are related to fast and slow muscle typing of chicken skeletal muscles. According to the invention, potential indicative genes closely related to chicken skeletal muscle slow muscle typing are found by analyzing a transcription expression map of skeletal muscle main structural proteins, and the genes comprise TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry breeding, and in particular to chicken skeletal muscle fast and slow muscle typing markers and applications thereof. Background Art

[0002] In recent years, with the development of high-throughput sequencing technology, transcriptome data on skeletal muscle tissue from various species have been gradually improved. In mammals, skeletal muscle fiber typing has been widely studied and is mainly divided into four types (types I, IIA, IIX, and IIB, corresponding to MYH7, MYH2, MYH1, and MYH4, respectively). However, the genetic information underlying skeletal muscle fiber typing in birds is currently unclear, and further in-depth research is needed to reveal the characteristics of the genes that characterize skeletal muscle fiber typing in birds. Summary of the Invention

[0003] To fill the gap in genetic information for avian skeletal muscle fiber typing, the present invention studied the expression profile of the MYH gene family and found that the expression patterns of MYH1E and MYH7B were significantly different from those of other MYH genes, showing a 0 or 1 relationship in two independent gene expression clustering patterns, thereby providing chicken skeletal muscle fast and slow muscle typing markers and their applications.

[0004] In order to achieve the above object, the technical solution of the present invention is: In a first aspect, the present invention provides chicken skeletal muscle fast and slow muscle typing markers, wherein the markers are MYH1E and / or MYH7B, wherein MYH1E is associated with a chicken skeletal muscle fast muscle characteristic indicator gene, and MYH7B is associated with a chicken skeletal muscle slow muscle characteristic indicator gene.

[0005] Preferably, the chicken skeletal muscle slow muscle characteristic indicator genes associated with the MYH7B include TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10.

[0006] In a second aspect, the present invention provides a chicken skeletal muscle slow muscle typing marker, wherein the marker is at least one of TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10.

[0007] In a third aspect, the present invention provides use of the marker described in the first or second aspect in preparing a product for fast and slow muscle typing of chicken skeletal muscle.

[0008] In a fourth aspect, the present invention provides a product for fast and slow muscle typing of chicken skeletal muscle, which comprises the markers described in the first aspect or the second aspect.

[0009] Preferably, the product also includes necessary reagents and tools for detecting the marker.

[0010] In a fifth aspect, the present invention provides a method for fast and slow muscle typing of chicken skeletal muscle, comprising: performing fast and slow muscle typing of chicken skeletal muscle by detecting the expression of the markers described in the first aspect or the second aspect in the fast and slow muscles of chicken skeletal muscle.

[0011] Preferably, the detection method is transcriptome sequencing.

[0012] Compared with the prior art, the present invention has the following beneficial effects: By analyzing the expression profile of the MYH gene family, the present invention has conducted in-depth research on the marker genes of chicken skeletal muscle fiber typing, and discovered two genes, MYH1E and MYH7B, which are related to the fast and slow muscle typing of chicken skeletal muscle. The present invention also discovered potential indicator genes closely related to the slow muscle typing of chicken skeletal muscle by analyzing the transcriptional expression profile of the main structural proteins of skeletal muscle. These genes include TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10. Overall, the present invention provides new insights into the typing of chicken skeletal muscle fibers, reveals the importance of MYH1E and MYH7B and major structural protein genes as marker genes in regulating skeletal muscle typing, and provides strong support for further exploring the molecular mechanism of muscle fiber type regulation. This also has potential application significance for understanding the biological characteristics of chicken skeletal muscle and optimizing meat quality in the breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 This is the MYH gene family expression profile in Example 1 of the present invention.

[0014] Figure 2 This is the transcriptional expression profile of the ACT gene family in Example 1 of the present invention in skeletal muscle of different parts of chicken.

[0015] Figure 3 This is the transcriptional expression map of the TPM gene family in skeletal muscle of different parts of chicken in Example 1 of the present invention.

[0016] Figure 4 This is the transcriptional expression map of the TNN gene family in skeletal muscle of different parts of chicken in Example 1 of the present invention.

[0017] Figure 5 This is the transcriptional expression map of the MYL gene family in skeletal muscle of different parts of chicken in Example 1 of the present invention.

[0018] Figure 6This is the functional enrichment of genes highly correlated with MYH1E (Pearson's r ≥ 0.7) in Example 1 of the present invention.

[0019] Figure 7 This is the functional enrichment of genes highly correlated with MYH7B (Pearson's r ≥ 0.7) in Example 1 of the present invention.

[0020] Figure 8 This figure illustrates the fast and slow muscle typing of skeletal muscle in Example 1 of the present invention. A represents the expression pattern of the gene set highly correlated with MYH1E and MYH7B in 61-week-old skeletal muscle. Color bars represent the mean expression level of the highly correlated gene set. Blue bars on the vertical axis represent the gene set highly correlated with MYH1E, and green bars represent the gene set highly correlated with MYH7B (same as B). B represents the expression pattern of the gene set highly correlated with MYH1E and MYH7B in 80-week-old skeletal muscle.

[0021] Figure 9 ATPase staining image of soleus muscle of white broiler chicken, spatial transcriptome type I and type II UMAP map and slow muscle marker gene location map.

[0022] Figure 10 ATPase staining image of the gastrocnemius muscle of white broiler chickens, spatial transcriptome type I and type II UMAP map and slow muscle marker gene location map. DETAILED DESCRIPTION

[0023] In the description of the present invention, it should be noted that if specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0024] In the examples of the present invention, five female white-feathered broiler chickens, aged 61 and 80 weeks, were used as experimental animals. The chickens were euthanized with 2% sodium pentobarbital (25 mg / kg body weight) via intravenous injection. Skeletal muscle tissue was collected from 11 sites, including the adductor mandibularis, longissimus dorsi, quadratus lumborum, semimembranosus, anterior iliotibial lateralis, posterior iliotibial lateralis, biceps femoris, gastrocnemius, soleus, tibialis anterior, and extensor digitorum longus, for a total of 55 samples. The skeletal muscle samples were rapidly frozen in liquid nitrogen and stored in a -80°C freezer for subsequent experiments.

[0025] In an embodiment of the present invention, the method for obtaining the MYH gene family expression map is: 1. Experimental Methods 1. Total RNA extraction and quality control of skeletal muscle tissue samples (1) Experimental preparation and sample processing: Pre-cool a sterile mortar and refrigerated centrifuge. Transfer the skeletal muscle tissue sample from a -80℃ freezer to liquid nitrogen. Take an appropriate amount of sample and place it in a mortar. Add liquid nitrogen and grind it into powder. Weigh 20-30 mg of sample and transfer it to a 1.5 mL RNase-free centrifuge tube pre-filled with 1 mL Trizol reagent. Let it stand at room temperature for 10 min. (2) Add 200 μL of chloroform to the lysate, invert and mix thoroughly. After mixing, place it on ice. After standing at room temperature for 3 min, transfer the centrifuge tube to a centrifuge and centrifuge at 4℃ and 12,000×g for 15 min. After centrifugation, the sample is divided into an upper colorless aqueous phase, a middle white protein layer, and a lower organic phase. RNA is located in the upper aqueous phase. (3) Use a pipette to transfer the upper colorless aqueous phase containing RNA to a 1.5 mL RNase-free centrifuge tube. Be careful not to aspirate the materials in the middle and lower layers. Add an equal volume of isopropanol to the centrifuge tube containing the supernatant, invert thoroughly to mix, and let it stand at room temperature for 10 minutes. After standing, transfer the centrifuge tube to a centrifuge and centrifuge at 4°C, 12,000 g for 10 minutes. At this time, a white RNA precipitate can be seen at the bottom of the centrifuge tube. (4) Use a pipette to aspirate the supernatant, being careful not to aspirate the RNA precipitate at the bottom. Add 1 mL of 75% ethanol to the centrifuge tube containing the RNA precipitate and gently invert the centrifuge tube to dissolve excess organic matter. After washing, place the centrifuge tube in a centrifuge and centrifuge at 4°C, 7500 g for 5 minutes. (5) Repeat step (4) once. (6) Open the centrifuge tube and place it in a clean bench. Dry the precipitate at room temperature for about 2 minutes. Add 20 to 40 µL of DEPC water to dissolve the precipitate. Use a pipette to gently pipette to promote RNA dissolution. (7) The RNA concentration and purity were tested by NanoDrop ND-2000. The A260 / A280 ratio of qualified RNA samples was 1.9 to 2.1. Then, the integrity of RNA was tested by 1% agarose gel electrophoresis. The qualified RNA samples were stored in a -80 ℃ refrigerator for future use. Afterwards, the integrity and concentration of RNA were tested by Agilent Bioanalyzer 2100. Samples with RIN values ​​> 7 were selected for transcriptome library construction and sequencing.

[0026] 2. Construction and sequencing of transcriptome library Qualified RNA samples were used to construct a common transcriptome library at Annoroad Gene Technology (Beijing) Co., Ltd. and high-throughput sequencing was performed using the MGI sequencing platform with a sequencing read length of PE150.

[0027] 3. Quality control, genome alignment and quantification of transcriptome data After obtaining RNA-seq sequencing data, reads containing adapter contamination greater than 5 bp, reads with a quality score of Q ≤ 19 representing more than 50% of the total bases, and reads containing more than 5% N were removed to generate clean data. The clean data were then aligned to the chicken reference genome, gallus_gallus6a, using Kallisto 0.44.0 for quantitative analysis. Note: The reference genome file (Gallus_gallus.GRCg6a.dna.toplev el.fa.gz) and genome annotation file (Gallus_gallus.GRCg6a.105.gtf.gz) were obtained from the Ensembl database (http: / / ensemblgenomes.org / ). Quantification was performed to obtain gene counts and TPM values ​​(transcripts per kilobase of exon model per million mapped reads). Finally, the R package tximport was used to construct the counts matrix and the TPM matrix. The quantitative data were corrected using the Trimmed Mean of M values ​​(TMM) method to eliminate technical bias between samples. TMM correction was implemented using edgeR v3.10 in the R language.

[0028] 4. Dimensionality reduction, clustering and correlation analysis of skeletal muscle gene expression profiles The corrected data were standardized using Log2 (TPM+1), and principal component analysis (PCA) was performed using the R package stats. UMAP dimensionality reduction analysis was performed using the R package umap. Pearson correlation and Spearman correlation analyses were performed using the R basic function cor.

[0029] 5. Functional enrichment analysis of differentially expressed genes in different parts of chicken skeletal muscle The R package edgeR v3.10 was used to analyze differentially expressed genes in skeletal muscle from different sites. Differentially expressed genes with a |log2 (fold change)| ≥ 1, a P value < 0.05, and an FDR < 0.01 were screened. These chicken differentially expressed genes were converted to human genes at a 1:1 ratio and then subjected to functional enrichment analysis. Functional enrichment analysis was performed using the Metascape online tool (http: / / metasc-ape.org) using the GO (Gene Ontology) and Kyoto Encyclopedia of Genes and Genomes (KEGG) framework. Pathways with Benjamini-adjusted P values ​​≤ 0.05 were considered significantly enriched.

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] Example 1

[0032] This example studies the typing of fast and slow skeletal muscle of chickens, as follows: (1) Typing and identification of the MYH gene The myosin heavy chain encoded by the MYHC gene plays a key role in the myosin molecule. In skeletal muscle tissue, there are multiple myosin heavy chain isoforms, which are encoded by different MYHC genes. In order to further explore the signature genes of chicken skeletal muscle fiber typing, this example is based on the expression map of the MYH gene family ( Figure 1 The results showed that the expression levels of the three genes MYH1C, MYH1A, and MYH9 were relatively high in all skeletal muscle samples. This suggests that these genes may have important functions or be involved in key physiological processes in chicken skeletal muscle. Furthermore, the expression patterns of MYH1E and MYH7B differed significantly from those of other MYH genes, exhibiting either a 0 or 1 relationship in two independent clustering patterns. Therefore, these two genes may be indicative of the fast and slow muscle types of chicken skeletal muscle.

[0033] (2) Transcription profile of structural protein genes In order to further discover potential indicative genes that may contribute to the classification of fast and slow skeletal muscle in chickens, this example also examined the transcriptional expression profiles of the main structural proteins of skeletal muscle based on the corrected TPM data, which mainly include the actin family (such as ACT, TPM), the troponin family (such as TNN), and the myosin light chain family (such as MYL). Figure 2 and Figure 3 As shown in the results, among the ACT and TPM actin families, ACTA1, ACTN2, TPM1, and TPM2 are highly expressed in all chicken skeletal muscles, suggesting that they play a key role in the biological functions of chicken skeletal muscles and may be essential for maintaining muscle structure and function. At the same time, it was also found that the expression pattern of TPM3 differed between fast and slow muscles ( Figure 3 ), that is, it is highly expressed in slow muscle and lowly expressed in fast muscle. Therefore, TPM3 is a potential indicator gene for slow muscle. In the TNN troponin family ( Figure 4 ), TNNT3, TNNI2 and TNNC2 are highly expressed in all chicken skeletal muscles, while the expression patterns of TNNI1, TNNC1 and TNNT1 are different between fast and slow muscles, that is, they are highly expressed in slow muscles and lowly expressed in fast muscles. Therefore, TNNI1, TNNC1 and TNNT1 are also potential indicator genes for slow muscles. Therefore, TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10 can be used as fast and slow muscle typing markers for chicken skeletal muscle fast and slow muscle typing, and can be developed into related typing products such as typing kits for practical applications. Primers for detecting the expression levels of these genes can be added to the product, for example: ATPase staining method is used to distinguish between type I and type II skeletal muscle fibers in the gastrocnemius and soleus muscles of white-feathered broilers ( Figure 9 and Figure 10 ). Then, after performing spatial transcriptome sequencing on the corresponding slices, the expression level of at least one of these genes in the spatial slices of chicken skeletal muscle can be detected, thereby determining whether the skeletal muscle belongs to fast muscle or slow muscle.

[0034] Similarly, in the transcriptional profile of the myosin light chain family (MYL) ( Figure 5 ), MYL1 and MYLPF are highly expressed in all chicken skeletal muscles, suggesting that they may play important roles in muscle structure, function, and physiology. However, the expression patterns of MYL3 and MYL10 differ between fast and slow muscles, with high expression in slow muscles and low expression in fast muscles. Therefore, it is speculated that MYL3 and MYL10 are also potential indicators of fast and slow muscle in chicken skeletal muscle.

[0035] Example 2

[0036] In order to further verify whether MYH1E and MYH7B are the marker genes for the fast and slow muscle typing of chicken skeletal muscle, the Pearson correlation coefficients of MYH1E and MYH7B with other genes were calculated using the corrected TPM data. Subsequently, genes with high correlation with MYH1E and MYH7B were screened out based on Pearson'sr ≥ 0.7. Among them, there were 116 genes with high correlation with MYH1E and 72 genes with high correlation with MYH7B. These highly correlated genes were mapped to 1:1 homologous human genes using the biomart tool in Ensembl, and metascape was used to perform functional enrichment analysis on these two highly correlated gene sets. The results are shown in Figure 2. Figure 6 As shown in the figure, most genes highly correlated with MYH1E were enriched in pathways such as carbohydrate metabolism (GO:0005975: carbohydrate metabolic process, n=1 6, LogP=-7.9), glycolysis (GO:0006096: glycolytic process, n=5, LogP =-6.6), glycogen metabolism (GO:0005977: glycogen metabolic process, n=7, LogP =-3.3), and microtubule cytoskeleton organization (GO:0000226: microtubule cytoskeleton organization, n=13, LogP=-2.7). Most genes highly correlated with MYH7B were enriched in ( Figure 7) pathways including muscle system process (GO:0003012: muscle system process, n=26, LogP=-17.2), regulation of heart contraction (GO:0008016: regulation of heart contraction, n=7, LogP=-6.8), regulation of striated muscle cell differentiation (GO:0051153: regulation of striated muscle cell differentiation, n=10, LogP=-5.3), and fatty acid biosynthetic process (GO:0006633: fatty acid biosynthetic process, n=3, LogP=-2.8). Fast twitch muscles are capable of rapid and intense contractions, primarily generating energy through anaerobic metabolism and relying more on carbohydrates for energy needs. Slow twitch muscles, on the other hand, contract at a slower pace, primarily generating energy through aerobic metabolism and relying more on oxygen and fat for energy needs. In this example, we observed that the pathways enriched in gene sets highly correlated with MYH1E and MYH7B showed a high degree of consistency with the characteristics of fast and slow twitch muscles, thus concluding that MYH1E and MYH7B are hallmark genes for the fast and slow twitch typing of chicken skeletal muscle. Specifically, MYH1E is highly correlated with fast twitch characteristics, and its highly correlated gene set is enriched in pathways associated with the "fermentative" characteristics of fast twitch muscles. MYH7B, on the other hand, is closely associated with slow twitch characteristics, and its highly correlated gene set is enriched in pathways associated with the "oxidative" characteristics of slow twitch muscles.

[0037] At the same time, the expression patterns of the MYH1E and MYH7B highly correlated gene sets were used to further identify the fast and slow muscle types of each chicken skeletal muscle site. Figure 8As shown, the longissimus dorsi, semimembranosus, adductor mandibularis, and biceps femoris all tend to be fast twitch; the soleus, tibialis anterior, and quadratus lumborum tend to be slow twitch; while the gastrocnemius and extensor digitorum longus tend to be mixed musculature. Notably, the expression patterns of the highly correlated gene sets in the anterior and posterior iliotibial lateralis muscles differ significantly, presumably due to fiber type conversion. Specifically, the anterior iliotibial lateralis muscle tends to be slow twitch at 61 weeks of age, but at 80 weeks of age, the expression of the gene set highly correlated with MYH1E increases significantly, making it more likely to be a mixed muscle. The opposite is true for the posterior iliotibial lateralis muscle: at 61 weeks of age, it tends to be fast twitch, but at 80 weeks of age, the expression of the gene set highly correlated with MYH1E decreases significantly, also making it more likely to be a mixed muscle. Therefore, MYH1E and MYH7B can be used as fast and slow muscle typing markers for chicken skeletal muscle typing. They can also be further used in combination with TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10 genes, and developed into related typing products such as typing kits for practical application. Among them, spatial transcriptome sequencing can be used to detect the expression level of MYH1E and / or MYH7B in chicken skeletal muscle, and then determine whether the skeletal muscle belongs to fast muscle or slow muscle ( Figure 9 and Figure 10 ).

[0038] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents. The embodiments described above merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent of the present invention. It should be pointed out that, without departing from the concept of the present invention, a number of variations and improvements may be made by those skilled in the art, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the appended claims.

Claims

1. A marker for fast and slow muscle typing of chicken skeletal muscle, characterized in that: The markers are MYH1E and / or MYH7B, wherein MYH1E is associated with a gene indicating fast muscle characteristics of chicken skeletal muscle, and MYH7B is associated with a gene indicating slow muscle characteristics of chicken skeletal muscle.

2. The marker according to claim 1, characterized in that Chicken skeletal muscle slow muscle characteristic indicator genes associated with the MYH7B include TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10.

3. A chicken skeletal muscle slow muscle typing marker, characterized in that: The marker is at least one of TPM3, TNNI1, TNNC1, TNNT1, MYL3 and MYL10.

4. Use of the marker according to any one of claims 1 to 3 in preparing a product for fast and slow muscle typing of chicken skeletal muscle.

5. Chicken skeletal muscle fast and slow muscle typing product, characterized in that: The product comprises the marker according to any one of claims 1 to 3.

6. The product according to claim 5, characterized in that The product also includes necessary reagents and tools for detecting the marker.

7. A method for fast and slow muscle typing of chicken skeletal muscle, characterized in that: include: Chicken skeletal muscle fast and slow muscle typing is performed by detecting the expression of the marker described in any one of claims 1 to 3 in the fast and slow muscles of chicken skeletal muscle.

8. The use according to claim 7, characterized in that The detection method is transcriptome sequencing.

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