CN117051118A

CN117051118A - Marker gene for muscle difference expression of hybrid culter No.1 under motion induction and application thereof

Info

Publication number: CN117051118A
Application number: CN202310918734.7A
Authority: CN
Inventors: 张浪; 喻亚丽; 王贵英; 李清; 何力; 甘金华; 孙艳红; 宋紫薇; 鲁晓蓉
Original assignee: Wuhan Xianfeng Aquatic Product Science & Technology Co ltd; Wuhan Academy of Agricultural Sciences; Yangtze River Fisheries Research Institute CAFS
Current assignee: Wuhan Xianfeng Aquatic Product Science & Technology Co ltd; Wuhan Academy of Agricultural Sciences; Yangtze River Fisheries Research Institute CAFS
Priority date: 2023-07-25
Filing date: 2023-07-25
Publication date: 2023-11-14
Anticipated expiration: 2043-07-25
Also published as: CN117051118B

Abstract

The application relates to a marker gene for expressing muscle difference under motion induction of hybrid culter No.1 and application thereof. The marker gene is selected from at least one of WNT2B, CDH1, MYCB, MYH1, MYH2, MYH6, MYH7, MYH15, MAVS, EPO, SKAP2, HK1, GAPDH, LDHA and PGK 1. These marker genes are identified as genes that are differentially expressed in muscle tissue of hybrid culter in both motor and non-motor states, correlating muscle traits, immune traits and/or energy metabolism traits of hybrid culter. The marker genes can be used for analyzing or identifying the muscle quality, the immunity and the energy utilization capacity of hybrid culter, and further provide important technical means support for variety breeding or improvement of hybrid culter.

Description

Marker gene for muscle difference expression of hybrid culter No.1 under motion induction and application thereof

Technical Field

The application relates to the technical field of hybrid culter muscle tissue differential expression genes, in particular to a marker gene for hybrid culter 'pioneer No. 1' muscle differential expression under motion induction and application thereof.

Background

The culter ilishaeformis belongs to carnivorous economic fishes in the genus of cyprinoid, cyprinoid and culter, has larger body type, but the artificial breeding feed has high cost, and is hard to transport in vivo due to dysphoria; the Erythroculter nigrocauda belongs to the species of Cyprinus, cypriidae and Erythroculter, and has the advantages of reduced feed cost by more than 40% compared with Erythroculter ilishaeformis, smooth temperament, easy living body transportation and smaller size. Thus, in aquaculture, researchers cultivate hybrid culter "pioneer 1" by distant hybridization of the culter ilishaeformis (female) and the erythroculter nigrocauda (male); the hybrid culter No.1 has the advantages of low cost of the culture feed (reduced by more than 40 percent compared with the culter, high growth speed, mild sex, easy living body transportation, strong stress resistance and the like, and is a novel variety of aquatic products for high-efficiency and excellent national examination in the culture industry. Nevertheless, there is still a lack of methods or means for breeding, detecting or identifying the muscle quality of the hybrid.

Disclosure of Invention

In the embodiment of the application, transcriptome sequencing is carried out by putting hybrid culter in a motion state and a non-motion state, the change of muscle transcriptome after different motion times is observed, and related histomorphological modification is studied. The research discovers a group of marker genes related to the muscle traits, the immune traits and the energy metabolism traits of the hybrid culter, and can analyze or identify the muscle quality, the immune ability and the energy utilization ability of the hybrid culter by detecting the expression quantity of the marker genes, thereby providing important technical means support for breeding the hybrid culter variety. For this purpose, the embodiment discloses at least the following technical scheme:

(1): an isolated marker genome comprising a gene identified as differentially expressed in muscle tissue of hybrid culter in a motor state and a non-motor state, the marker genome comprising: a gene WNT2B encoding WNT family member 2B protein; a gene CDH1 encoding a cadherein 1 protein; a gene MYCB encoding bHLH transcription factor b; a gene MYH1 encoding myosin heavy chain-1; a gene MYH2 encoding myosin heavy chain-2; a gene MYH6 encoding myosin heavy chain-6; a gene MYH7 encoding myosin heavy chain-7; a gene MYH15 encoding myosin heavy chain-15; a gene MAVS encoding a mitochondrial antiviral signal protein; a gene EPO encoding erythropoietin; gene SKAP2, which encodes src kinase-associated phosphoprotein 2; gene HK1, which encodes hexaphosphatase 1; a gene GAPDH encoding glycerol-3-phosphate dehydrogenase; gene LDHA, which encodes lactate dehydrogenase a; a gene PGK1 encoding phosphoglycerate kinase 1; or at least one of the specific oligonucleotides that recognize the gene.

(2): (1) Use of the marker genome in the preparation of a kit for predicting a muscle trait, an immune trait and/or an energy metabolism trait of hybrid culter by comparing the differential expression of a marker gene selected from any one of the marker genomes of (1), or for selecting a variety resource of hybrid culter based on the muscle trait, the immune trait and/or the energy metabolism trait.

(3): an RT-qPCR kit for predicting muscle traits, immune traits and/or energy metabolism traits of hybrid culters by comparing differential expression of a marker gene selected from any one of the marker genomes described in (1), or for selecting variety resources of hybrid culters according to the muscle traits, the immune traits and/or the energy metabolism traits.

Drawings

Fig. 1 is a schematic diagram of a short-term micro-flow water movement experimental device and a scheme provided by an embodiment of the application.

FIG. 2 is a graph showing the results of the short-term micro-running water motion versus the time dynamic change of the muscle properties of hybrid culter in accordance with the embodiment of the present application; FIG. 2A is a graph showing muscle stiffness results; FIG. 2B is a graph showing the result of muscle elasticity; fig. 2C is a muscle chewiness result; fig. 2D is a muscle resilience result. FIG. 2E is a muscle tight connectivity result; fig. 2F is a muscle viscosity result.

FIG. 3 is a graph of HE staining of short-term micro-water movement versus hybrid culter muscle fiber morphology provided by an embodiment of the present application; FIG. 3A is a lengthwise arrangement of muscle fibers of 0d, 4d, 8d and 12 d; fig. 3B is a radial cross-sectional view of muscle fibers of 0d, 4d, 8d, and 12d.

FIG. 4 is a graph showing the effect of short-term micro-running water on the diameter (A) and density (B) of the hybrid culter muscle fiber according to the present application.

FIG. 5 shows the results of the de novo assembly of independent genes from hybrid culter provided in the examples of the present application.

FIG. 6 shows GO annotation results of the assembled independent genes of hybrid culter according to the present application.

FIG. 7 shows the results of KEGG annotation of the assembled independent genes of hybrid culter according to the present application.

FIG. 8 is EggNOG annotation of the independent genes assembled by hybrid culter in accordance with the present application.

Fig. 9 is a graph of the results of principal coordinate analysis (PCoA) of samples of the control group (CK), the T1 group, the T2 group, and the T3 group according to the embodiment of the present application.

FIG. 10 shows the results of Differential Expression Genes (DEGs) and cluster analysis in the hybrid culter muscle provided by the examples of the application; FIG. 10A is a schematic diagram of the DEGs volcanic diagrams of the T1 group and the control group; FIG. 10B is a schematic diagram of the DEGs volcanic of the T2 group and the control group; FIG. 10C is a schematic diagram of the DEGs volcanic of the T3 group and the control group; FIG. 10D is a Wen diagram of the T1 and control, the T2 and control, and the T3 and control; FIG. 10E shows the different expression patterns of the DEGs found.

FIG. 11 is a GO enrichment analysis of hybrid culter muscle DEGs provided by an embodiment of the present application; FIG. 11A is a graph of GO enrichment for the T1 group versus the control group; FIG. 11B is a graph of GO enrichment of the T2 group versus the control group; FIG. 11C is a graph of GO enrichment of the T3 group versus the control group; fig. 11D is a cross GO enrichment profile of T1 and control, T2 and control, and T3 and control.

FIG. 12 is a KEGG enrichment analysis of hybrid culter muscle DEGs provided by an embodiment of the application; FIG. 12A is a KEGG enrichment profile for the T1 group and the control group; FIG. 12B is a KEGG enrichment profile for the T2 group and the control group; FIG. 12C is a KEGG enrichment profile for the T3 group and the control group; FIG. 12D is a cross-KEGG enrichment plot of group T1 and control, group T2 and control, and group T3 and control.

FIG. 13 is a signal path associated with a Hippo signal path provided in accordance with an embodiment of the present application.

FIG. 14 is a thermal graph analysis of the hardening-associated DEGs in the hybrid culter muscle tissue according to an embodiment of the present application; FIG. 14A is a heat map of the hybrid culter muscle tissue contraction-related DEGs; FIG. 14B is a heat map of hybrid culter muscle tissue protein-related DEGs; FIG. 14C is a heat map of the hybrid culter muscle tissue tight junction related DEGs.

FIG. 15 is a heat map analysis of immune defenses and glucose metabolism related DEGs in hybrid culter muscle tissue provided by an embodiment of the present application; FIG. 15A is a heat map of the hybrid culter muscle tissue immune response associated DEGs; FIG. 15B is a heat map of DEGs related to the immune system process of the hybrid culter muscle tissue; FIG. 15C is a heat map of the related DEGs of the AMPK signaling pathway of the hybrid culter muscle tissue; FIG. 15D is a heat map of the glycolysis/gluconeogenesis related DEGs of hybrid culter muscle tissue.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

An "isolated marker genome" herein refers to a set of genes that can be used to classify or categorize the muscle traits, immune traits and/or energy metabolism traits of a hybrid culter according to the application. A "differentially expressed" gene is one that is expressed at a higher or lower level in the motile or non-motile state of the hybrid culter. By "specific oligonucleotides" is meant that these oligonucleotides are unique to each gene, for example, so that a fragment of the gene uniquely identifies the gene.

An embodiment of the present application discloses an isolated marker genome comprising a gene identified as differentially expressed in muscle tissue of a hybrid culter in a motor state versus a non-motor state, the marker genome comprising: a gene WNT2B encoding WNT family member 2B protein; a gene CDH1 encoding a cadherein 1 protein; a gene MYCB encoding bHLH transcription factor b; a gene MYH1 encoding myosin heavy chain-1; a gene MYH2 encoding myosin heavy chain-2; a gene MYH6 encoding myosin heavy chain-6; a gene MYH7 encoding myosin heavy chain-7; a gene MYH15 encoding myosin heavy chain-15; a gene MAVS encoding a mitochondrial antiviral signal protein; a gene EPO encoding erythropoietin; gene SKAP2, which encodes src kinase-associated phosphoprotein 2; gene HK1, which encodes hexaphosphatase 1; a gene GAPDH encoding glycerol-3-phosphate dehydrogenase; gene LDHA, which encodes lactate dehydrogenase a; a gene PGK1 encoding phosphoglycerate kinase 1; or at least one of the specific oligonucleotides that recognize the gene.

The application can obtain the muscle character, the immunity character and the energy metabolism character by detecting at least one of the marker genome of the hybrid culter under the movement state and the non-movement state, and can analyze or identify the muscle quality, the immunity capability and the energy utilization capability of the hybrid culter, thereby providing important technical means support for breeding or improving the hybrid culter variety.

In some embodiments, the marker genome comprises at least one of the gene WNT2B, the gene CDH1, the gene MYCB, the gene MYH1, the gene MYH2, the gene MYH6, the gene MYH7, and the gene MYH 15. These genes are shown to be higher in the motor state of hybrid culter, and further analyze or identify the muscle traits of hybrid culter.

In some embodiments, the marker genome comprises at least one of the gene MAVS, the gene EPO, and the gene SKAP 2. These genes are shown to be higher in the motile state of hybrid culter, and thus analyze or identify the immune traits of hybrid culter.

In some embodiments, the marker genome comprises at least one of the gene HK1, the gene GAPDH, the gene LDHA, and the gene PGK 1. These genes are shown to be higher in the motor state of hybrid culter, and further the energy metabolism traits of hybrid culter are analyzed or identified.

In some embodiments, the specific oligonucleotide is selected from any one of SEQ ID NOS.33-47. Wherein, the specific oligonucleotide shown as WNT2B in SEQ ID NO.33, the specific oligonucleotide shown as CDH1 in SEQ ID NO.34, the specific oligonucleotide shown as MYH1 in SEQ ID NO.35, the specific oligonucleotide shown as MYH2 in SEQ ID NO.36, the specific oligonucleotide shown as MYH6 in SEQ ID NO.37, the specific oligonucleotide shown as MYH7 in SEQ ID NO.38, the specific oligonucleotide shown as MYH15 in SEQ ID NO.39, the specific oligonucleotide shown as MYH15 in SEQ ID NO.40, the specific oligonucleotide shown as MAVS in SEQ ID NO.41, the specific oligonucleotide shown as EPO in SEQ ID NO.42, the specific oligonucleotide shown as SKAP2 in SEQ ID NO.43, the specific oligonucleotide shown as HK1 in SEQ ID NO.44, the specific oligonucleotide shown as GAPDH in SEQ ID NO.45, the specific oligonucleotide shown as GAPDH in SEQ ID NO.46, and the specific oligonucleotide shown as PGID NO. 47.

The embodiment of the application discloses application of a marker genome in preparation of a kit, wherein the kit is used for predicting muscle traits, immune traits and/or energy metabolism traits of hybrid culters by comparing differential expression of any marker gene selected from the marker genome or is used for breeding or improving variety resources of hybrid culters according to the muscle traits, the immune traits and/or the energy metabolism traits.

In some embodiments, the comparison is performed by RNA-seq. For example, the Illumina Hi-Seq 4000 sequencing platform was used to observe changes in muscle transcriptome of hybrid culter in non-motile or different motile states and to analyze differentially expressed genes based on the changes in transcriptome. In some embodiments, the comparison is performed by RT-qPCR.

In some embodiments, the muscle trait is selected from at least one of muscle stiffness, muscle fiber diameter, muscle fiber density, inter-muscle fiber gap, spatial height, muscle fiber type, and muscle texture. In some embodiments, the immune trait is selected from immune resistance and/or immune defenses. In some embodiments, the energy metabolism trait is selected from at least one of muscle glycogen content, muscle glucose content, and insulin secretion.

The embodiment of the application discloses an RT-qPCR kit which is used for predicting muscle traits, immune traits and/or energy metabolism traits of hybrid culters by comparing the differential expression of any marker gene selected from the marker genome or is used for breeding or improving variety resources of hybrid culters according to the muscle traits, the immune traits and/or the energy metabolism traits.

In some embodiments, the RT-qPCR kit comprises a primer combination for performing RT-qPCR, the primer combination selected from at least one of: primer pairs shown in SEQ ID NO. 1-2; primer pairs shown in SEQ ID NO. 3-4; primer pairs shown in SEQ ID NO. 5-6; primer pairs shown in SEQ ID NO. 7-8 and primer pairs shown in SEQ ID NO. 9-10; primer pairs shown in SEQ ID NO. 11-12; primer pairs shown in SEQ ID NO. 13-14; primer pairs shown in SEQ ID NO. 15-16; primer pairs shown in SEQ ID NO. 17-18; primer pairs shown in SEQ ID NO. 19-20; primer pairs shown in SEQ ID NO. 21-22; primer pairs shown in SEQ ID NO. 23-24; primer pairs shown in SEQ ID NO. 25-26; primer pairs shown in SEQ ID NO. 27-28; primer pairs shown in SEQ ID NO. 29-30; primer pairs shown in SEQ ID NOS.31-32.

In some embodiments, the RT-qPCR kit predicts muscle properties of hybrid culters by comparing the differential expression of marker genes selected from any of the marker genomes. The marker genome comprises at least one of the gene WNT2B, the gene CDH1, the gene MYCB, the gene MYH1, the gene MYH2, the gene MYH6, the gene MYH7, and the gene MYH 15. The primer combination comprises: primer pairs shown in SEQ ID NO. 1-2 for detecting specific oligonucleotides shown in SEQ ID NO. 33; primer pairs shown in SEQ ID NO. 3-4 for detecting specific oligonucleotides shown in SEQ ID NO. 34; primer pairs shown in SEQ ID NO. 5-6 for detecting specific oligonucleotides shown in SEQ ID NO. 35; primer pairs shown in SEQ ID NO. 7-8 for detecting specific oligonucleotides shown in SEQ ID NO. 36; primer pairs shown in SEQ ID NO. 9-10 for detecting specific oligonucleotides shown in SEQ ID NO. 37; primer pairs shown in SEQ ID NO. 11-12 for detecting specific oligonucleotides shown in SEQ ID NO. 38; primer pairs shown in SEQ ID NO. 13-14 for detecting specific oligonucleotides shown in SEQ ID NO. 39; at least one of the primer pairs shown in SEQ ID NOS.15-16 for detecting a specific oligonucleotide shown in SEQ ID NO. 40.

In some embodiments, the RT-qPCR kit predicts the immune trait of hybrid culter by comparing the differential expression of marker genes selected from any of the marker genomes. The marker genome comprises at least one of the gene MAVS, the gene EPO and the gene SKAP 2. The primer combination comprises: primer pairs shown in SEQ ID NO. 17-18 for detecting the specific oligonucleotide shown in SEQ ID NO. 41; primer pairs shown in SEQ ID NO. 19-20 for detecting specific oligonucleotides shown in SEQ ID NO. 42; at least one of the primer pairs shown in SEQ ID NOS.21 to 22 for detecting a specific oligonucleotide shown in SEQ ID NO. 43.

In some embodiments, the RT-qPCR kit predicts the energy metabolism trait of hybrid culter by comparing the differential expression of marker genes selected from any of the marker genomes. The marker genome includes at least one of the gene HK1, the gene GAPDH, the gene LDHA, and the gene PGK 1. The primer combination comprises: primer pairs shown in SEQ ID NO. 23-24 for detecting the specific oligonucleotides shown in SEQ ID NO. 44; primer pairs shown in SEQ ID NO. 25-26 for detecting specific oligonucleotides shown in SEQ ID NO. 45; primer pairs shown in SEQ ID NO. 27-28 for detecting specific oligonucleotides shown in SEQ ID NO. 46; at least one of the primer pairs shown in SEQ ID NOS.29 to 30 for detecting a specific oligonucleotide shown in SEQ ID NO. 47.

The application will now be further illustrated with reference to non-limiting examples. It will be understood by those skilled in the art that the following examples, while indicating preferred embodiments of the application, are given by way of illustration only and that all reagents used are commercially available.

1. Short-term micro-flow water movement experiment

The hybrid culter used in this study, "pioneer 1" was widely cultivated in China, as a non-protected or endangered species. Before the start of the experiment, randomly selected hybrid culter fish were acclimatized in a 1000L flow tank for two weeks (temperature 21.0.+ -. 1.0 ℃ C.; dissolved oxygen >6.8mg/L; pH 7.2.+ -. 0.2; natural photoperiod). To eliminate the effects of female oviposition and sex variation, only healthy male fish of similar size (155.37 ±14.98g per tail) were used in this experiment. 90 hybrid fish were randomly selected, divided into 3 circular swim grooves (n=30, height=100 cm; diameter=150 cm), and after allowing the fish to adapt for 7 days in a new environment without directional flow, swimming experiments were performed.

The speeds of the three swim tanks were steadily increased to the required level (1.2 BL/s) before the start of the experiment and remained unchanged throughout the experiment. The fish were fed commercial feed containing 30% protein (sea da limited, jiangsu province, china) once daily until significant satiety was observed. The water flow was stopped every morning for 2 hours to ensure complete digestion of the feed provided. In addition, water quality parameters were measured once daily for 12 days (nitrate 0.009-0.029 mM, NH 30.003-0.008 mM, dissolved oxygen >6.9mg/L, nitrite 0.0009-0.0019 mM, and pH 7.1.+ -. 0.3) throughout the exercise experiment.

The experiments were divided into 4 groups: control, no movement or movement 0d; group T1, motion 4d; group T2, motion 8d; t3 group, motion 12d. Anesthesia was performed using MS-222 (150 mg/L) at each time point (0, 4, 8, 12 d). 3 fish were selected for each group, and first, the muscle properties of the fish were analyzed by dissecting the muscle into smaller cubes (1.0X1.0X1.0 cm). Dissecting muscle (0.5X0.5X0.5 cm), soaking in 4% paraformaldehyde PBS for 24h, preserving with 70% ethanol, preparing paraffin blocks and slicing for histological examination.

Simultaneously, 3 fish were harvested, their muscles were mixed as a biological repeat, and immediately frozen in liquid nitrogen, stored at-80 ℃ and waited for ribonucleic acid (RNA) extraction for real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNA-seq). Each group contained 3 biological replicates in 3 swimming pools, all sample acquisitions were performed on an ultra clean bench, and the experimental setup and protocol is shown in fig. 1.

2. Analysis of muscle section texture characteristics of hybrid culter No.1 and results thereof

Texture is an important intrinsic aspect in determining fish meat quality, and is often assessed by muscle properties such as elasticity, hardness and chewiness. The back muscle properties of each group of fish were analyzed using an XT2i surface analyzer.

The results are shown in FIG. 2, where the elasticity, hardness and chewiness of the T2 and T3 groups are significantly higher than the control group, thus demonstrating that short-term swimming movements can improve the muscle texture and fillet quality of hybrid culters.

3. Morphological analysis of hybrid culter under motion-induced "pioneer No. 1" myofibers

Skeletal muscle cells, including myofiber properties and muscle connective tissue, are the main determinants of fish muscle texture. The increased muscle stiffness and mastication is generally caused by high fiber density, small fiber diameter, high collagen levels, and narrow fibromuscular interstices. The muscle samples of hybrid culter "pioneer 1" were dehydrated through a series of different percentages of ethanol, paraffin embedded, sectioned, and observed with hematoxylin-eosin (H & E) staining to determine myofiber characteristics.

As shown in fig. 3A and 3B, the myofibers of the T2 group and the T3 group are more closely arranged, and the diameter of the myofibers of the sports group is smaller than that of the control group. . As shown in fig. 4, the longer the exercise time, the smaller the diameter of the muscle fiber, the higher the density of the muscle fiber, the shortest the diameter of the muscle fiber of group 12d, and the greatest the density of the muscle fiber (P < 0.05).

In this study, the short-term micro-aquaculture for 12d increased the muscle elasticity of the hybrid culter "pioneer 1" by 28.57% and the muscle fiber density by 54.12%. Thus, swimming may enhance its muscle properties by increasing the density of the hybrid culter muscle fibers and decreasing the diameter of the muscle fibers. The above results indicate that 12d movement is an effective way to enhance the muscle properties of hybrid culter. The pattern of changes in muscle properties (characteristics of muscle texture and tissue morphology, etc.) suggests that longer movements may be more efficient and that further investigation of optimal movement times and velocities is required.

4. RNA sequencing and transcriptome assembly

As shown in Table 1, a total of 12 cDNA libraries were constructed and 37,207,706 ～ 49,336,860 Raw Reads were obtained from the RNA-seq. After data quality screening, a total of 34,178,758 to 45,354,210 clean reads (90.72% to 92.01% of the original data) were generated. Sequences were assembled using clean reads obtained from 12 libraries, as in FIG. 5, to yield 45071 independent genes from head assembly. The average length of the independent genes was 1244.88bp (n50=2155 bp), and the longest sequence length was 45416bp.

TABLE 1 statistics of sequencing data of muscle transcriptomes of hybrid culter

cDNA library	Raw Reads	Clean Reads	Clean Reads(％)	Q20(％)	Q30(％)
						Ctrl-1	45,087,366	40,906,472	90.72	97.89	94.41
Ctrl-2	37,207,706	34,178,758	91.85	97.96	94.56
						Ctrl-3	49,336,860	45,354,210	91.92	97.44	93.13
T1-1	47,507,114	43,629,530	91.83	98.62	95.99
						T1-2	44,713,320	40,807,126	91.26	97.98	94.66
T1-3	44,392,122	40,629,742	91.52	97.86	94.32
						T2-1	40,997,546	37,557,866	91.61	98	94.67
T2-2	39,828,204	36,647,468	92.01	98.02	94.73
						T2-3	40,262,108	36,903,650	91.65	98.1	94.89
T3-1	39,171,306	35,887,268	91.61	98.01	94.67
						T3-2	39,938,842	36,642,246	91.74	98	94.66
T3-3	38,521,912	35,306,596	91.65	97.94	94.52

5. Analysis and identification of differential expression genes related to muscle traits of hybrid culter 'pioneer No. 1' under motion induction

By comparing the independent genes of the different genes, the genetic functions were comprehensively identified, and the results are shown in Table 2.

TABLE 2 statistics of independent Gene annotation of muscle transcriptomes of hybrid culter

Database for storing data	Annotation quantity	Percentage (%)
			NR	23119	51.29
GO	7648	16.97
			KEGG	14125	31.34
Pfam	15932	35.35
			eggNOG	21785	48.33
Swissprot	18807	41.73

Basic Local Alignment Search Tool (BLAST) results showed that 23119, 7648, 14125, 21785, 15932 and 18807 matching annotation sequences in NR, GO, KEGG, eggNOG, pfam and Swissprot databases, respectively, were matched. The functional classifications of GO, KEGG and EggNOG are shown in FIGS. 6, 7 and 8.

As shown in fig. 9, specimens from groups 0, 4, 8 and 12d were dispersed into four independent fractions according to the PCoA analysis.

The 4 groups were subjected to Differential Expression Gene (DEG) analysis to investigate the molecular processes of this hybrid culter, which were behind short-term movement, and the results are shown in FIG. 10.

As shown in fig. 10A, the T1 group obtained 2056 DEGs,991 up-regulated, 1065 down-regulated compared to the control group. As shown in fig. 10B, the T2 group found 2414 DEGs up-regulated, 1304 up-regulated, 1110 down-regulated compared to the control group. As shown in fig. 10C, the T3 group found 3366 DEGs,1820 up-regulated, 1546 down-regulated compared to the control group. The results show that the effect of motion on molecular changes increases with increasing motion time.

FIG. 10D depicts a Venn diagram showing 594 DEGs co-expressed in all three comparisons (Ctrl vs T1, ctrl vs T2 and Ctrl vs T3), 920 DEGs co-expressed in Ctrl and T1 groups, 742 DEGs co-expressed in Ctrl and T2 groups, 1658 DEGs co-expressed in Ctrl and T3 groups. While figure 10E depicts the different expression patterns of these DEGs.

6. GO enrichment analysis of differentially expressed genes

GO enrichment analysis was performed on DEGs to find Biological Processes (BP), molecular Functions (MF) and Cellular Components (CC) that were significantly enriched (FDR +.0.05).

As shown in fig. 11A, BP comprises contracting skeletal muscle contractions, slow contractions, skeletal muscle fiber contractions, skeletal muscle contractions, voluntary skeletal muscle contractions, and multicellular biological movements; CC contains Major Histocompatibility Complex (MHC) protein complexes, troponin complexes, myofilaments, striated myofilaments and actin cytoskeleton; MF contains peptide antigen binding, amide binding and actin binding.

As shown in fig. 11B, BP comprises skeletal muscle contraction, skeletal muscle fiber contraction, skeletal muscle contraction, multicellular body movement, voluntary skeletal muscle contraction; CC contains striated muscle filaments, troponin complexes, myofilaments and actin cytoskeleton; MF contains endopeptidase inhibitor activity, endopeptidase modulating activity, actin binding and cytoskeletal protein binding.

As shown in fig. 11C, BP comprises skeletal muscle contraction, skeletal muscle slow contraction skeletal muscle fiber contraction, voluntary skeletal muscle contraction, multicellular body movement, and skeletal muscle contraction; CC contains striated muscle filaments, troponin complexes, muscle filaments, mitochondrial envelopes and actin cytoskeleton; MF contains endopeptidase inhibitor activity, actin binding and cytoskeletal protein binding.

As shown in fig. 11D, in the sports group (T1, T2, T3), genes are mostly enriched for muscle contraction functions (skeletal muscle contraction, slow contraction skeletal muscle fiber contraction, actin cytoskeleton and muscle contraction) and immune defense functions (immune response, immune system processes, antigen processing and presentation, MHC protein complexes). Furthermore, GO-enriched DEGs associated with muscle contraction function increases with prolonged exercise time.

7. KEGG cascade, signal path and heat map analysis of DEGs

As shown in fig. 12A, DEGs of the T1 group are enriched in antigen processing and presentation, pentose phosphate pathway, myocardial contraction, fructose and mannose metabolism, tight junctions, and Amp-activated protein kinase (AMPK) signaling pathways. As shown in fig. 12B, DEGs of T2 group are enriched in antigen processing and presentation, pentose phosphate pathway, myocardial contraction, fructose and mannose metabolism, tight junctions, AMPK signaling pathway, glycolysis/gluconeogenesis, etc. As shown in fig. 12C, DEGs of T3 group are enriched in antigen processing and presentation, pentose phosphate pathway, myocardial contraction, tight junctions, proteasome, complement and coagulation cascade, and glycolysis/gluconeogenesis, among others. Figure 12D shows that the signal pathways due to enrichment in T1, T2, T3 are mainly related to the immune system, muscle contraction and energy metabolism. As shown in fig. 13, T3 group induced DEGs was enriched in Hippo signaling pathway.

Thus summarizing the crossover of the enrichment KEGG pathway as follows: 1) Muscle contraction (tight junctions, cardiomyocyte adrenergic signaling and myocardial contraction); 2) The immune system (hematopoietic cell lineage, antigen processing and presentation); 3) Energy metabolism (starch and sucrose metabolism, AMPK signaling pathway, fructose and mannose metabolism, glycolysis/gluconeogenesis). Furthermore, the number of related genes and the degree of enrichment of KEGG (myocardial contraction, tight junctions, AMPK signaling pathway, glycolysis/gluconeogenesis) steadily increased with prolonged exercise time (4, 8, 12 d). The study also analyzed the DEGs for association with muscle contraction (fig. 14A-C), immune system (fig. 15A-B), and energy metabolism (fig. 15C-D).

8、RT-qPCR

Total RNA was extracted from Ctrl, T1, T2 and T3 samples using TRIzol (Invitrogen, waltham, mass., USA) and transcriptome sequenced. And comparing the sequencing result with a control group respectively, and carrying out functional enrichment analysis on the differential genes. After total RNA extraction, DNA was removed using DNase I, and qPCR was performed using SYBR Green with beta-actin as an internal reference gene for verification of RNA-seq data. Table 3 shows the list of primer sequences used. Finally, adopt 2 ^-ΔΔCt The relative gene expression level was calculated by the method.

TABLE 3 primer information for real-time fluorescent quantitative PCR analysis

9. Comparison of RNA-seq and RT-qPCR results

22 DEGs related to muscle properties, immune defense properties and energy metabolism properties are selected for RT-qPCR verification, and RT-qPCR results are compared with NA-seq results, and when the results are shown in the table 4 and the expression multiple of the table 4 is more than 1, the expression multiple of T1/T2/T3 is higher than that of a control group; "fold expression" +.1 value, it means that the fold expression of T1/T2/T3 is lower than that of the control group.

TABLE 4 comparison of RNA-seq and RT-qPCR results

As can be seen from Table 4, the expression levels of MYH1, MYH6, MYH7, MYH15, MYH2, WNT2B, CDH1 and MYCB were significantly increased in the T1\T2\T3 group, CLDN4 was significantly decreased, and ACTN2 and CLDND did not show significant increases in the T1\T2\T3 group. From this, MYH1, MYH6, MYH7, MYH15, MYH2, WNT2B, CDH1, MYCB and CLDN4 showed significant expression differences from the hybrid culter in both the motile and non-motile states.

As can be seen from Table 4, the MAVS, EPO and SKAP2 gene expression levels of T1/T2/T3 groups were significantly increased, and SPPL2A, IRF1, SPI1 and NFIL3 were significantly decreased. From this, MAVS, EPOSKAP2, SPPL2A, IRF1, SPI1 and NFIL3 showed significant differences in expression between the hybrid culter in the motile and non-motile states.

As can be seen from Table 4, the HK1, GAPDH, LDHA and PGK1 gene expression levels of the T1\T2\T3 groups were significantly increased. From this, HK1, GAPDH, LDHA and PGK1 were significantly different from the hybrid culter in terms of expression in both the motile and non-motile state.

In addition, table 4 shows that the mutation trend of these genes is consistent with the RNA-seq data, demonstrating the reliability and accuracy of the RNA-seq analysis.

10. Correlation analysis of motor-induced hybrid culter muscle traits and marker genes

The transcriptome analysis of non-motile and motile hybrid culter in the examples of the present application uses RNA-seq and RT-qPCR to analyze muscle traits. Muscle stiffness is reported to be highly related to muscle fiber diameter, muscle fiber density, inter-muscle fiber gap and space, and muscle fiber density is related to hyperplasia (increase in the number of muscle fibers in life).

The results presented in the examples show that genes that are significantly upregulated in the motor status, such as WNT2B (WNT family member 2B), CDH1 (cadherein 1) and MYCB (MYC protooncogene, bHLH transcription factor B), are abundantly present in the hippo signaling pathway (fig. 13), and are critical for regulating differentiation and proliferation of adult stem cells. The hippo signaling pathway includes a highly conserved cascade of core kinases that limit organ size by managing the dynamic balance of apoptosis and proliferation. Cell proliferation and apoptosis are regulated by Wnt2B in the Wnt/β -catenin pathway. Alterations in CDH1 activity are thought to be the basis for cell proliferation. MYC is a transcription factor family member, including MYCL, MYCN, and MYCB, responsible for metabolism, proliferation, and growth of cells. Thus, the results of this study may suggest that the significant up-regulated genes (WNT 2B, CDH1 and MYCB) concentrated in the hippo signaling pathway are critical in the molecular mechanisms of motor-induced muscle hardening.

Muscle fiber type is another key component that has a long-term impact on meat quality (e.g., texture, taste, and color). The myosin heavy chain gene family (MYHs) is a polygene family in the vertebrate genome whose polymorphisms are associated with the molecular basis of these different fiber types. Generally, aquatic species possess more MYHs than mammals. Furthermore, myofiber types are affected by biological evolution; thus, systematically relating one MyHC isomer to the fiber type of aquatic species is a challenge, as there are multiple MyHC in a given fiber type.

In the above examples, several up-regulating genes responsible for muscle contraction and muscle fiber differentiation, such as myosin heavy chain-1 (MYH 1), myosin heavy chain-2 (MYH 2), myosin heavy chain-6 (MYH 6), myosin heavy chain-7 (MYH 7), myosin heavy chain-15 (MYH 15) were enriched for down-regulation under motor induction. MYH1 is a skeletal muscle-specific myophilic gene, essential for muscle function and architecture. Meanwhile, MYH2 is a typical myosin heavy chain protein, involved in muscle contraction. Missense mutations in MYH2 are associated with progressive muscular dystrophy, muscle weakness, and abnormalities in myosin fiber type. Furthermore, MYH6 and MYH7 are cardiac muscle protein isoforms expressed in mammalian heart and specialized skeletal muscle. In addition, MYH7 is the major isomer released in slow muscle fibers (sometimes referred to as type I fibers). MYH15 is a slow myosin that is involved in cytoskeletal remodeling and muscle contraction.

As can be seen from the results of fig. 2, 3, 4 and table 4, the T2 and T3 groups have higher myofiber density, smaller myofiber diameter, and higher elasticity, hardness and chewiness than the control group. From this, it is known that MYH1, MYH6, MYH7, MYH15, MYH2, WNT2B, CDH1 and MYCB can determine the myofiber density and myofiber diameter of hybrid culter, and by detecting the expression levels of these marker genes, the hybrid culter myofiber density and myofiber diameter, and thus the elasticity, hardness and chewiness of the muscles can be known, thereby providing a molecular index for improving the muscle quality of hybrid culter by exercise training, and providing a technical support for domesticating or breeding hybrid culters with excellent muscle texture and fish fillet quality.

The tight junction complex is primarily responsible for regulating and maintaining intimate contact between adjacent cells. The increase in tight junctions results in a decrease in the intercellular space of the mice and a more robust structure. As shown in fig. 12D, the relevant genes and KEGG enrichment of the tight junction pathway gradually increased with increasing movement time, suggesting that activation of the tight junction pathway may help to reduce the intercellular space of the stalk-like hybrid muscle and further promote higher firmness. Taken together, the results indicate that exercise can activate the hippo pathway, increasing myofiber density; regulating expression of MYHs, and changing muscle fiber type; the tight connection signal path is activated, the space between muscle fibers is reduced, and the fish hardness is further improved.

11. Hybrid culter under motion induction immune defense changes

Mitochondrial antiviral signaling proteins (MAVS) cause immune activation and inflammation by activating the NF- κB pathway, which is critical in preventing excessive detrimental immune responses. Erythropoietin (EPO) is produced by monocytes and has functions other than erythropoietin, including immunomodulation. Neutrophil recruitment and activation are part of the primary innate immune response to viral and bacterial infections. src kinase-associated phosphoprotein 2 (SKAP 2) plays an important role in neutrophil recruitment and integrin activation. It was found that expression of genes related to immune defense of hybrid culter can be affected by swimming, and thus immunity can be provided.

As can be seen in fig. 15 and table 4, the MAVS, EPO, and SKAP2 genes were significantly up-regulated in the sports group. Therefore, MAVS, EPO and SKAP2 can determine the immunity of hybrid culter, and the hybrid culter immunity can be obtained by detecting the expression quantity of the marker genes, so that molecular indexes are provided for improving the quality of hybrid culter muscle through exercise training, and technical support is provided for domesticating or breeding hybrid culter with strong immunity and excellent development.

12. Energy metabolism change of hybrid culter under motion induction

Glucose metabolism includes glycolysis and glucose production. Glycolysis is the basic metabolic mechanism of energy production in an organism. Hexaphosphatase 1 (HK 1) is a ubiquitously expressed phosphofructokinase that acts as a catalyst during the rate limiting and irreversible initial stages of glycolysis. In addition, glycerol-3-phosphate dehydrogenase (GAPDH) is one of the most critical enzymes in cellular energy metabolism, which promotes the reversible conversion of glycerol-3-phosphate (G-3-P) to glycerol-1, 3-diphosphate. In aerobic glycolysis, lactate dehydrogenase a (LDHA) retains pyruvate in the cytoplasm and converts it to lactate. In the aerobic glycolysis process of cells, the only two enzymes that govern ATP synthesis are phosphoglycerate kinase 1 (PGK 1) and pyruvate kinase M2 (PKM 2). Studies have reported that AMPK can alter the activity of insulin receptor tyrosine kinase and increase the number of Insulin Receptors (IR). Once AMPK is activated, this molecule catalyzes a range of physiological effects related to glucose and lipid metabolism, including: 1) Stimulating glucose uptake and stimulating glycolysis of skeletal muscle; 2) Preventing glucose production in the liver; 3) Increase the activity of islet beta cells and regulate insulin secretion.

FIG. 15C/D and the results of Table 4 show that the expression of HK1, GAPDH, LDHA and PGK1 were significantly up-regulated in the sports group, whereas the AMPK pathway and glycolysis/glucose production were enriched in the sports group (8 days and 12 days). Thus, it can be inferred that changes in gene expression of these pathways may affect the energy utilization efficiency of fish muscle.

From this, it is known that HK1, GAPDH, LDHA and PGK1 can determine the muscle energy utilization efficiency of hybrid culter, and by detecting the expression levels of these marker genes, the muscle energy utilization efficiency of hybrid culter can be known, thereby providing a molecular index for improving the muscle quality of hybrid culter by exercise training, and providing a technical support for domesticating or breeding hybrid culter with excellent muscle texture and fish filet quality.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims

1. An isolated marker genome comprising a gene identified as differentially expressed in muscle tissue of hybrid culter in a motor state and a non-motor state, the marker genome comprising:

a gene WNT2B encoding WNT family member 2B protein;

a gene CDH1 encoding a cadherein 1 protein;

a gene MYCB encoding bHLH transcription factor b;

a gene MYH1 encoding myosin heavy chain-1;

a gene MYH2 encoding myosin heavy chain-2;

a gene MYH6 encoding myosin heavy chain-6;

a gene MYH7 encoding myosin heavy chain-7;

a gene MYH15 encoding myosin heavy chain-15;

a gene MAVS encoding a mitochondrial antiviral signal protein;

a gene EPO encoding erythropoietin;

gene SKAP2, which encodes src kinase-associated phosphoprotein 2;

gene HK1, which encodes hexaphosphatase 1;

a gene GAPDH encoding glycerol-3-phosphate dehydrogenase;

gene LDHA, which encodes lactate dehydrogenase a;

a gene PGK1 encoding phosphoglycerate kinase 1;

or at least one of the specific oligonucleotides that recognize the gene.

2. The marker genome of claim 1, optionally comprising the gene WNT2B, the gene CDH1, the gene MYCB, the gene MYH1, the gene MYH2, the gene MYH6, the gene MYH7, and the gene MYH15, the marker genome being associated with a muscle trait of the hybrid culter;

optionally, the marker genome comprises the gene MAVS, the gene EPO and the gene SKAP2, and the marker genome is associated with the immune trait of the hybrid culter;

alternatively, the marker genome comprises the gene HK1, the gene GAPDH, the gene LDHA and the gene PGK1, the marker genome being associated with an energy metabolism trait of the hybrid culter.

3. The marker genome of claim 1, wherein the specific oligonucleotide is selected from any one of SEQ ID NOS.33 to 47.

4. Use of the marker genome of claim 1 in the preparation of a kit for predicting a muscle trait, an immune trait and/or an energy metabolism trait of hybrid culter by comparing the differential expression of a marker gene selected from any of the marker genomes of claims 1 to 3, or for breeding or improving a variety resource of hybrid culter based on the muscle trait, the immune trait and/or the energy metabolism trait.

5. The use according to claim 4, wherein the comparison is performed by RNA-seq.

6. The use according to claim 4, wherein the comparison is performed by RT-qPCR.

7. The use according to claim 4, optionally the muscle trait is selected from at least one of muscle stiffness, muscle fiber diameter, muscle fiber density, inter-muscle fiber space, space height, muscle fiber type and muscle texture;

optionally, the immune trait is selected from immune resistance and/or immune defenses;

optionally, the energy metabolism trait is selected from at least one of muscle glycogen content, muscle glucose content, and insulin secretion.

8. An RT-qPCR kit for predicting muscle traits, immune traits and/or energy metabolism traits of hybrid culters by comparing the differential expression of marker genes selected from any one of the marker genomes of claims 1 to 3, or for selecting variety resources of hybrid culters according to the muscle traits, the immune traits and/or the energy metabolism traits.

9. The RT-qPCR kit of claim 8 comprising a primer combination for performing RT-qPCR selected from at least one of: primer pairs shown in SEQ ID NO. 1-2; primer pairs shown in SEQ ID NO. 3-4; primer pairs shown in SEQ ID NO. 5-6; primer pairs shown in SEQ ID NO. 7-8 and primer pairs shown in SEQ ID NO. 9-10; primer pairs shown in SEQ ID NO. 11-12; primer pairs shown in SEQ ID NO. 13-14; primer pairs shown in SEQ ID NO. 15-16; primer pairs shown in SEQ ID NO. 17-18; primer pairs shown in SEQ ID NO. 19-20; primer pairs shown in SEQ ID NO. 21-22; primer pairs shown in SEQ ID NO. 23-24; primer pairs shown in SEQ ID NO. 25-26; primer pairs shown in SEQ ID NO. 27-28; primer pairs shown in SEQ ID NO. 29-30; primer pairs shown in SEQ ID NOS.31-32.

10. The RT-qPCR kit of claim 8, optionally wherein the primer combination comprises: primer pairs shown in SEQ ID NO. 1-2 for detecting specific oligonucleotides shown in SEQ ID NO. 33; primer pairs shown in SEQ ID NO. 3-4 for detecting specific oligonucleotides shown in SEQ ID NO. 34; primer pairs shown in SEQ ID NO. 5-6 for detecting specific oligonucleotides shown in SEQ ID NO. 35; primer pairs shown in SEQ ID NO. 7-8 for detecting specific oligonucleotides shown in SEQ ID NO. 36; primer pairs shown in SEQ ID NO. 9-10 for detecting specific oligonucleotides shown in SEQ ID NO. 37; primer pairs shown in SEQ ID NO. 11-12 for detecting specific oligonucleotides shown in SEQ ID NO. 38; primer pairs shown in SEQ ID NO. 13-14 for detecting specific oligonucleotides shown in SEQ ID NO. 39; at least one of the primer pairs shown in SEQ ID NO. 15-16 for detecting the specific oligonucleotide shown in SEQ ID NO. 40;

optionally, the primer combination comprises: primer pairs shown in SEQ ID NO. 17-18 for detecting the specific oligonucleotide shown in SEQ ID NO. 41; primer pairs shown in SEQ ID NO. 19-20 for detecting specific oligonucleotides shown in SEQ ID NO. 42; at least one of the primer pairs shown in SEQ ID NOS.21 to 22 for detecting a specific oligonucleotide shown in SEQ ID NO. 43;

optionally, the primer combination comprises: primer pairs shown in SEQ ID NO. 23-24 for detecting the specific oligonucleotides shown in SEQ ID NO. 44; primer pairs shown in SEQ ID NO. 25-26 for detecting specific oligonucleotides shown in SEQ ID NO. 45; primer pairs shown in SEQ ID NO. 27-28 for detecting specific oligonucleotides shown in SEQ ID NO. 46; at least one of the primer pairs shown in SEQ ID NOS.29 to 30 for detecting a specific oligonucleotide shown in SEQ ID NO. 47.