CN117051118B - Marker gene for muscle difference expression of hybrid culter No.1 under motion induction and application thereof - Google Patents
Marker gene for muscle difference expression of hybrid culter No.1 under motion induction and application thereof Download PDFInfo
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Abstract
The application relates to a marker gene for expressing muscle difference under motion induction of hybrid culter 'pioneer 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 PGK1. 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
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 1' muscle differential expression under motion induction and application thereof.
Background
Erythroculter ilishaeformis (Erythroculter ilishaeformis), belongs to a large-sized and economic-value fleshy variety of fish of Erythroculter genus of Cypriorder, but has high artificial breeding cost and is difficult to transport in vivo. The Erythroculter nigrocauda is a member of the genus Erythroculter of the order Cyprinus, the family Cypriidae, the Erythroculter nigrocauda, but is easy to capture and transport due to small size, soft holding and early sexual maturity, but has economic value obviously lower than the Erythroculter ilishaeformis. Thus, in aquaculture, researchers have performed distant hybridization (i.e., culter nigrum is female x culter ilishaeformis) with culter nigrum to obtain a hybrid culter "pioneer 1". The hybrid culter No.1 has high stress resistance and high disease resistance, has high growth speed, is easy to capture and transport, and is suitable for pond culture. Nevertheless, there is still a lack of methods or means for breeding, detecting or identifying the muscle quality of such hybrid varieties.
Disclosure of Invention
The examples herein performed transcriptome sequencing by placing hybrid culter in the motile and non-motile state, observing changes in muscle transcriptome after different motile times, and studying the relevant histomorphological modifications. 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-fluidic motion experimental apparatus and a scheme provided in an embodiment of the present application.
FIG. 2 is a graph showing the results of the short-term micro-running water movement versus the time dynamic change of the muscle properties of hybrid culter in 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 in the examples herein; 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 diameter (A) and density (B) of hybrid culter muscle fibers according to the examples of the present application.
FIG. 5 shows the results of the de novo assembly of independent genes from hybrid culter as provided in the examples of the present application.
FIG. 6 shows GO annotation results of the individual genes assembled by hybrid culter, as provided in the examples of the present application.
FIG. 7 shows the KEGG annotation result of the independent genes assembled by hybrid culter in the examples of the present application.
FIG. 8 is EggNOG annotation of individual genes assembled by hybrid culter, provided in the examples herein.
Fig. 9 is a graph of the results of principal coordinate analysis (PCoA) of the Control (CK), T1, T2, and T3 samples provided in the examples of the present application.
FIG. 10 shows the results of Differential Expression Genes (DEGs) and cluster analysis in the hybrid culter muscle provided in the examples of the present 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 in the examples herein; 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 in the examples herein; 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 the Hippo signal path provided in an embodiment of the present application.
FIG. 14 is a thermal image analysis of the hardening-associated DEGs in the hybrid culter muscle tissue provided in the examples herein; 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 in the examples herein; 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 further described in detail with reference to examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the present application. Reagents not specifically and individually described in this 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 muscle traits, immune traits and/or energy metabolism traits of hybrid culter according to the present 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.
The present embodiments disclose an isolated marker genome comprising a gene identified as differentially expressed in muscle tissue of 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 muscle property, the immunity property and the energy metabolism property can be known by detecting at least one of the marker genomes of the hybrid culter under the movement state and the non-movement state, and the muscle quality, the immunity capability and the energy utilization capability of the hybrid culter can be analyzed or identified, so that important technical means support is provided for variety breeding of the hybrid culter.
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 SKAP2. 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 PGK1. 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 application discloses application of the 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 selecting 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, 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 selecting 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 SKAP2. 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 PGK1. 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 present application will be further illustrated with reference to the following non-limiting examples. It will be understood by those skilled in the art that the following examples, while indicating preferred embodiments of the present 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 movement of 12d significantly reduced the diameter of the muscle fibers, increasing the density of the muscle fibers, and thus increasing the stiffness of the muscle. 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. Furthermore, these 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, adoptThe 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; when the expression multiple is less than or equal to 1, the expression multiple of T1/T2/T3 is lower than that of a 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 TI\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 gene expression levels of HK1, GAPDH, LDHA and PGK1 in the TI\T2\T3 group 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 present examples analyze the transcriptome analysis of non-motile and motile hybrid culter, and analyze muscle traits using RNA-seq and RT-qPCR. 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 foregoing is merely a preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present application should be covered by the scope of the present application.
Claims (9)
1. Application of a reagent for detecting the expression level of gene combinations, namely MYH1, MYH6, MYH7, MYH15, MYH2, WNT2B, CDH1 and MYCB, in preparing a kit for predicting the diameter and/or the density of muscle fibers of hybrid culter.
2. The use of claim 1, wherein the reagent comprises a primer combination for performing RT-qPCR, the primer combination comprising:
primer pairs shown in SEQ ID NO. 1-2 for amplifying the MYH1 gene;
primer pairs shown in SEQ ID NO. 3-4 for amplifying the MYH6 gene;
primer pairs shown in SEQ ID NO. 5-6 for amplifying the MYH7 gene;
primer pairs shown in SEQ ID NO. 7-8 for amplifying the MYH15 gene;
primer pairs shown in SEQ ID NO. 9-10 for amplifying the MYH2 gene;
primer pairs shown in SEQ ID NO. 11-12 for amplifying the WNT2B gene;
primer pairs shown in SEQ ID NO. 13-14 for amplifying the CDH1 gene;
and a primer pair shown in SEQ ID NO. 15-16 for amplifying the MYCB gene.
3. The use according to claim 2, wherein,
the primer pair shown as SEQ ID NO. 1-2 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 33;
the primer pair shown as SEQ ID NO. 3-4 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 34;
primer pairs shown as SEQ ID NO. 5-6 are used for detecting specific oligonucleotides shown as SEQ ID NO. 35;
the primer pair shown as SEQ ID NO. 7-8 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 36;
the primer pair shown as SEQ ID NO. 9-10 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 37;
the primer pair shown as SEQ ID NO. 11-12 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 38;
the primer pair shown as SEQ ID NO. 13-14 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 39;
the primer pair shown as SEQ ID NO. 15-16 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 40.
4. The application of a reagent for detecting the expression level of a gene combination in preparing a kit for predicting the immunity intensity of hybrid culter, wherein the gene combination is MAVS, EPO and SKAP2.
5. The use according to claim 4, the reagent comprising a primer combination for performing RT-qPCR, the primer combination comprising:
primer pairs shown in SEQ ID NO. 17-18 for amplifying MAVS gene;
primer pairs shown in SEQ ID NO. 19-20 for amplifying EPO gene; and
primer pairs shown in SEQ ID NO. 21-22 for amplifying SKAP2 gene.
6. The use according to claim 5, wherein,
the primer pair shown as SEQ ID NO. 17-18 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 41;
the primer pair shown as SEQ ID NO. 19-20 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 42;
the primer pairs shown as SEQ ID NO. 21-22 are used for detecting the specific oligonucleotides shown as SEQ ID NO. 43.
7. The application of a reagent for detecting the expression quantity of gene combinations, namely HK1, GAPDH, LDHA and PGK1, in the preparation of a kit for predicting the energy utilization efficiency of hybrid culter.
8. The use of claim 7, the reagent comprising a primer combination for performing RT-qPCR, the primer combination comprising:
primer pairs shown in SEQ ID NO. 23-24 for amplifying HK1 genes;
primer pairs shown in SEQ ID NO. 25-26 for amplifying GAPDH genes;
primer pairs shown in SEQ ID NO. 27-28 for amplifying LDHA genes; and
the primer pair is used for amplifying PGK1 genes shown as SEQ ID NO. 29-30.
9. The use according to claim 8, wherein,
the primer pair shown as SEQ ID NO. 23-24 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 44;
the primer pair shown as SEQ ID NO. 25-26 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 45;
the primer pair shown as SEQ ID NO. 27-28 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 46;
the primer pair shown as SEQ ID NO. 29-30 is used for detecting the specific oligonucleotide shown as SEQ ID NO. 47.
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