A method for constructing a zebrafish model for regulating muscle development

Abstract

The application discloses a kind of muscle development regulation zebra fish model construction method, it includes the following steps: by feeding N-acetylglucosamine to zebra fish, constructs O-GlcNAc glycosylation activation zebra fish model;And, by microinjection to zebra fish fertilized egg containing O-GlcNAc glycosidase gene fragment vector, constructs muscle-specific overexpression O-GlcNAc glycosidase zebra fish model.The application is verified bidirectionally to O-GlcNAc glycosylation function by constructing O-GlcNAc glycosylation activation model and muscle-specific OGA overexpression model, and it is revealed that O-GlcNAc glycosylation promotes muscle fiber thickening and whole fish protein deposition by up-regulating protein synthesis gene and muscle development regulatory factor.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing a zebrafish model of muscle development regulation. Background Technology

[0002] Protein metabolism is a core process in the growth and development of organisms, involving not only protein synthesis, folding, degradation, and homeostasis, but also cell growth, differentiation, and tissue repair. Given its central role in the nutritional metabolic system of organisms, in-depth exploration of its functions and metabolic regulatory mechanisms is beneficial for a more comprehensive understanding of the principles of nutritional balance and metabolic regulation in the body. Furthermore, proteins play a vital role in the growth and development of fish and are one of the essential nutrients for them.

[0003] O-linked N-acetylglucosamine (O-GlcNAc) modification is a post-translational modification (PTM) in which N-acetylglucosamine is covalently linked to a serine or threonine residue of a protein via an O-glycosidic bond. In recent years, with the deepening research on the mechanism of O-GlcNAc glycosylation modification, its function in the regulation of fish development has gradually attracted attention.

[0004] Studies have found that O-GlcNAc glycosylation modification participates in the regulation of multiple stages of early embryonic development and organogenesis in fish by modulating transcription factors and signaling pathway proteins. However, existing research mainly focuses on developmental regulation and neurodegenerative diseases. Research on the role of O-GlcNAc glycosylation modification in the regulation of protein metabolism in fish is still relatively lacking, especially the mechanism of action in muscle development and protein deposition has not been systematically elucidated. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for constructing a zebrafish model of muscle development regulation. By constructing an O-GlcNAc glycosylation activation model and a muscle-specific OGA overexpression model, the function of O-GlcNAc glycosylation is verified bidirectionally. This systematically reveals the key role of O-GlcNAc glycosylation in promoting muscle fiber thickening and whole-fish protein deposition by upregulating protein synthesis genes and muscle development regulatory factors.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing a zebrafish model of muscle development regulation is provided, which includes the following: (1) Weigh 1% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activation group during the week and 0.4% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activation group during the week and mix them to obtain the experimental group feed. Each time feeding was carried out, the experimental group was fed first, followed by the regular feed, which was 3% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activated group for that week, in order to construct the O-GlcNAc glycosylation activated zebrafish model. (2) Using zebrafish whole fish cDNA as a template, PCR amplification was performed using upstream primer mylpfa-F and downstream primer mylpfa-R. The amplification products were subjected to agarose gel electrophoresis to verify the size of the PCR amplification products. The gel was then cut, recovered, and purified to obtain the mylpfa gene promoter fragment. The nucleotide sequences of upstream primer mylpfa-F and downstream primer mylpfa-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Using zebrafish whole-fish cDNA as a template, PCR amplification was performed using upstream primer OGA-F and downstream primer OGA-R. The amplification products were then subjected to agarose gel electrophoresis to verify the size of the PCR amplification products. The gel was then excised and purified to obtain the zebrafish O-GlcNAc glycosidase gene fragment. The nucleotide sequences of upstream primer OGA-F and downstream primer OGA-R are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequence of the zebrafish O-GlcNAc glycosidase gene fragment is shown in SEQ ID NO.6. The pTOL2 plasmid vector was double-digested to divide it into two fragments of different sizes, and the larger fragment was recovered by gel extraction. The recovered large fragment, mylpfa gene promoter fragment, and zebrafish O-GlcNAc glycosidase gene fragment were homologously recombined using homologous recombinase to obtain recombinant plasmids. The recombinant plasmid was transformed into competent Escherichia coli DH5α, and the single colonies with correct sequencing results after transformation were expanded and cultured. After the expansion culture was completed, the endotoxin-free plasmid was extracted to obtain a muscle-specific overexpression O-GlcNAc glycosidase vector. A microinjection system containing a muscle-specific overexpression O-GlcNAc glycosidase vector was prepared, and the microinjection system was injected into fertilized eggs of wild-type zebrafish of the AB line. After the fertilized eggs hatch into zebrafish larvae, they are observed under a fluorescence microscope. Zebrafish larvae with fluorescent muscles are selected and cultured until they grow into sexually mature adults to construct a zebrafish model that specifically overexpresses O-GlcNAc glycosidase.

[0007] The present invention has at least the following beneficial effects:

[0008] This invention achieves bidirectional verification of the function of O-GlcNAc glycosylation through a dual strategy of constructing an O-GlcNAc glycosylation activation model and a muscle-specific OGA overexpression model. It systematically reveals the key role of O-GlcNAc glycosylation in promoting muscle fiber thickening and whole-fish protein deposition by upregulating protein synthesis genes and muscle development regulators. This provides a reliable research tool for in-depth analysis of the molecular mechanisms of fish nutrition metabolism and muscle development, and also provides a new theoretical basis for improving fish growth performance and muscle quality through nutritional intervention strategies in aquaculture. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0010] Figure 1 The body condition, feed efficiency, shell displacement ratio, and weight gain rate of zebrafish in the control group and the O-GlcNAc glycosylation activation group of this invention are shown.

[0011] Figure 2 The crude protein content of whole fish and crude protein content of muscle in zebrafish in the control group and the O-GlcNAc glycosylation activation group of this invention;

[0012] Figure 3 These are H&E stained sections of zebrafish muscle from the control group and the O-GlcNAc glycosylation activation group of this invention.

[0013] Figure 4 This is the frequency distribution of cross-sectional area of ​​muscle fibers in zebrafish in the control group and the O-GlcNAc glycosylation activation group of this invention.

[0014] Figure 5 The diameter of the muscle fibers of zebrafish in the control group and the O-GlcNAc glycosylation activation group of this invention;

[0015] Figure 6 This refers to the cross-sectional area of ​​zebrafish muscle fibers in the control group and the O-GlcNAc glycosylation activation group of this invention.

[0016] Figure 7 The expression levels of HBP pathway-related genes in the control group and the O-GlcNAc glycosylation activation group of this invention are shown.

[0017] Figure 8 The expression level of N-acetylglucosamine kinase gene in zebrafish muscle in the control group and the O-GlcNAc glycosylation activation group of this invention;

[0018] Figure 9This refers to the expression level of the O-GlcNAc transferase gene in zebrafish muscle in the control group and the O-GlcNAc glycosylation activation group of this invention.

[0019] Figure 10 The expression levels of protein catabolism-related genes in zebrafish muscle in the control group and the O-GlcNAc glycosylation activation group of this invention;

[0020] Figure 11 The expression levels of protein synthesis-related genes in zebrafish muscle in the control group and the O-GlcNAc glycosylation activation group of this invention;

[0021] Figure 12 These are H&E stained sections of zebrafish muscle from the control group and OGA overexpression group of this invention.

[0022] Figure 13 These are H&E stained sections of adult zebrafish muscle from the control group and OGA overexpression group of this invention.

[0023] Figure 14 The frequency distribution, diameter, and cross-sectional area of ​​muscle fibers in the muscle of zebrafish juveniles in the control group and OGA overexpression group of this invention are shown.

[0024] Figure 15 The frequency distribution, diameter, and cross-sectional area of ​​muscle fibers in adult zebrafish muscle in the control group and OGA overexpression group of this invention are shown.

[0025] Figure 16 Images of OGT, OGA, and O-GlcNAc protein bands in the muscle of adult zebrafish in the control group and OGA overexpression group of this invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1:

[0028] This embodiment provides a method for constructing a zebrafish model of muscle development regulation based on O-GlcNAc glycosylation, which includes constructing an O-GlcNAc glycosylation activated zebrafish model and constructing a zebrafish model of muscle-specific overexpression of OGA (i.e., O-GlcNAc glycosidase).

[0029] Specifically, the construction of the O-GlcNAc glycosylation activated zebrafish model includes the following steps:

[0030] S1. Disinfect the breeding container with potassium permanganate. After disinfection, remove all 2-3 month old zebrafish ( Danio rerio The animals were transferred to breeding containers for temporary rearing for two weeks. During this period, air stones were added to the breeding containers to ensure sufficient oxygen. The water temperature in the breeding containers was maintained at 28±1℃ using heating rods and other equipment. The animals were fed commercial feed twice a day during the temporary rearing period.

[0031] S2. After the temporary holding period, healthy zebrafish were selected and divided into a control group and an experimental group (i.e., the "O-GlcNAc glycosylation activation group"). Each treatment group had 3 replicates, and each replicate contained 50 zebrafish. The initial average weight of the zebrafish in each replicate was 0.2826±0.0005 g / fish.

[0032] S3. Zebrafish in each treatment group were cultured for 4 weeks to conduct O-GlcNAc glycosylation activation culture experiment. During the activation culture experiment, the zebrafish in each treatment group were weighed weekly, and 1 / 3 of the water volume in the culture container was changed every two days. They were fed twice a day at 9:00 am and 5:00 pm. At the same time, before each feeding, the feces at the bottom of the culture container were sipped with a pipette to keep the water clean.

[0033] Zebrafish in the O-GlcNAc glycosylation activation group were fed the following method:

[0034] Weigh 1% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activation group during the week and mix it with 0.4% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activation group during the week to obtain the experimental group feed.

[0035] Each time feeding, the experimental group feed was given first, followed by the regular feed, which was 3% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activation group for that week.

[0036] The zebrafish in the control group were fed as follows:

[0037] Each time the fish were fed, they were first fed a regular diet consisting of 1% of the total body weight of the control group zebrafish for that week, and then fed a regular diet consisting of 3% of the total body weight of the control group zebrafish for that week.

[0038] The formulation of the conventional feed is shown in Table 1:

[0039] Table 1. Conventional Feed Formulation Table

[0040]

[0041] Note: Vitamin premix contains 500,000 mg of vitamin A, 10,000 mg of vitamin C, 50,000 mg of vitamin D3, 2,500 mg of vitamin E, 1,000 mg of vitamin K3, 5,000 mg of vitamin B1, 5,000 mg of vitamin B2, 5,000 mg of vitamin B6, and 5,000 μg of vitamin B1 per kg. 12 25,000 mg inositol, 10,000 mg pantothenic acid, 100,000 mg choline, 25,000 mg niacin, 1,000 mg folic acid, 250 mg biotin, with the remainder being wheat bran.

[0042] The mineral premix contains 314.0g of calcium carbonate, 469.3g of potassium dihydrogen phosphate, 147.4g of magnesium sulfate, 49.8g of sodium chloride, 10.9g of ferric gluconate (II), 4.67g of zinc sulfate, 3.12g of manganese sulfate, 0.62g of copper sulfate, 0.16g of potassium iodide, 0.08g of cobalt chloride, 0.06g of ammonium molybdate, and 0.02g of sodium selenite per 1kg, with the remainder being wheat bran.

[0043] After the S4, O-GlcNAc glycosylation activation culture experiment, all zebrafish were subjected to a 12-hour overnight fasting treatment. After the overnight fasting treatment, zebrafish in each parallel were anesthetized by placing them in an ice-water mixture, drying off their body surface moisture, weighing them quickly, and measuring their body length. Then, zebrafish in each parallel were dissected and sampled, and muscle samples were separated to obtain muscle samples from each treatment group of zebrafish.

[0044] Muscle samples from each treatment group were frozen in liquid nitrogen and stored at -80 ℃ for subsequent testing; meanwhile, a portion of whole zebrafish samples from each treatment group were retained for whole fish crude protein content testing.

[0045] S5. Statistical analysis was performed on the growth performance indicators and biochemical indicators of zebrafish in each treatment group. The growth performance indicators included weight gain rate, condition factor, body size ratio and feed efficiency.

[0046] It should be noted that all data results in this embodiment are expressed in the form of mean ± standard error (Mean ± SEM), and the independent samples t-test is used to test the significant differences between two groups, while one-way ANOVA is used to test the significant differences between multiple groups; wherein, " " indicates p < 0.05, " indicates p < 0.01", " indicates p < 0.001, " indicates that p < 0.0001.

[0047] The results of statistical analysis of the growth performance indicators of zebrafish in each treatment group are as follows: Figure 1 As shown in the figure, compared with the control group, the weight gain rate, condition factor, body size ratio and feed efficiency of zebrafish in the O-GlcNAc glycosylation activation group were significantly increased, indicating that adding GlcNAc to the conventional feed can promote the growth of zebrafish.

[0048] Specifically, the biochemical indicators of zebrafish in each treatment group were statistically analyzed, including the following:

[0049] 1. Crude protein content detection

[0050] The crude protein content in whole zebrafish samples and muscle samples from the control group and the O-GlcNAc glycosylation activation group was determined by the Kjeldahl method. The results are as follows: Figure 2 As shown.

[0051] 2. Tissue H&E staining

[0052] Fresh zebrafish muscle samples from each treatment group were fixed in muscle fixative for 24 hours, followed by paraffin embedding, sectioning, and staining to observe zebrafish muscle fibers. The stained sections are shown below. Figure 3 As shown.

[0053] Simultaneously, data analysis was performed on the stained images using tools such as GraphPad Prism 9.0 software to obtain, respectively, Figure 4-6 The frequency distribution of muscle fiber cross-sectional area, muscle fiber diameter, and muscle fiber cross-sectional area are shown.

[0054] from Figure 2 It can be seen that, compared with the control group, the crude protein content in the whole fish and muscle of zebrafish in the O-GlcNAc glycosylation activation group was significantly increased. Meanwhile, from... Figure 3 The H&E staining results showed that, compared with the control group, the muscle fibers of zebrafish in the O-GlcNAc glycosylation activation group were significantly thicker. Simultaneously, the muscle fiber diameter and cross-sectional area of ​​the O-GlcNAc glycosylation activation group were significantly increased compared with the control group. Furthermore, the statistical distribution of muscle fiber area frequency also showed that the O-GlcNAc glycosylation activation group mainly consisted of muscle fibers with larger cross-sectional areas, while the control group mainly consisted of muscle fibers with smaller cross-sectional areas.

[0055] The results indicate that activation of O-GlcNAc glycosylation can promote increased protein deposition in the whole zebrafish and muscle, and that O-GlcNAc glycosylation may play an important role in zebrafish muscle development and protein synthesis.

[0056] 3. Gene expression analysis

[0057] 3.1 Detection of expression levels of genes related to the HBP pathway (Hexosamine biosynthesis pathway), N-acetylglucosamine kinase, and O-GlcNAc transferase genes.

[0058] RNA was extracted from muscle samples using extraction kits such as MagZolReagent (Meiji Biotechnology, China). The specific steps were performed according to the instructions of the extraction kit.

[0059] RNA of suitable quality and concentration was reverse transcribed to synthesize cDNA. Primer sequences for the following enzymes were designed and synthesized using the NCBI website and Oligo 7 software: glucose-6-phosphate isomerase, glutamine-6-phosphate fructose acyltransferase 1, glutamine-6-phosphate fructose acyltransferase 2, glucosamine-6-phosphate acetyltransferase 1, acetylglucosamine phosphate mutase 3, UDP-N-acetylglucosamine synthetase 1 (these enzyme genes are related to the HBP pathway), as well as N-acetylglucosamine kinase and O-GlcNAc transferase. The specific primer sequences are shown in Table 2.

[0060] Table 2 Primer Sequence List for Real-Time Quantitative PCR

[0061]

[0062] A 10 μL real-time quantitative PCR reaction system was prepared using SYBR Mix (Nanjing Novizan Biotechnology, China). The real-time quantitative PCR reaction system included 5 μL of SYBR, 0.5 μL of upstream primer F, 0.5 μL of downstream primer R, 1 μL of cDNA, and 3 μL of enzyme-free water.

[0063] Mix the above real-time quantitative PCR reaction system, add it to a 96-well plate, centrifuge, and then place it in a CFX ConnectReal Time System (Bio-Rad, USA) instrument for real-time quantitative PCR reaction.

[0064] After the reaction, the specificity of the primers was tested using melting curves. β-actin and elongation factor 1-alpha were used as internal reference genes. The relative expression levels of HBP pathway-related genes, as well as N-acetylglucosamine kinase and O-GlcNAc transferase, were calculated. The results are shown below. Figure 7-9 As shown.

[0065] Real-time quantitative PCR revealed that, compared with the control group, the expression of O-GlcNAc transferase (OGT) in zebrafish from the O-GlcNAc glycosylation activation group was significantly increased, and the expression of HBP pathway-related genes was also increased. Simultaneously, the expression of N-acetylglucosamine kinase gene in the muscle of zebrafish from the O-GlcNAc glycosylation activation group was also significantly increased compared with the control group.

[0066] O-GlcNAc transferase is responsible for catalyzing the addition of GlcNAc to Ser / Thr residues of protein substrates. Significantly elevated OGT levels in muscle indicate that supplementing a conventional diet with GlcNAc promotes O-GlcNAc glycosylation in zebrafish muscle. Simultaneously, increased expression of the N-acetylglucosamine kinase gene in muscle allows O-GlcNAc to enter the HBP pathway via N-acetylglucosamine kinase, further efficiently converting O-GlcNAc to UDP-GlcNAc through the HBP pathway. This provides substrate support for O-GlcNAc glycosylation, thereby promoting its occurrence.

[0067] 3.2 Detection of expression levels of genes related to protein catabolism and protein anabolism

[0068] The expression levels of genes related to protein catabolism and protein synthesis in zebrafish muscle samples were also obtained using real-time quantitative PCR. (For gene names, please refer to [link to relevant documentation]). Figure 10-11 The amplification primer sequences for the x-axis were also designed and synthesized using the NCBI website and Oligo 7 software. The preparation and reaction steps for quantitative real-time PCR were the same as in 3.1, and will not be repeated here. The gene expression level detection results are as follows: Figure 10-11 As shown.

[0069] Compared with the control group, the expression of genes related to protein synthesis and metabolism was significantly increased in the zebrafish muscle of the O-GlcNAc glycosylation activation group, while there was no significant difference in the expression of genes related to protein catabolism between the two groups. This indicates that the increase in the level of O-GlcNAc glycosylation modification can promote the synthesis of zebrafish muscle proteins, thereby promoting protein deposition.

[0070] Therefore, this embodiment successfully constructed an O-GlcNAc glycosylation-activated zebrafish model by adding GlcNAc to conventional feed. This model can be used to accurately evaluate the effects of GlcNAc on zebrafish growth performance and O-GlcNAc glycosylation levels.

[0071] The construction of the muscle-specific OGA overexpression zebrafish model includes the following steps:

[0072] S1. Using zebrafish whole-fish cDNA as a template, PCR amplification was performed using upstream primer mylpfa-F and downstream primer mylpfa-R (nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively). The amplification products were subjected to agarose gel electrophoresis to verify the size of the PCR amplification products. The gel was then excised and purified to obtain the mylpfa gene promoter fragment (this mylpfa gene promoter fragment is the zebrafish muscle-specific overexpression OGA promoter fragment). The nucleotide sequence of the mylpfa gene promoter fragment is shown in SEQ ID NO.3, and each end of the mylpfa gene promoter fragment has a corresponding homologous recombination site.

[0073] Furthermore, using zebrafish whole-fish cDNA as a template, PCR amplification was performed using upstream primer OGA-F and downstream primer OGA-R (nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO.5, respectively). The amplification products were subjected to agarose gel electrophoresis to verify the size of the PCR amplification products. The gel was then excised and purified to obtain the zebrafish OGA gene fragment. The nucleotide sequence of the zebrafish OGA gene fragment is shown in SEQ ID NO.6, and each end of the zebrafish OGA gene fragment corresponds to a homologous recombination site.

[0074] Specifically, prepare the PCR amplification system according to Table 3 and complete the amplification according to the following procedure: 98℃ for 10 sec, 55℃ for 15 sec, 68℃ for 1 min / kb, 35 cycles; incubate at 4℃.

[0075] Table 3 PCR System

[0076]

[0077] S2. The pTOL2 plasmid vector was double-digested with XHOI and SACI to divide it into a large fragment of 4718bp and a small fragment of 297bp, and the large fragment was recovered by gel extraction.

[0078] S3. The recovered large fragment, mylpfa gene promoter fragment, and OGA gene fragment are homologously recombined using a homologous recombinase to obtain a recombinant plasmid. In this embodiment, the homologous recombinase is Novitan homologous recombinase, and the reaction system is prepared according to the product instructions.

[0079] S4. Transform the recombinant plasmid into competent Escherichia coli DH5α, which specifically includes the following steps:

[0080] 10 μL of recombinant plasmid and 100 μL of competent Escherichia coli DH5α were mixed to obtain a mixture. The mixture was then placed in an ice bath for 30 min, followed by heat shock at 42 °C for 45 s, and then in an ice bath for 2 min.

[0081] Add 500 μL of antibiotic-free LB liquid medium to the mixture, place it on a shaker, and incubate at 200 rpm for 1 h. After the incubation is complete, spread the mixture evenly on LB solid medium plates with Kana resistance and incubate at 37°C overnight.

[0082] S5. Select a single colony from the culture medium plate and perform PCR amplification using upstream primer Test-F and downstream primer Test-R (nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8). Perform gel electrophoresis on the amplification products and select the band with a fragment size of approximately 5974 bp for sequencing. If the sequencing results are correct, add the single colony corresponding to the band (i.e., the single colony corresponding to the band with a fragment size of approximately 5974 bp) to 100 mL of LB liquid medium with kana resistance for expansion culture for 12-14 h. After the expansion culture is completed, extract the endotoxin-free plasmid to obtain the muscle-specific overexpression OGA vector.

[0083] S6. Prepare a microinjection system with a total volume of 3 μL, wherein the final concentration of muscle-specific overexpression OGA vector is 50 ng / μL, the concentration of Tol 2 mRNA is 20 ng / μL, and phenol red staining solution is included as an indicator.

[0084] S7. Select healthy AB-type wild-type zebrafish and divide them into a control group and an OGA overexpression group. Each treatment group has 3 replicates, and each replicate contains 50 zebrafish, and each replicate contains both female and male zebrafish. The zebrafish in the control group and the OGA overexpression group are fed the same conventional diet and in the same way.

[0085] After the male and female AB wild-type zebrafish in the OGA overexpression group mate and produce fertilized eggs, the microinjection system is injected into the animal pole of the fertilized eggs in the OGA overexpression group at a volume of 1-2 nl. After injection, the fertilized eggs are cultured at 37℃. After the male and female AB wild-type zebrafish in the control group mate and produce fertilized eggs, they are not subjected to microinjection treatment and are allowed to hatch naturally.

[0086] During the hatching process of fertilized eggs, dead fertilized eggs were removed every 4 hours. After the fertilized eggs in the OGA overexpression group hatched into zebrafish fry, they were observed under a fluorescence microscope. Zebrafish fry with fluorescent muscles were selected and continued to be cultured until the zebrafish grew into sexually mature adult fish. After the fertilized eggs in the control group hatched into zebrafish fry, they were allowed to grow into sexually mature adult fish naturally.

[0087] After obtaining juvenile and adult zebrafish in the control group and OGA overexpression group, all zebrafish were subjected to a 12-hour overnight fasting treatment. After the overnight fasting treatment, muscle samples were collected from zebrafish in each parallel, and the muscle samples were subjected to tissue H&E staining and protein expression analysis.

[0088] The tissue H&E staining method is the same as in section "2. Tissue H&E Staining", and will not be repeated here. The sample staining results are as follows: Figure 12-13 As shown. Similarly, data analysis was performed on the stained images to obtain the results as follows. Figure 14 The frequency distribution of cross-sectional area of ​​muscle fibers in juvenile fish, muscle fiber diameter, and muscle fiber cross-sectional area are shown, as well as, for example, Figure 15 The frequency distribution of cross-sectional area of ​​muscle fibers in adult fish, muscle fiber diameter, and muscle fiber cross-sectional area are shown.

[0089] like Figure 12-13 As shown, the muscle fibers of juvenile and adult fish in the OGA overexpression group were thinner, while the muscle fibers of juvenile and adult fish in the control group were thicker. Furthermore, as... Figure 14-15 As shown, compared with the control group, the diameter and cross-sectional area of ​​muscle fibers in juvenile and adult fish in the OGA overexpression group were significantly reduced. The frequency distribution of muscle fiber cross-sectional area also showed that the OGA overexpression group mainly had muscle fibers with smaller cross-sectional areas, while the control group mainly had muscle fibers with larger cross-sectional areas.

[0090] O-GlcNAc glycosidase (OGA) is responsible for removing GlcNAc molecules linked to the Ser / Thr residues of O-GlcNAc glycosylated proteins to inhibit O-GlcNAc glycosylation. The results show that muscle-specific overexpression of OGA to inhibit O-GlcNAc glycosylation significantly reduced the diameter of zebrafish muscle fibers, indicating that O-GlcNAc glycosylation plays a key role in zebrafish muscle development and maintenance, and further confirming that activation of O-GlcNAc glycosylation promotes zebrafish growth.

[0091] Furthermore, in this embodiment, Western blotting was performed on muscle samples from adult fish in the control group and the OGA overexpression group to obtain protein bands of OGT protein, OGA protein, and O-GlcNAc protein (e.g., Figure 16 As shown in the figure, the process is as follows:

[0092] Weigh an appropriate amount of muscle sample and place it in a 1.5 mL enzyme-free EP tube containing homogenization beads for homogenization. Add the pre-prepared lysis buffer, which is prepared according to the product instructions of Beyotime Biotechnology Co., Ltd. by mixing RIPA lysis buffer (Beyotime Biotechnology, China) and PMSF (Beyotime Biotechnology, China) at a volume ratio of 100:1.

[0093] After homogenization, place the mixture on ice in an ice bath, then centrifuge. Take the supernatant and mix it with 5×SDS protein loading buffer. Place the mixture in a boiling water bath for 10 min to denature the protein and prepare the protein sample to be tested.

[0094] Protein samples were separated by SDS-PAGE electrophoresis, then transferred to a nitrocellulose membrane for transfer, followed by blocking with a rapid blocking buffer. The blocking buffer was then recovered, and the primary antibody to be detected was added. The mixture was incubated overnight at 4 °C on a shaker. The next day, the samples were washed three times with 1×TBST solution containing 1% Tween for 10 min each time. Rabbit or mouse secondary antibody was then selected according to the species of the primary antibody and incubated on a shaker at an appropriate speed in the dark for 1 h. After incubation, the secondary antibody was recovered and washed three times with 1×TBST solution containing 1% Tween for 10 min each time.

[0095] Finally, the target protein band was scanned using an Odyssey imaging system (Li-Cor Biotechnology, USA) or a chemiluminescence imaging system (BioRad, USA) to obtain images such as... Figure 16 The target protein band is shown.

[0096] from Figure 16As can be seen, the O-GlcNAc protein band in the control group was thicker, indicating that its content was significantly higher than that in the OGA overexpression group. The OGA protein content was significantly lower than that in the OGA overexpression group, indicating that the O-GlcNAc glycosylation level in the zebrafish muscle of the control group was significantly higher than that in the OGA overexpression group. However, there was no significant difference in OGT expression, indicating that overexpression of OGA in zebrafish muscle can significantly reduce the O-GlcNAc glycosylation level.

[0097] In summary, current research on the role of O-GlcNAc glycosylation in the regulation of protein metabolism in fish is still relatively lacking, especially regarding its mechanism of action in muscle development and protein deposition, which has not yet been systematically elucidated. This invention utilizes a dual strategy—constructing an O-GlcNAc glycosylation activation model and a muscle-specific OGA overexpression model—to achieve bidirectional verification of the function of O-GlcNAc glycosylation. It systematically reveals the crucial role of O-GlcNAc glycosylation in promoting muscle fiber thickening and whole-fish protein deposition by upregulating protein synthesis genes and muscle development regulators.

[0098] Therefore, this invention not only provides a reliable research tool for in-depth analysis of the molecular mechanisms of fish nutrient metabolism and muscle development, but also provides a new theoretical basis for improving fish growth performance and muscle quality through nutritional intervention strategies in aquaculture.

[0099] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for constructing a zebrafish model of muscle development regulation, characterized in that, Includes the following: (1) Weigh 1% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activation group during the week and 0.4% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activation group during the week and mix them to obtain the experimental group feed. Each time feeding was carried out, the experimental group was fed first, followed by the regular feed, which was 3% of the total body weight of the zebrafish in the O-GlcNAc glycosylation activated group for that week, in order to construct the O-GlcNAc glycosylation activated zebrafish model. (2) Using zebrafish whole fish cDNA as a template, PCR amplification was performed using upstream primer mylpfa-F and downstream primer mylpfa-R. The amplification products were subjected to agarose gel electrophoresis to verify the size of the PCR amplification products. The gel was then cut, recovered, and purified to obtain the mylpfa gene promoter fragment. The nucleotide sequences of upstream primer mylpfa-F and downstream primer mylpfa-R are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Using zebrafish whole-fish cDNA as a template, PCR amplification was performed using upstream primer OGA-F and downstream primer OGA-R. The amplification products were then subjected to agarose gel electrophoresis to verify the size of the PCR amplification products. The products were then excised, recovered, and purified to obtain the zebrafish O-GlcNAc glycosidase gene fragment. The nucleotide sequences of upstream primer OGA-F and downstream primer OGA-R are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively, and the nucleotide sequence of the zebrafish O-GlcNAc glycosidase gene fragment is shown in SEQ ID NO.

6. The pTOL2 plasmid vector was double-digested to divide it into two fragments of different sizes, and the larger fragment was recovered by gel extraction. The recovered large fragment, mylpfa gene promoter fragment, and zebrafish O-GlcNAc glycosidase gene fragment were homologously recombined using homologous recombinase to obtain recombinant plasmids. The recombinant plasmid was transformed into competent Escherichia coli DH5α, and the single colonies with correct sequencing results after transformation were expanded and cultured. After the expansion culture was completed, the endotoxin-free plasmid was extracted to obtain a muscle-specific overexpression O-GlcNAc glycosidase vector. A microinjection system containing a muscle-specific overexpression O-GlcNAc glycosidase vector was prepared, and the microinjection system was injected into fertilized eggs of wild-type zebrafish of the AB line. After the fertilized eggs hatch into zebrafish larvae, they are observed under a fluorescence microscope. Zebrafish larvae with fluorescent muscles are selected and cultured until they grow into sexually mature adults to construct a zebrafish model that specifically overexpresses O-GlcNAc glycosidase.

2. The zebrafish model construction method as described in claim 1, characterized in that, By weight percentage, the conventional feed comprises: 35% casein, 8.88% gelatin, 30% corn starch, 7% soybean oil, 0.4% vitamin premix, 0.4% mineral premix, 0.2% choline chloride, 0.02% 2,6-di-tert-butylhydroxytoluene, 0.1% thiamethoxam dimethyl-β-propionate, 1% calcium dihydrogen phosphate, 2% carboxymethyl cellulose, and 15% cellulose.

3. The zebrafish model construction method as described in claim 1, characterized in that, During the rearing of zebrafish with the O-GlcNAc glycosylation activation group, they were fed twice a day, at 9:00 AM and 5:00 PM.

4. The zebrafish model construction method as described in claim 1, characterized in that, In the microinjection system, the final concentration of muscle-specific overexpression of O-GlcNAc glycosidase vector was 50 ng / μL, and the concentration of Tol2 mRNA was 20 ng / μL.

5. The zebrafish model construction method as described in claim 1, characterized in that, The microinjection system is injected into the animal pole of the zebrafish fertilized egg.

6. The zebrafish model construction method as described in claim 5, characterized in that, The injection volume of the microinjection system is 1-2 nl.

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