Method for relieving fatty liver of lateolabrax japonicus and improving muscle quality based on MSTN gene interference

Abstract

The invention belongs to the technical field of development of molecular nutrition and microcapsule sustained-release particles of aquatic animals, and discloses a method for relieving fatty liver of lateolabrax japonicus and improving muscle quality based on MSTN (myostatin) gene interference, which comprises the following steps: designing a specific fragment according to an MSTN gene coding sequence of lateolabrax japonicus, inserting the specific fragment into an RNA (Ribonucleic Acid) interference vector L4440, constructing a recombinant plasmid, and transforming the recombinant plasmid into HT115 (DE3) escherichia coli; the preparation method comprises the following steps: carrying out multiplication culture in a culture medium containing double antibodies, carrying out IPTG induced expression to generate double-stranded RNA (dsRNA), collecting expressed thalli, and carrying out treatment inactivation with 75% ethanol to prepare microcapsule sustained-release particles. After the microcapsule sustained-release particles are orally delivered, the dsRNA carried by the microcapsule sustained-release particles is subjected to enzyme digestion in the body of lateolabrax japonicus to generate siRNA, and then gene silencing is induced, so that the expression level of the lateolabrax japonicus is remarkably reduced. The interference effect can adjust lipid metabolism distribution between fish body muscles and livers, reduce liver lipid deposition and promote lipid accumulation in the muscles, so that the dual purposes of relieving fatty liver and improving muscle quality are achieved.

Description

Technical Field

[0001] This invention belongs to the field of molecular nutrition and microcapsule sustained-release particle development technology for aquatic animals, and particularly relates to a method for alleviating fatty liver of spotted sea bass and improving muscle quality based on MSTN gene interference. Background Technology

[0002] Spotted sea bass ( Lateolabrax maculatus The spotted sea bass (Siniperca chuatsi) is an important marine aquaculture fish in my country, characterized by its rapid growth and high market value. In recent years, with the widespread use of high-energy feeds and the promotion of high-density intensive aquaculture, lipid metabolism disorders have become increasingly prominent in spotted sea bass farming, manifesting as abnormal lipid accumulation in the liver, fatty degeneration, and an increased incidence of fatty liver. Under these metabolically abnormal conditions, the fish's functions of lipid absorption, transport, and distribution are disrupted, leading to excessive lipid deposition in the liver and insufficient lipid deposition in peripheral tissues such as skeletal muscle, resulting in a metabolic characteristic of "lipid imbalance." This not only disrupts the fish's metabolic homeostasis but also severely affects the fatty acid composition, texture, and flavor characteristics of muscle tissue, thereby reducing the quality of the fish meat and its overall commercial value.

[0003] Currently, aquaculture practices largely rely on feed nutrition regulation or functional additives to prevent fatty liver disease. For example, some patents propose improving liver lipid deposition and enhancing lipid metabolism efficiency by adding functional factors such as bile acids, taurine, and β-glucan (CN104171648A); others use compound components such as chitosan oligosaccharide, rare earth chelate salts, and milk thistle extract to enhance antioxidant capacity and achieve liver protection and metabolic regulation (CN114365802A). Although these strategies have some alleviating effect on fatty liver, most are still limited to the level of nutritional regulation and fail to precisely intervene in lipid metabolism pathways at the molecular level. In addition, most existing technologies focus on reducing liver lipid accumulation, paying insufficient attention to lipid deposition in muscle tissue and improving meat quality, and lacking systematic lipid redistribution regulation schemes. In actual aquaculture, fish metabolic health and muscle quality are equally important, and a strategy of simply inhibiting liver lipid accumulation is difficult to comprehensively improve overall aquaculture efficiency. At the same time, current additive methods still face problems such as low absorption rate, poor bioavailability, and unclear mechanisms of action, making it difficult to achieve long-term and efficient metabolic regulation.

[0004] Recent studies have revealed that myostatin (MSTN) not only plays a negative regulatory role in muscle development but also participates in regulating lipid metabolism. Existing research has shown that knocking out or downregulating MSTN gene expression can achieve a dual effect of muscle hyperplasia and lipid redistribution in multiple species. However, there is currently no research on lipid metabolism regulation based on MSTN gene interference technology for spotted sea bass, and published literature and patents lack molecular strategies to achieve "reduced liver lipids and increased muscle fat" using this target. Therefore, there is an urgent need to develop a molecular regulation method based on MSTN gene interference and to achieve oral delivery using a feedable and safe carrier, thereby establishing an efficient, safe, and controllable precise intervention method for lipid metabolism in spotted sea bass. This invention addresses the aforementioned technological gaps and industry needs, proposing an innovative, practical, and scalable solution that provides new ideas and pathways for healthy aquaculture.

[0005] Based on the above analysis, the problems and shortcomings of the existing technology are as follows: (1) Most existing technologies focus on reducing lipid accumulation in the liver, but pay insufficient attention to lipid deposition in muscle tissue and improvement of meat quality, and lack systematic lipid redistribution regulation schemes.

[0006] (2) In actual aquaculture, fish metabolic health is just as important as muscle quality, and a strategy of simply inhibiting hepatic lipid accumulation is unlikely to comprehensively improve the overall aquaculture efficiency. At the same time, current additive methods still face problems such as low absorption rate, poor bioavailability, and unclear mechanism of action, making it difficult to achieve long-term and efficient metabolic regulation. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a method for alleviating fatty liver in sea bass and improving muscle quality based on MSTN gene interference.

[0008] This invention is implemented as follows: A method for alleviating fatty liver and improving muscle quality in spotted bass based on MSTN gene interference includes: Step 1: Design specific primers based on the coding sequence (CDS) of the MSTN gene of the spotted bass to amplify the target fragment of 500-600 bp in the conserved functional region and insert it into the RNA interference vector L4440 with a double T7 promoter to construct a recombinant plasmid targeting MSTN. Step 2: The constructed recombinant plasmid was transformed into RNase III-deficient Escherichia coli HT115(DE3), and positive clones were screened in LB liquid medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL); when the bacterial culture optical density (OD) reached a certain level, the positive clones were selected. 600When the expression level reaches 0.4–0.6, add 0.6 mmol / L isopropyl-β-D-thiogalactoside (IPTG) and induce culture at 37°C for 5–6 hours to obtain the optimal dsRNA expression level. Step 3: After collecting the expression product, the bacterial cells are inactivated with 75% ethanol to ensure biosafety and maintain the structural stability of dsRNA. The inactivated bacterial cells can be stored for a long time at -80℃, or freeze-dried with the addition of a protectant and then transported at room temperature. Step 4: Purify the total RNA of IPTG-induced HT115 cells using an RNase-free process; encapsulate the dsRNA into 200–300 μm microspheres using a chitosan-sodium tripolyphosphate ionogel method, and then spray a liposome-palmitic acid membrane (waterproof and trypsin-resistant) on the outside of the chitosan layer to obtain microcapsule sustained-release granules; the drug loading is 2% (w / w), that is, 1g of microcapsule sustained-release granules contains 20mg of dsRNA; after vacuum drying at 40℃, store; sprinkle directly on the surface of floating feed at a rate of 2.5g of microcapsule sustained-release granules / kg of fish body weight, and the granules will adhere by "splashing and rolling" when the fish are feeding; feeding once a week can maintain an MSTN inhibition rate of over 70%.

[0009] Furthermore, the MSTN interference fragment is designed based on the coding sequence (CDS) of the MSTN gene of the spotted bass, preferably with a length of 500-600 bp, located in a conserved functional region of the gene, to ensure interference efficiency and target specificity.

[0010] Furthermore, the RNA interference vector L4440 contains a dual T7 promoter structure, which can bidirectionally transcribe to form stable dsRNA for targeted interference with the MSTN gene.

[0011] Furthermore, the host bacteria of the recombinant engineered bacteria are selected from HT115(DE3), which is an RNase III-deficient Escherichia coli that can stably express exogenous dsRNA and is suitable for large-scale bacterial cell production.

[0012] Furthermore, the recombinant engineered bacteria were amplified and cultured in a double-antibiotic medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL). When the bacterial culture OD... 600 When the expression level reaches 0.4–0.6, 0.6 mmol / L IPTG is added for induction, and the cells are induced at 37°C for 5–6 hours to obtain the optimal dsRNA expression level.

[0013] Furthermore, the recombinant bacteria, after collection and inactivation with 75% ethanol, can be stored for a long time at -80℃, or freeze-dried with added protectant and transported at room temperature, maintaining the integrity of the bacterial morphology and the stability of the dsRNA structure, which facilitates large-scale feed application.

[0014] Another objective of this invention is to provide a system for alleviating fatty liver in sea bass and improving muscle quality based on MSTN gene interference, comprising: The amplification module is used to design specific primers based on the coding sequence (CDS) of the MSTN gene of the spotted bass, amplify the target fragment of 500-600 bp in the conserved functional region, and insert it into the RNA interference vector L4440 with a double T7 promoter to construct a recombinant plasmid targeting MSTN. The recombinant module is used to transform the constructed recombinant plasmid into RNase III-deficient *Escherichia coli* HT115(DE3), and screen for positive clones in LB liquid medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL); when the bacterial culture optical density (OD) reaches a certain level... 600 When the expression level reaches 0.4–0.6, add 0.6 mmol / L isopropyl-β-D-thiogalactoside (IPTG) and induce culture at 37°C for 5–6 hours to obtain the optimal dsRNA expression level. The inactivation module is used to inactivate the bacterial cells with 75% ethanol after collecting the expression products, ensuring biosafety and maintaining the structural stability of dsRNA. The inactivated bacterial cells can be stored for a long time at -80℃, or freeze-dried with the addition of a protectant and then transported at room temperature. The microcapsule sustained-release particle preparation module is used to purify IPTG-induced total RNA from HT115 using an RNase-free process. The dsRNA is encapsulated into 200–300 μm microspheres using a chitosan-sodium tripolyphosphate ionogel method. A liposome-palmitic acid membrane (waterproof and trypsin-resistant) is then sprayed onto the chitosan layer to obtain the microcapsule sustained-release particles. The drug loading is 2% (w / w), meaning 1g of microcapsule sustained-release particles contains 20mg of dsRNA. After vacuum drying at 40℃, the particles are stored. They are then sprinkled directly onto the surface of floating feed at a rate of 2.5g of microcapsule sustained-release particles per kg of fish body weight, where they adhere due to the fish's "splashing and rolling" action when feeding. Feeding once a week is sufficient to maintain an MSTN inhibition rate of over 70%.

[0015] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: 1. Innovative Bidirectional Lipid Metabolism Regulation Mechanism Based on MSTN Gene: This invention is the first to design an interference fragment targeting the conserved functional region of the MSTN gene in sea bass, achieving long-term inhibition of MSTN expression via oral administration, thus constructing a novel reversible and controllable lipid metabolism regulation strategy. Unlike traditional nutritional regulation methods that can only unidirectionally inhibit hepatic lipid deposition, this invention can reduce hepatic lipid droplet accumulation while promoting rational lipid deposition in muscle tissue, thereby optimizing lipid redistribution and energy utilization efficiency at the overall metabolic level.

[0016] 2. Construction of a safe and efficient oral delivery system for dsRNA: This invention uses HT115 (DE3) as the expression host, leveraging its RNase III deficiency to stably express exogenous MSTN-targeting dsRNA. The RNA is inactivated by 75% ethanol to ensure biosafety and RNA structural integrity. The dsRNA microcapsules, formed using a chitosan-sodium tripolyphosphate ionogel method, are further coated with a liposome-palmitic acid composite membrane, achieving waterproofing, protection against trypsin degradation, and sustained-release absorption. This design allows for stable release of RNA interference substances in the intestine, producing systemic interference without microinjection or cell transfection, significantly simplifying operation, reducing costs, and exhibiting good biosafety and feed applicability.

[0017] 3. Significant physiological improvement effects and application prospects: MSTN gene expression levels can be stably downregulated by approximately 60%–70%, liver lipid droplet area is significantly reduced, muscle lipid content is significantly increased, and fish growth rate and muscle quality are both improved. These results verify the effectiveness of the method of this invention in lipid metabolism remodeling, fatty liver relief, and meat quality improvement, proving its application value in actual aquaculture.

[0018] 4. High-expression-level and stable dsRNA recombinant bacterial system: The HT115(DE3) recombinant bacterial expression system constructed in this invention exhibits high expression levels and good stability. Through optimization of induction conditions (OD... 600 The maximum dsRNA yield was obtained by using a concentration of 0.4–0.6 mmol / L IPTG and an induction time of 5–6 hours. The resulting bacterial cells could be stored for a long time at -80°C after inactivation with 75% ethanol, and the RNA structure remained intact. The yield fluctuations between batches were small, which significantly improved the stability, controllability and biosafety of the system in the industrial production stage.

[0019] 5. Simple feed preparation process and controllable drug loading: The microcapsule sustained-release granule preparation process proposed in this invention is simple to operate. RNase-free extracted dsRNA can be encapsulated into 200-300 μm microspheres by chitosan-sodium tripolyphosphate iontophoresis. After being sprayed with a liposome-palmitic acid protective film, it can be directly and uniformly mixed with conventional floating feed. It has good bioactivity, sustained-release characteristics and economic applicability, and is suitable for large-scale preparation and aquaculture application.

[0020] 6. Standardizable and scalable microcapsule product system: The technical route proposed in this invention can be directly transformed into standardized "microcapsule sustained-release granule products", which are easy to promote to large-scale aquaculture systems and have significant commercial application potential and market transformation prospects.

[0021] As further supporting evidence of the inventiveness of this invention, the following important aspects are also reflected: (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: This invention achieves bidirectional regulation of lipid metabolism in fish through MSTN gene interference, which has the effects of alleviating hepatic lipid deposition and promoting muscle fat accumulation, breaking through the unidirectional limitation of traditional nutritional regulation. Targeted dsRNA is expressed using HT115(DE3) engineered bacteria, and after ethanol inactivation and microencapsulation, a safe and stable oral delivery system is formed. This method is simple, low-cost, and can be mass-produced, applicable to various perciformes fish, and has significant application value and promotion prospects in the prevention and control of fatty liver and the improvement of muscle quality.

[0022] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: Currently, the prevention and control of fatty liver in fish mainly relies on feed nutrition optimization or the addition of antioxidants, which makes it difficult to simultaneously improve muscle quality. There are no mature gene-level regulation strategies at home and abroad that can simultaneously solve the dual problems of "excessive liver lipid deposition and insufficient muscle lipid deposition". This invention proposes for the first time to use MSTN gene interference to achieve liver-muscle lipid redistribution and achieve long-term effects through a microcapsule oral delivery system, filling the technical gap in molecular nutritional intervention for the prevention and control of fatty liver in fish and the improvement of muscle quality.

[0023] (3) Does the technical solution of this invention solve a long-standing technical problem that people have long desired to solve but have never been able to successfully address? For a long time, high-density farmed fish such as perch have generally suffered from problems such as high incidence of fatty liver, decreased muscle quality, and low feed conversion rate. Existing nutritional intervention measures are difficult to achieve a reasonable distribution of fat between the liver and muscle. This invention, by targeting the interference of the MSTN gene, achieves a rebalancing of lipid synthesis and transport at the metabolic regulation level, significantly reduces the accumulation of lipid droplets in the liver and increases the lipid content in the muscle, fundamentally solving the technical problem that fatty liver in fish is difficult to effectively control and that muscle quality is difficult to improve simultaneously.

[0024] (4) Does the technical solution of this invention overcome technical bias? It is generally believed in the prior art that the application of RNA interference in aquatic animals is limited by problems such as low oral delivery efficiency, easy degradation of dsRNA, and unstable interference effects. Therefore, it is generally believed in the industry that oral RNA interference is difficult to achieve long-term and effective gene suppression in fish. This invention, by constructing a high-expression dsRNA recombinant bacterial system and combining it with a bilayer sustained-release technology of chitosan-sodium tripolyphosphate ion gel and liposome composite coating, has for the first time achieved efficient oral delivery of dsRNA and sustained in vivo interference effects. This technical solution breaks through the long-standing technical bias in the industry that "oral RNA interference is not feasible in fish," providing a stable and scalable technical path for molecular nutritional regulation in aquatic animals. Attached Figure Description

[0025] Figure 1The flowchart illustrates a method for alleviating fatty liver and improving muscle quality in sea bass based on MSTN gene interference, as provided in an embodiment of the present invention.

[0026] Figure 2 This is a system structure diagram for alleviating fatty liver in sea bass and improving muscle quality based on MSTN gene interference, as provided in an embodiment of the present invention.

[0027] Figure 3 The diagram provided in this embodiment of the invention illustrates the changes in the expression level of the target gene after MSTN gene interference.

[0028] Figure 4 The diagram provided in this embodiment of the invention illustrates the changes in lipid accumulation in the muscle of sea bass after MSTN interference treatment.

[0029] Figure 5 The diagram provided in this embodiment of the invention illustrates the effect of MSTN interference treatment on reducing lipid droplet deposition in the liver of sea bass.

[0030] Figure 6 This image shows the detection results of dsRNA expression in L4440-MSTN recombinant bacteria provided in this embodiment of the invention. After inducing recombinant bacterial expression with different IPTG concentrations (0.6 mmol / L, 0.8 mmol / L, and 1.0 mmol / L), the dsRNA expression level was detected by agarose gel electrophoresis to verify the expression efficiency.

[0031] Figure 7 The image showing the results of MSTN gene expression level detection in sea bass provided in this embodiment of the invention demonstrates the detection of the relative expression level of MSTN using two methods: real-time quantitative PCR (qPCR) and transcriptome sequencing (FPKM), reflecting the gene interference effect.

[0032] Figure 8 These are HE-stained micrographs of individual morphology and muscle tissue sections of spotted bass from the control group (si-EGFP) and experimental group (si-MSTN) provided in this embodiment of the invention. The left side shows the differences in individual appearance, and the right side shows the hematoxylin-eosin (HE) stained micrographs of the corresponding muscle tissue sections, used to observe changes in muscle structure.

[0033] Figure 9 This is a statistical chart of the body length and weight of the spotted bass provided in an embodiment of the present invention. The left side of the chart shows the statistical results of the body length of the spotted bass in different treatment groups, and the right side shows the statistical results of the body weight. The control group is si-EGFP, and the experimental group is si-MSTN.

[0034] Figure 10This is a statistical chart showing the number and average diameter of muscle fibers in sea bass provided in an embodiment of the present invention. The left side of the chart shows the statistical results of the total number of muscle fibers in sea bass in different treatment groups, and the right side shows the statistical results of the average muscle fiber diameter. The control group was si-EGFP, and the experimental group was si-MSTN.

[0035] Figure 11 This is a micrograph of a sea bass muscle tissue section stained with Oil Red O, provided in an embodiment of the present invention. The upper part of the image shows the location of muscle tissue sampling and sectioning; the lower part shows the Oil Red O staining image of the sea bass muscle tissue section, used to demonstrate lipid deposition in the sea bass muscle tissue of different treatment groups.

[0036] Figure 12 The images provided in this embodiment of the invention show the quantitative statistical results of the staining of sea bass muscle tissue sections. The left side shows the statistical results of the absolute lipid droplet area in the Oil Red O staining image, and the right side shows the statistical results of the relative lipid droplet area.

[0037] Figure 13 This is a micrograph of a sea bass liver tissue section stained with Oil Red O, provided in an embodiment of the present invention. The top row is the control group (si-EGFP), and the bottom row is the experimental group (si-MSTN).

[0038] Figure 14 This is a quantitative statistical analysis of the stained images of sea bass liver tissue sections provided in this embodiment of the invention. The left side shows the statistical results of the absolute lipid droplet area, and the right side shows the statistical results of the relative lipid droplet area. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] like Figure 1 As shown in the figure, an embodiment of the present invention provides a method for alleviating fatty liver of sea bass and improving muscle quality based on myostatin (MSTN) gene interference, which includes the following steps: S101. Based on the coding sequence (CDS) of the MSTN gene of the spotted bass, specific primers were designed to amplify the target fragment of 500-600 bp in the conserved functional region and insert it into the RNA interference vector L4440 with a double T7 promoter to construct a recombinant plasmid targeting MSTN. S102, the constructed recombinant plasmid was transformed into RNase III-deficient Escherichia coli HT115(DE3), and positive clones were screened in LB liquid medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL); when the bacterial culture optical density (OD) reached a certain level, the positive clones were selected. 600 When the expression level reaches 0.4–0.6, add 0.6 mmol / L isopropyl-β-D-thiogalactoside (IPTG) and induce culture at 37°C for 5–6 hours to obtain the optimal expression level of double-stranded RNA (dsRNA). S103, after collecting the expression product, the bacterial cells were inactivated with 75% ethanol to ensure biosafety and maintain the structural stability of dsRNA; the inactivated bacterial cells can be stored for a long time at -80℃, or freeze-dried with the addition of a protectant and then transported at room temperature. S104 uses RNase-free technology to purify total RNA from IPTG-induced HT115 cells. Using a chitosan-sodium tripolyphosphate ionogel method, dsRNA is encapsulated into 200–300 μm microspheres. A liposome-palmitic acid membrane (waterproof and trypsin-resistant) is then sprayed onto the chitosan layer to obtain microcapsule sustained-release granules. The drug loading is 2% (w / w), meaning 1g of microcapsule sustained-release granules contains 20mg of dsRNA. After vacuum drying at 40℃, the granules are stored. They are then sprinkled directly onto the surface of floating feed at a rate of 2.5g of microcapsule sustained-release granules per kg of fish body weight, allowing them to adhere by splashing and rolling when the fish feed. Feeding once a week is sufficient to maintain an MSTN inhibition rate of over 70%.

[0041] The MSTN interference fragment provided in this embodiment of the invention is designed based on the coding sequence (CDS) of the MSTN gene of sea bass, preferably with a length of 500-600 bp, located in a conserved functional region of the gene, to ensure interference efficiency and target specificity.

[0042] The RNA interference vector L4440 provided in this embodiment of the invention contains a dual T7 promoter structure, which can bidirectionally transcribe to form stable dsRNA for targeted interference with the MSTN gene.

[0043] The host bacteria of the recombinant engineered bacteria provided in this embodiment of the invention are selected from HT115 (DE3), which is an RNase III-deficient Escherichia coli that can stably express exogenous dsRNA and is suitable for large-scale bacterial production.

[0044] The recombinant engineered bacteria provided in this embodiment of the invention were amplified and cultured in a double-antibiotic medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL). When the bacterial culture OD... 600 When the expression level reaches 0.4–0.6, 0.6 mmol / L IPTG is added for induction, and the cells are induced at 37°C for 5–6 hours to obtain the optimal dsRNA expression level.

[0045] The recombinant bacteria provided in this invention are inactivated by 75% ethanol after collection and can be stored for a long time at -80℃, or freeze-dried with added protectant and transported at room temperature, maintaining the integrity of the bacterial morphology and the stability of the dsRNA structure, which is convenient for large-scale feed application.

[0046] like Figure 2 As shown, an embodiment of the present invention provides a system for alleviating fatty liver in sea bass and improving muscle quality based on MSTN gene interference, comprising: The amplification module is used to design specific primers based on the coding sequence (CDS) of the MSTN gene of the spotted bass, amplify the target fragment of 500-600 bp in the conserved functional region, and insert it into the RNA interference vector L4440 with a double T7 promoter to construct a recombinant plasmid targeting MSTN. The recombinant module is used to transform the constructed recombinant plasmid into RNase III-deficient *Escherichia coli* HT115(DE3), and screen for positive clones in LB liquid medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL); when the bacterial culture optical density (OD) reaches a certain level... 600 When the expression level reaches 0.4–0.6, add 0.6 mmol / L isopropyl-β-D-thiogalactoside (IPTG) and induce culture at 37°C for 5–6 hours to obtain the optimal dsRNA expression level. The inactivation module is used to inactivate the bacterial cells with 75% ethanol after collecting the expression products, ensuring biosafety and maintaining the structural stability of dsRNA. The inactivated bacterial cells can be stored for a long time at -80℃, or freeze-dried with the addition of a protectant and then transported at room temperature. The microcapsule sustained-release particle preparation module is used to purify IPTG-induced total RNA from HT115 using an RNase-free process. The dsRNA is encapsulated into 200–300 μm microspheres using a chitosan-sodium tripolyphosphate ionogel method. A liposome-palmitic acid membrane (waterproof and trypsin-resistant) is then sprayed onto the chitosan layer to obtain the microcapsule sustained-release particles. The drug loading is 2% (w / w), meaning 1g of microcapsule sustained-release particles contains 20mg of dsRNA. After vacuum drying at 40℃, the particles are stored. They are then sprinkled directly onto the surface of floating feed at a rate of 2.5g of microcapsule sustained-release particles per kg of fish body weight, where they adhere due to the fish's "splashing and rolling" action when feeding. Feeding once a week is sufficient to maintain an MSTN inhibition rate of over 70%.

[0047] The system provided in this invention, based on MSTN gene interference, alleviates fatty liver in sea bass and improves muscle quality. With RNA interference biological regulation as its core, the system achieves continuous inhibition of the MSTN gene in sea bass through the coordinated operation of multiple functional modules such as amplification, expression, stabilization treatment and delivery protection, thereby simultaneously improving lipid metabolism and muscle growth performance.

[0048] During system operation, the amplification module first designs specific primers based on functionally conserved regions in the MSTN gene coding sequence of the spotted sea bass to amplify the target fragment and construct a recombinant plasmid. By placing this fragment under the control of a bidirectional transcriptional structure, subsequent expression stages can form complementary transcripts on the same template, providing a molecular basis for the stable generation of dsRNA. This structure ensures the sequence specificity and interference efficiency of dsRNA, which is a key prerequisite for achieving targeted gene silencing.

[0049] The recombinant module amplifies and expresses the recombinant plasmid by introducing it into a nuclease-deficient host bacterium, preventing intracellular degradation of the dsRNA and achieving efficient transcription under induction conditions, thereby obtaining dsRNA with a complete double-stranded structure. This module combines nucleic acid engineering expression with biosafety control, providing a stable source for subsequent large-scale preparation.

[0050] The inactivation module processes the engineered bacteria after expression, causing the bacteria to lose their biological activity while maintaining the integrity of the cell structure and dsRNA. This fundamentally eliminates the risks of environmental release and food safety. At the same time, the bacterial structure provides primary physical protection for the dsRNA, which is beneficial for subsequent processing and storage.

[0051] The microcapsule sustained-release particle preparation module encapsulates dsRNA in multiple layers using an ionomer gel and an outer hydrophobic membrane structure, protecting it from hydrolysis and enzymatic degradation in the aquatic environment and fish digestive tract, and allowing for gradual release after feeding. This module, combined with the feeding method, enables dsRNA to be absorbed through the intestines and continuously exert its RNA interference effect in vivo.

[0052] Through the synergistic operation of the above modules, dsRNA effectively inhibits MSTN gene expression in sea bass, reduces its negative regulatory effect on muscle growth, improves lipid metabolism imbalance, and reduces abnormal fat deposition in the liver, thereby achieving the dual technical effects of alleviating fatty liver and improving muscle quality.

[0053] Example 1: Construction of a lipid metabolism regulation system for spotted bass based on MSTN gene interference This invention provides a lipid metabolism regulation system for spotted sea bass based on MSTN gene interference, mainly comprising: recombinant bacterial construction, induced expression, inactivation treatment, and feed application. In the construction of the recombinant bacterial strain, healthy spotted sea bass muscle tissue is first used as material. Total RNA is extracted using the TRIzol method, and cDNA is synthesized via reverse transcription as a template. Specific primers are designed based on the conserved functional regions of the spotted sea bass MSTN gene to perform PCR amplification of the target fragment, ensuring the specificity and efficiency of the interference. After purification, the PCR product is ligated into the double-enzyme-digested linearized RNA interference vector L4440. The target sequence is inserted into the multiple cloning site (MCS) region between the two T7 promoters of this vector, constructing the recombinant plasmid L4440-MSTN containing the MSTN target sequence. After sequencing verification confirms the sequence and insertion direction are correct, it is ready for use. In induced expression, the recombinant plasmid L4440-MSTN is transformed into RNase III-deficient Escherichia coli HT115(DE3). This strain, lacking dsRNA degrading enzymes, has the ability to stably express exogenous dsRNA. The recombinant bacteria were inoculated into double-antibiotic LB medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL) and cultured with shaking at 37℃ and 220 rpm for later use.

[0054] Example 2: Extraction of recombinant bacterial dsRNA and preparation of microcapsule sustained-release granules HT115(DE3) recombinant bacteria, induced by IPTG expression, were inactivated with 75% ethanol. Total RNA was extracted and purified using an RNase-free process to obtain high-purity dsRNA. The extracted dsRNA solution was added to a chitosan solution, and sodium tripolyphosphate was added dropwise under stirring to form chitosan-sodium tripolyphosphate gel microspheres with a particle size of approximately 200–300 μm. The microspheres were then coated with a liposome-palmitic acid outer membrane to form a bilayer sustained-release structure with waterproof and trypsin-resistant properties. The resulting microcapsule sustained-release particles had a drug loading of approximately 2% (w / w), exhibited good stability after vacuum drying at 40°C, and could be directly and evenly sprinkled onto the surface of floating feed for oral delivery, achieving sustained release and efficient in vivo absorption of dsRNA.

[0055] Example 2: Verification of dsRNA expression effect and MSTN gene knockdown effect like Figure 3 As shown, under different IPTG concentrations (0.6 mmol / L, 0.8 mmol / L, and 1.0 mmol / L), recombinant plasmids L4440-MSTN1 (457 bp) and L4440-MSTN2 (613 bp) successfully expressed dsRNA products with clear bands, indicating good expression efficiency and high stability of the constructed system. Among these, the 0.6 mmol / L IPTG induction condition resulted in the highest dsRNA expression level and the clearest target band, indicating the optimal induction effect and highest dsRNA expression efficiency at this concentration. Therefore, 0.6 mmol / L IPTG was determined as the optimal concentration for induction in this system. Further in vivo validation was performed by oral delivery of dsRNA via microcapsule sustained-release granules (e.g., ...). Figure 4 As shown in the figure, after si-MSTN treatment in the experimental group, the expression level of MSTN gene in muscle tissue decreased by approximately 67.57% in qPCR detection and approximately 57.19% in transcriptome sequencing (FPKM). This result clearly demonstrates that recombinant bacteria delivered orally can produce a significant gene knockdown effect in fish, with high interference efficiency, and possesses good biological effects and application potential.

[0056] Example 3: Improved growth performance and optimized muscle tissue structure like Figure 5 As shown, the si-MSTN group of spotted bass exhibited superior growth characteristics in terms of morphology, with a significantly fuller body and thicker trunk, showing better body shape characteristics than the control group (si-EGFP). Figure 6 Statistical results showed that after 90 days of continuous feeding, the average body length of the fish in the si-MSTN group was significantly higher than that in the control group, and the average weight also increased accordingly. These data indicate that MSTN gene interference can significantly promote the overall growth performance of spotted sea bass and has a good growth-promoting effect.

[0057] Regarding muscle tissue structure, HE staining results ( Figure 5 (Right) This shows that the muscle fibers in the si-MSTN group are more tightly and evenly arranged, and the intermuscular spaces are significantly reduced; while the muscle tissue structure in the control group is relatively loose. Figure 7 Quantitative analysis further validated that the total number of muscle fibers in the si-MSTN group significantly increased, while the average diameter of individual muscle fibers decreased. This indicates that the muscle growth pattern in the si-MSTN group is primarily "proliferative," meaning an increase in the number of muscle fibers, rather than "hypertrophic," meaning an increase in the diameter of individual muscle fibers. This proliferative muscle characteristic can improve the firmness, texture consistency, and mouthfeel of fish meat, contributing to enhanced product market competitiveness. In summary, targeted interference with the MSTN gene demonstrates a significant multidimensional regulatory effect on growth promotion and muscle quality improvement, providing a scientifically feasible technical means for promoting fish growth and optimizing meat quality.

[0058] Example 4: Analysis of Changes in Muscle Lipid Accumulation To assess the effect of MSTN interference on muscle lipid deposition, Oil Red O staining analysis was performed on the dorsal muscle tissue of si-MSTN and control group sea bass (e.g., ...). Figure 8 (As shown). The control group showed almost no obvious lipid droplet deposition, while the si-MSTN group showed a significant increase in the number of lipid droplets in the muscles, mainly concentrated in the subcutaneous tissue and near the spine, indicating obvious lipid deposition.

[0059] like Figure 9 The quantitative analysis results shown indicate that the absolute area of ​​lipid droplets in the muscle tissue of the si-MSTN group was approximately 1.1 × 10⁻⁶. 6 μm 2 The value was significantly higher than that of the control group (1.5 × 10⁻⁶). 5 μm 2 (p < 0.01); the relative area percentage also increased from approximately 0.4% in the control group to 3.0% (p < 0.01), a highly statistically significant difference. These results indicate that MSTN gene interference can effectively promote lipid deposition in muscle, helping to improve the flavor, tenderness, and nutritional value of fish meat, thus meeting the dual demands of the mid-to-high-end aquatic product market for both texture and nutrition.

[0060] Example 5: Relief effect of hepatic lipid deposition To further evaluate the effect of MSTN gene interference on lipid metabolism in the liver of spotted sea bass, this example performed Oil Red O staining and lipid droplet quantification analysis on liver tissues from si-MSTN and control groups. Figure 10 As shown, a large number of red lipid droplets accumulated in hepatocytes of the si-EGFP group, indicating significant lipid deposition; while lipid droplets in liver tissue of the si-MSTN group were significantly reduced, sparsely distributed, clearly structured, and more discretely distributed, indicating a significant decrease in lipid deposition level.

[0061] Figure 11 Quantitative data showed that the absolute lipid droplet area in the si-MSTN group was approximately 6.2 × 10⁻⁶. 3 μm 2 The value was significantly lower than that of the control group (1.6 × 10⁻⁶). 4 μm 2 (p < 0.001); the relative lipid droplet area also decreased from 20% in the control group to approximately 9% (p < 0.001). The results clearly show that MSTN gene interference can effectively inhibit excessive lipid deposition in the liver, alleviate the pathological state of fatty liver, and improve liver metabolic function.

[0062] In summary, by constructing and applying microcapsule sustained-release particles, not only can bidirectional regulation of lipid distribution in muscle and liver be achieved, but also a synergistic optimization effect can be generated from multiple levels such as individual growth, tissue structure, and metabolic health, providing an innovative molecular nutrition strategy for the prevention and control of metabolic diseases and the improvement of meat quality in aquatic animals.

[0063] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for alleviating fatty liver and improving muscle quality in spotted bass based on MSTN gene interference, characterized in that, This includes the following steps that have a collaborative relationship: Specific dsRNA was obtained by constructing a bidirectional transcription dsRNA expression system targeting the MSTN gene of spotted sea bass; The engineered bacteria expressing dsRNA were inactivated to eliminate biological activity while preserving the complete structure of the dsRNA; The dsRNA was administered orally to the spotted bass, thus preventing the dsRNA from being digested and degraded in the intestine and allowing it to be absorbed. The expression of MSTN gene in sea bass was inhibited by RNA interference mechanism, thereby simultaneously reducing abnormal lipid deposition in the liver and promoting skeletal muscle growth and improving muscle quality. Among them, the stability maintenance of dsRNA, the protection of intestinal delivery, and the gene interference effect work synergistically to achieve long-term effective MSTN inhibition.

2. The method according to claim 1, characterized in that, The dsRNA is derived from a conserved functional region in the MSTN gene coding sequence of the spotted bass, and the dsRNA is 500 to 600 units in length.

3. The method according to claim 1, characterized in that, The dsRNA is driven by a bidirectional promoter to form a complementary transcription product, enabling the dsRNA to be stably expressed in a double-stranded structure within the engineered bacteria.

4. A method for oral delivery of dsRNA for implementing MSTN gene interference, characterized in that, Including the following collaborative technology mechanisms: The engineered bacteria expressing MSTN-targeting dsRNA were inactivated, so that the bacteria lost their ability to reproduce but maintained the integrity of their cell structure. A multilayer protective structure is formed outside the inactivated bacterial cells or their extracted dsRNA to block hydrolysis and enzymatic degradation in the gastrointestinal tract; The protected dsRNA was delivered into the intestine of the spotted bass via oral administration and absorbed. The multi-layered protective structure works in conjunction with the inactivated bacterial cells to maintain the structural stability of dsRNA in the intestinal environment and ensure its continuous release, thereby guaranteeing the persistence of the RNA interference effect.

5. The method according to claim 4, characterized in that, The engineered bacteria are RNase-deficient strains, which prevent the dsRNA from being degraded by endogenous nucleases during expression.

6. The method according to claim 4, characterized in that, The inactivation process uses ethanol to achieve biosafety without damaging the bacterial cell structure and the dsRNA double-stranded structure.

7. The method according to claim 4, characterized in that, The multilayer protective structure includes a polymer coating layer formed by ionogel and an outer membrane structure with hydrophobic barrier function, which is used to resist hydrolysis and digestive enzyme degradation at the same time.

8. A feed administration method based on a microencapsulation sustained-release mechanism to achieve sustained inhibition of the MSTN gene, characterized in that, include: The dsRNA targeting the MSTN gene was prepared into microcapsule sustained-release particles, which kept the dsRNA stable in the aquatic environment and the fish digestive system. The microcapsule slow-release granules are attached to the surface of floating feed and fed periodically. The sustained-release effect of microcapsules allows dsRNA to exert a continuous RNA interference effect in the sea bass. Thus, without changing the feed formulation structure, long-term inhibition of the MSTN gene can be achieved, and fat metabolism and muscle development can be improved.

9. The method according to claim 8, characterized in that, The drug loading ratio of the microcapsule sustained-release particles is 2, which enables the continuous release of dsRNA during the feeding cycle.

10. The method according to claim 8, characterized in that, The feeding method employs low-frequency, cyclical feeding to establish a stable interference level of dsRNA in vivo, thereby avoiding short-term high-dose fluctuations.

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