Application of fish miRNA in regulation and control of hypoxia resistance of pelteobagrus vachelli

Abstract

The invention relates to the technical field of biotechnology and aquaculture, and mainly relates to an application of fish miRNA in regulation and control of hypoxia tolerance of pelteobagrus vachelli, the critical suffocation point of the pelteobagrus vachelli can be reduced by inhibiting miR-135b-5p expression through an inhibitor, and the hypoxia tolerance of the pelteobagrus vachelli can be remarkably improved; the critical suffocation point is improved by activating miR-135b-5p expression through an agonist, so that the hypoxia resistance of the pelteobagrus vachelli is remarkably weakened, the limitation of a traditional means for coping with hypoxia stress in existing aquaculture is effectively overcome, mechanical oxygenation equipment limited by environmental conditions (such as water temperature and water flow) is not needed, and the cost is reduced. And the risk that chemical substance residues in the water body possibly caused by a chemical oxygen producer affect the quality safety of aquatic products is avoided, and the development requirements of current green and efficient aquaculture are completely met.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and aquaculture technology, and mainly to the application of a fish miRNA in regulating the hypoxia tolerance of yellow catfish. Background Technology

[0002] The yellow catfish (Pelteobagrus vachelli) is a high-quality fish species with significant economic value in my country's freshwater aquaculture sector. Its flesh is tender, nutritious, and has a unique flavor. It also has a wide adaptability, making it one of the core aquaculture species in the Yangtze River basin and many southern regions. In recent years, with the increasing intensification and high-density development of aquaculture, coupled with problems such as fluctuations in dissolved oxygen levels due to climate change and bottom hypoxia caused by eutrophication, hypoxia stress has become a key environmental factor restricting the improvement of the quality and efficiency of the yellow catfish farming industry.

[0003] Hypoxia has a significant negative impact on the physiological metabolism, survival, and reproduction of yellow catfish. On the one hand, hypoxia inhibits the efficiency of aerobic respiration in the gills, causing the body's energy metabolism pathway to shift from aerobic oxidation to anaerobic glycolysis. This leads to the accumulation of lactic acid in muscles and blood, resulting in metabolic acidosis, which in turn reduces the fish's swimming ability and appetite, and in the long term, significantly reduces its growth rate. On the other hand, hypoxia disrupts the homeostasis of the yellow catfish's immune system, inhibits the activity of immune cells (such as macrophages and lymphocytes), reduces the body's resistance to pathogens (such as Flavobacterium columnare and Edwardsiella tarda), and increases the risk of diseases such as bacterial septicemia and gill rot. In severe cases, it can lead to large-scale mortality in farmed fish populations, causing heavy economic losses to fish farmers.

[0004] Currently, traditional methods for dealing with hypoxia stress in aquaculture mainly fall into two categories: physical aeration and water quality control. Physical aeration technologies (such as impeller aerators, jet aerators, and bottom aeration devices) increase the dissolved oxygen content in the water mechanically and are the most widely used emergency measures. However, these methods have significant limitations—not only are the equipment purchase and operating costs high, but the aeration effect is also easily affected by factors such as water depth, water flow conditions, and weather conditions, making it difficult to achieve uniform and continuous control of dissolved oxygen in the aquaculture water. In particular, in the bottom area of ​​high-density ponds, "oxygen debt" is still easily accumulated. Water quality control methods (such as adding oxygenating agents, controlling the structure of plankton communities, and regular water changes) can improve the dissolved oxygen environment in the water to a certain extent, but the effect is short-lived and unstable. Furthermore, excessive use of chemical oxygenating agents may lead to chemical residues in the water, affecting the quality and safety of aquatic products, which does not meet the development needs of green aquaculture. In addition, while screening hypoxia-tolerant yellow catfish strains through traditional breeding methods can improve the hypoxia tolerance of the species at the genetic level, this process requires multiple generations of selection and takes 3-5 years. Furthermore, it is constrained by factors such as genetic trait segregation and low selection efficiency, making it difficult to quickly meet the current industry's urgent need for hypoxia-tolerant varieties.

[0005] Furthermore, with the deepening research into biomolecular regulatory mechanisms, it has been discovered that microRNAs (miRNAs) play a crucial role in the biological response to environmental stress. Under hypoxic stress, the expression profile of miRNAs in fish undergoes significant changes, and some miRNAs are involved in the fish's response to and adaptation to hypoxia. However, the specific applications of particular miRNAs in regulating the hypoxia tolerance of fish are not yet clearly defined in current technologies. Therefore, regulating these miRNAs to enhance or weaken the hypoxia tolerance of fish has significant theoretical and practical application value. Summary of the Invention

[0006] The purpose of this invention is to provide an application of fish miRNA in regulating the hypoxia tolerance of yellow catfish. This invention is the first to discover the positive or negative regulatory role of miR-135b-5p in the hypoxia adaptation of yellow catfish, providing a new molecular target and regulatory means for the breeding of hypoxia-resistant aquatic varieties.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides the application of fish miRNA in regulating the hypoxia tolerance of yellow catfish, wherein the fish miRNA is miR-135b-5p, and its base sequence is shown in SEQ ID NO.1.

[0008] The present invention also provides a reagent for regulating the hypoxia tolerance of yellow catfish, the reagent comprising a miR-135b-5p inhibitor or a miR-135b-5p agonist.

[0009] Furthermore, the miR-135b-5p inhibitor comprises a single-stranded oligonucleotide complementary to the miR-135b-5p sequence; the miR-135b-5p agonist comprises a double-stranded RNA mimic containing the mature miR-135b-5p sequence.

[0010] Furthermore, the single-stranded oligonucleotide complementary to the miR-135b-5p sequence or the double-stranded RNA mimic of the mature miR-135b-5p sequence has also undergone 2'-O-methylation modification, phosphate thioester backbone modification, and cholesterol coupling modification.

[0011] Furthermore, the miR-135b-5p inhibitor or miR-135b-5p agonist is purified by high performance liquid chromatography, and the purity after purification is ≥95%; the concentration of the miR-135b-5p inhibitor or miR-135b-5p agonist is 0.3 nmol / μL.

[0012] The present invention also provides the application of the miR-135b-5p inhibitor or miR-135b-5p agonist in regulating the hypoxia tolerance of yellow catfish.

[0013] Furthermore, the miR-135b-5p inhibitor or miR-135b-5p agonist is administered via intraperitoneal injection at a dose of 1 nmol / g.

[0014] The present invention also provides an application of miR-135b-5p in the preparation of products that regulate the hypoxia tolerance of yellow catfish.

[0015] Furthermore, the product contains the reagent described above for regulating the hypoxia tolerance of yellow catfish; Furthermore, the regulatory effect of the product was verified by measuring the critical asphyxiation point of the yellow catfish. The method for measuring the critical asphyxiation point is as follows: the yellow catfish treated with the product are transferred to a closed low-oxygen treatment system, allowing the dissolved oxygen in the water to decrease naturally. When 50% of the experimental fish stop moving their gill covers, the measured dissolved oxygen concentration in the water is the critical asphyxiation point.

[0016] The beneficial effects of this invention are as follows: This invention is the first to clearly reveal the regulatory function of miR-135b-5p on the hypoxia tolerance of yellow catfish. Experiments have confirmed that inhibiting the expression of this miRNA can significantly reduce the critical asphyxiation point of yellow catfish and effectively improve its hypoxia tolerance, while activating the expression of this miRNA will increase the critical asphyxiation point and weaken its hypoxia tolerance. This fills the technical gap in the prior art where the specific application of a particular miRNA in the regulation of hypoxia tolerance in fish has not been clearly defined, and provides a clear new direction and reliable experimental evidence for in-depth research on the molecular mechanism of hypoxia adaptation in fish.

[0017] The miR-135b-5p inhibitor and agonist designed in this invention possess high stability and precise regulatory capabilities. The inhibitor is a single-stranded oligonucleotide complementary to the miR-135b-5p sequence, and the agonist is a double-stranded RNA mimic containing the mature sequence of this miRNA. Both are modified by 2'-O-methylation, phosphate thioester backbone modification, and cholesterol coupling treatment, and then purified by high-performance liquid chromatography (HPLC) to ensure a purity ≥95%. They are prepared at a concentration of 0.3 nmol / μL and administered via intraperitoneal injection at a dose of 1 nmol / g body weight. This method can efficiently and stably regulate the expression level of miR-135b-5p, avoiding the problems of unstable effects and unclear mechanisms of traditional regulatory methods.

[0018] This invention effectively overcomes the limitations of traditional methods for dealing with hypoxia stress in existing aquaculture. It does not rely on mechanical aeration equipment that is constrained by environmental conditions (such as water temperature and water flow), and it also avoids the risks of chemical residues in the water that may be caused by chemical oxygenating agents, which could affect the quality and safety of aquatic products. It is in line with the current development needs of green and efficient aquaculture. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a comparison diagram of the critical asphyxiation points of each group of yellow catfish under hypoxic stress in Example 2 of the present invention. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Hypoxia-responsive miRNA (miR-135b-5p) in Yellow Catfish (Pelteobagrus vannamei) This embodiment aims to identify key miRNAs in yellow catfish involved in hypoxia response, providing molecular targets for subsequent regulation of its hypoxia tolerance.

[0027] The specific method is as follows: After treating yellow catfish with a low-oxygen environment, liver tissue and extracellular vesicles of liver tissue were obtained. Transcriptome sequencing and miRNA sequencing were performed on the two types of samples respectively, and omics joint analysis was carried out.

[0028] Analysis revealed that under hypoxic conditions, the expression level of extracellular vesicles miR-135b in the liver tissue of *Pelteobagrus vachelli* was significantly downregulated. Simultaneously, several target genes with significantly upregulated expression, including HK1, PCK2, TRIAP1, ADIPOR2, and KDM4B, were identified in the liver tissue. Among these, HK1 and PCK2 are involved in the regulation of glycolysis / gluconeogenesis, TRIAP1 is related to cell survival, and ADIPOR2 and KDM4B are involved in biological processes such as angiogenesis. These target genes collectively influence the survival ability of *Pelteobagrus vachelli* in hypoxic environments by regulating related physiological processes.

[0029] In this embodiment, the nucleotide sequence of miR-135b-5p is as shown in SEQ ID NO.1, specifically: 5′-TATGGCTTTTTATTCCTATCTGA-3′.

[0030] Example 2: Method for regulating the hypoxia tolerance of yellow catfish using miR-135b-5p This embodiment aims to provide a specific operational method for regulating the hypoxia tolerance of yellow catfish based on miR-135b-5p inhibitors or agonists, including the following steps: 1. Preparation of miR-135b-5p inhibitors and agonists Preparation of the inhibitor (miR-135b-5p Antagomir): Based on the miR-135b-5p sequence determined in Example 1, a single-stranded oligonucleotide completely complementary to the miR-135b sequence was chemically synthesized; the synthesized single-stranded oligonucleotide was modified by means of 2'-O-methylation, phosphate thioester backbone modification and cholesterol coupling; the modified oligonucleotide was purified by high performance liquid chromatography (HPLC) to ensure that the purity of the purified inhibitor was ≥95%; the purified inhibitor was prepared into a working solution with a concentration of 0.3 nmol / μL.

[0031] Preparation of agonist (miR-135b-5p Agomir): A double-stranded RNA mimic containing the mature miR-135b sequence was chemically synthesized (the mimic contains a functional strand and a complementary strand); the double-stranded RNA mimic was modified using the same modification method as the inhibitor (2'-O-methylation modification, thiophosphate backbone modification, and cholesterol coupling); the modified double-stranded RNA mimic was purified by HPLC to ensure a purity ≥95%; the purified agonist was prepared as a working solution with a concentration of 0.3 nmol / μL.

[0032] 2. Experimental grouping and treatment Healthy yellow catfish (purchased from Sichuan Rongsen Fishery Co., Ltd.) with a weight of 15±0.94g were selected. The experiment was set up with 3 treatment groups, each with 3 replicates, and each replicate containing 10 yellow catfish. The specific grouping and treatment methods are as follows: Control group: Yellow catfish were injected intraperitoneally with PBS buffer at a volume of 3.33 μL / g (based on fish body weight).

[0033] Agonist group: Yellow catfish were injected intraperitoneally with the miR-135b-5pAgomir solution prepared above, and the injection dose was 1 nmol / g (based on fish body weight), i.e., the injection volume was 3.33 μL / g.

[0034] Inhibitor group: Yellow catfish were injected intraperitoneally with miR-135b-5pAntagomir solution prepared above, and the injection dose was 1 nmol / g (based on fish body weight), i.e., the injection volume was 3.33 μL / g.

[0035] 3. Determination of the critical asphyxiation point The three groups of treated yellow catfish were transferred to a closed hypoxic treatment system to allow the dissolved oxygen in the water to decrease naturally. During the process of decreasing dissolved oxygen, the behavioral characteristics of each group of yellow catfish were continuously observed and recorded. When 50% of the experimental fish in a certain group showed signs of cessation of gill cover movement, the dissolved oxygen concentration of that group was immediately measured. This concentration was the critical asphyxiation point for that group of yellow catfish.

[0036] 4. Experimental Results The results of the critical asphyxiation point determination for each group of yellow catfish are as follows: Figure 1 As shown, the specific results are as follows: The critical asphyxiation point for the control group (injected with PBS buffer) was 0.33 mg / L; The critical asphyxiation point in the agonist group (injection of miR-135b-5pAgomir) was 0.42 mg / L, which was about 27% higher than that in the control group, and the difference was statistically significant (P < 0.05). The critical asphyxiation point in the inhibitor group (injection of miR-135b-5pAntagomir) was 0.25 mg / L, which was about 24% lower than that in the control group, and the difference was statistically significant (P < 0.05).

[0037] The above results indicate that miR-135b-5p, as a key molecule regulating the hypoxia tolerance of *Pelteobagrus vachelli*, can directly affect the organism's adaptation to hypoxic stress by altering its expression level. Existing studies on fish miRNAs have shown that the expression profile of miRNAs in fish changes significantly under hypoxic stress, and some miRNAs participate in the hypoxia response and adaptation process. This invention further confirms through experiments that miR-135b-5p has a clear "negative regulatory effect" on the hypoxia tolerance of *Pelteobagrus vachelli*. This property is the core mechanism by which its inhibitors can increase and its agonists can decrease the hypoxia tolerance of *Pelteobagrus vachelli*. In this invention, after treating *Pelteobagrus vachelli* with a hypoxic environment, miRNA profiling analysis of its liver tissue and extracellular vesicles showed... The expression of miR-135b-5p was significantly downregulated, while the expression of its target genes HK1, PCK2 (involved in glycolysis / gluconeogenesis), TRIAP1 (related to cell survival), ADIPOR2, and KDM4B (involved in angiogenesis) was significantly upregulated (data from our group's omics experiments, unpublished). This change is a characteristic manifestation of the body's autonomous initiation of hypoxia tolerance adaptation, fully demonstrating that normal expression of miR-135b-5p inhibits the activation of hypoxia tolerance mechanisms, essentially having the biological function of weakening the hypoxia tolerance of *Pelteobagrus vachelli*. The miR-135b-5p prepared in this invention, modified with 2'-O-methylation, thiophosphate backbone, and cholesterol coupling, with a purity ≥95%, and prepared at a concentration of 0.3 nmol / μL, was further analyzed. When a miR-135b-5p inhibitor or agonist is injected intraperitoneally at a dose of 1 nmol / g into *Pelteobagrus vachelli*, the reagent acts on the physiological processes related to hypoxia adaptation. The inhibitor is a single-stranded oligonucleotide complementary to the miR-135b-5p sequence, which specifically binds to and inhibits its activity or expression, relieving the inhibition of downstream hypoxia-related target genes such as HK1, PCK2, and TRIAP1, allowing the target genes to express normally. This helps improve hypoxia tolerance by regulating physiological processes such as energy metabolism and cell survival. The agonist is a double-stranded RNA mimic containing the mature miR-135b-5p sequence, which specifically activates or enhances its expression and activity, strengthening molecules that hinder the initiation of hypoxia tolerance mechanisms, further exacerbating the inhibition of downstream target genes, leading to... The body is unable to effectively activate adaptive mechanisms such as energy metabolism adjustment and cell protection, thereby weakening its hypoxia tolerance. At the same time, both inhibitors and agonists directly affect the body's hypoxia tolerance level by precisely regulating miR-135b-5p expression. These molecular regulations jointly determine the hypoxia tolerance performance of yellow catfish, which is ultimately reflected in the fact that the critical asphyxiation point of the inhibitor group (0.25 mg / L) is significantly lower than that of the PBS control group (0.33 mg / L), and the critical asphyxiation point of the agonist group (0.42 mg / L) is significantly higher than that of the control group (both differences are statistically significant, P < 0.05). This confirms the regulatory role of miR-135b-5p from the perspective of molecular regulation and functional verification, and also provides direct experimental support for the effectiveness of the technical solution of this invention.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of a fish miRNA in regulating the hypoxia tolerance of the yellow catfish (Pelteobagrus vachelli), characterized in that, The fish miRNA is miR-135b-5p, and its base sequence is shown in SEQ ID NO.

1.

2. A reagent for regulating the hypoxia tolerance of yellow catfish, characterized in that, The reagent contains either a miR-135b-5p inhibitor or a miR-135b-5p agonist.

3. The reagent according to claim 2, characterized in that, The miR-135b-5p inhibitor comprises a single-stranded oligonucleotide complementary to the miR-135b-5p sequence; the miR-135b-5p agonist comprises a double-stranded RNA mimic containing the mature miR-135b-5p sequence.

4. The reagent according to claim 3, characterized in that, The single-stranded oligonucleotide complementary to the miR-135b-5p sequence or the double-stranded RNA miR-135b-5p mature sequence were also modified with 2'-O-methylation, phosphate thioester backbone modification and cholesterol coupling modification.

5. The reagent according to claim 2, characterized in that, The miR-135b-5p inhibitor or miR-135b-5p agonist is purified by high performance liquid chromatography, and the purity after purification is ≥95%; the concentration of the miR-135b-5p inhibitor or miR-135b-5p agonist is 0.3 nmol / μL, and the reagent is a lyophilized powder.

6. The application of the miR-135b-5p inhibitor or miR-135b-5p agonist as described in claim 2 in regulating the hypoxia tolerance of yellow catfish.

7. The application according to claim 6, characterized in that, The miR-135b-5p inhibitor or miR-135b-5p agonist is administered via intraperitoneal injection at a dose of 1 nmol / g.

8. Application of miR-135b-5p in the preparation of products that regulate the hypoxia tolerance of yellow catfish.

9. The application according to claim 8, characterized in that, The product comprises the reagent for regulating the hypoxia tolerance of yellow catfish as described in any one of claims 2-5.

10. The application according to claim 8, characterized in that, The regulatory effect of the product was verified by measuring the critical asphyxiation point of yellow catfish. The method for measuring the critical asphyxiation point is as follows: yellow catfish treated with the product are transferred to a closed low-oxygen treatment system to allow the dissolved oxygen in the water to decrease naturally. When 50% of the experimental fish stop moving their gill covers, the measured dissolved oxygen concentration in the water is the critical asphyxiation point.

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