Biological preparation for improving quality of weever and application

Abstract

The invention relates to a biological preparation for improving quality of weever, the biological preparation comprises an injection and an edible agent, the injection comprises chemically modified chemically synthesized single-stranded small RNA, Rev-erbalpha protein and Per1 protein, and the edible agent comprises Clock core peptide 1, Clock core peptide 2 and taurine. Skeletal muscle development, liver lipid metabolism and generation of cell inflammatory factors of the weever are influenced by adding and injecting the feed, so that the quality of the weever is improved, intramuscular fat deposition is promoted, the taste and flavor of fish meat are improved, the energy reserve of the weever can be improved, and the culture cycle is shortened. The improvement of cell inflammatory factors shows that the disease resistance of the weever is improved, the survival rate in the breeding process is further improved, and the use amount of antibiotics is reduced. The method provides an important theoretical basis for improving fish meat quality in China and promoting high-quality, efficient and healthy development of aquaculture, and has a wide market prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquaculture feed additives, and relates to a biological preparation for improving the quality of perch, its application, and also relates to key aquaculture methods. Background Art

[0002] Perch is a special freshwater fish that has developed rapidly in recent years. It has the characteristics of delicious meat and no intermuscular spines. With a large market demand, high economic value, and continuously expanding breeding scale, it has become a breeding variety with good development prospects.

[0003] The circadian rhythm is a biological cycle of approximately 24 hours, and its function is to prepare organisms for daily environmental changes. Almost all organisms, from single-celled bacteria to plants and animals, exhibit behavioral, physiological, and biochemical rhythms, called "circadian rhythms". The molecular mechanism behind circadian rhythms is a gene regulatory network composed of transcriptional-translational feedback loops, called the "core clock". Clock, Rev-erbα, and Per1b are all core circadian clock genes that make up the core clock. They can regulate the expression of genes that do not have a timing function in transcription, and these genes are called clock-controlled genes, such as the expression of the transcriptional regulator in skeletal muscle (MyoD) or the protein in the rate-limiting step of cell physiology (such as the rate-limiting enzyme PBEF in the salvage pathway). Fish muscle is both the structural tissue and power organ of fish, and at the same time, it is also one of the important protein sources for humans. The growth and development of muscle directly affect the growth rate and muscle quality of fish, and also bring excellent economic benefits to the artificial aquaculture industry. The formation of multinucleated myofibers is crucial for fish muscle development. Currently known tissue-specific clock-controlled genes in the skeletal muscle circadian transcriptome include MyoD, Ucp3, Fbxo32 / Atrogin, and Myh1, etc. McCarthy et al. found that in the muscles of Clock mutant mice, the expression of approximately 35% of all expressed genes was different, indicating that the disruption of the core clock mechanism had a significant impact on gene expression and affected normal cell function and health. MyoD was identified to be expressed in a circadian manner, and it is expressed in a circadian manner in muscle tissue and is directly controlled by Clock. Clock binds to the core enhancer of the MyoD promoter in a rhythmic manner. Lipidomics analysis of the livers of wild-type and Per1-deficient mice showed that there were changes in the oscillation phases of 16 - 17% of the lipids (including triglycerides) between the two mouse strains, which means that the feeding / fasting rhythm can affect liver lipid accumulation. Rev-erbα knockout leads to lipid metabolism disorders and affects the expression of the liver lipid signaling protein - apolipoprotein CIII (ApoC3). + The muscle of fish is not only the structural tissue and power organ of fish, but also one of the important protein sources for humans. The growth and development of muscle directly affect the growth rate and muscle quality of fish, and also bring excellent economic benefits to the artificial aquaculture industry. The formation of multinucleated myofibers is crucial for fish muscle development. Currently known tissue-specific clock-controlled genes in the skeletal muscle circadian transcriptome include MyoD, Ucp3, Fbxo32 / Atrogin, and Myh1, etc. McCarthy et al. found that in the muscles of Clock mutant mice, the expression of approximately 35% of all expressed genes was different, indicating that the disruption of the core clock mechanism had a significant impact on gene expression and affected normal cell function and health. MyoD was identified to be expressed in a circadian manner, and it is expressed in a circadian manner in muscle tissue and is directly controlled by Clock. Clock binds to the core enhancer of the MyoD promoter in a rhythmic manner. Lipidomics analysis of the livers of wild-type and Per1-deficient mice showed that there were changes in the oscillation phases of 16 - 17% of the lipids (including triglycerides) between the two mouse strains, which means that the feeding / fasting rhythm can affect liver lipid accumulation. Rev-erbα knockout leads to lipid metabolism disorders and affects the expression of the liver lipid signaling protein - apolipoprotein CIII (ApoC3).

[0004] There is currently a large amount of evidence indicating that immune activation and inflammatory responses during infection are controlled by the circadian rhythm. Many immune-related genes are regulated by the molecular clock. Mutation of the zebrafish circadian rhythm gene Per1b leads to reduced phosphorylation of extracellular signal-regulated kinase (ERK), followed by reduced degradation of IkB and activation of p65, ultimately resulting in reduced NF-kB signaling and decreased production of TNF-a, IL-1b, and IL-8 after tail injury. Restoring Per1b can induce ERK phosphorylation in fish and thereby increase TNF-a production, confirming the positive regulation of Per1 on inflammatory cytokines.

[0005] Taurine, a non-protein amino acid that does not participate in the composition of proteins and enzymes, can improve the food conversion rate and growth rate of fish, maintain the osmotic balance and stability of the body, promote fish growth, improve lipid metabolism, and reduce oxidative stress and inflammatory responses. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a biological preparation and a key aquaculture method for improving the quality of bass, and their applications in the artificial aquaculture industry of bass.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A biological preparation for improving the quality of bass, the biological preparation comprising an injection, the injection comprising a chemically modified chemically synthesized single-stranded small RNA, Rev-erbα protein, and Per1 protein; the sequence of the single-stranded small RNA is: 5′-CAGUUUUGCAUGGGUUUGCAC-3′.

[0009] Furthermore, the biological preparation further comprises an edible agent, the edible agent being Clock core peptide 1, Clock core peptide 2, and taurine, the amino acid sequence of Clock core peptide 1 being: LIATVRLAKPQFIKEMCT; the amino acid sequence of Clock core peptide 2 being: LAQNTGPNTPAVATFAQ.

[0010] Furthermore, in the biological preparation for improving the quality of bass, the 5′ end of the single-stranded small RNA is modified with 2 phosphorothioate backbones, the 3′ end is modified with 4 phosphorothioate backbones, the 3′ end is modified with cholesterol, and the entire sequence is modified with base methylation, and the sequence is: 5′-CsAsGUUUUGCAUGGGUUUGsCsAsCs-Chol-3′.

[0011] In the biological agent for further improving the quality of bass, the preparation methods of Rev-erbα and Per1 proteins are as follows: Collect early embryos of bass and break them. Centrifuge the embryo lysate at 1200-1500 r / min for 10-15 min. After standing, take the supernatant and pass it through a chromatography column containing Rev-erbα or Per1 monoclonal antibody to obtain purified Rev-erbα and Per1 proteins respectively.

[0012] 2. The application of the biological agent described in any of the above solutions in fish feed or fish growth additives is also within the scope of protection of the present invention.

[0013] Further, the fish is bass.

[0014] 3. The third technical solution provided by the present invention is a key method for improving the quality of bass. When the bass is 1-6 months old, single-stranded small RNA, Rev-erbα protein and Per1 protein are injected every 10-15 days.

[0015] The sequence of the single-stranded small RNA is: 5′-CsAsGUUUUGCAUGGGUUUGsCsAsCs-Chol-3′.

[0016] The 5′ end of the single-stranded small RNA is modified with 2 phosphorothioate backbones, the 3′ end is modified with 4 phosphorothioate backbones, the 3′ end is modified with cholesterol, and the entire sequence is modified with base methylation.

[0017] Further, the single-stranded small RNA in the injection part is formulated into a solution with a concentration of 2.5-10 mg / L.

[0018] Further, the solutions of Rev-erbα and Per1 for injection are solutions with a concentration of 1 mg / ml.

[0019] Further, by weight, the injection dose ratio is 5-10 μl / g.

[0020] Further, in the premixed feed of the basic diet of bass, Clock core peptide 1, Clock core peptide 2 and taurine are added. The amino acid sequence of Clock core peptide 1 is: LIATVRLAKPQFIKEMCT; the amino acid sequence of Clock core peptide 2 is: LAQNTGPNTPAVATFAQ.

[0021] Further, the addition amount of Clock core peptide 1 is 100-200 mg / kg; the addition amount of Clock core peptide 2 is 100-200 mg / kg; the addition amount of taurine is 1-1.5 g / kg.

[0022] The beneficial effects of the present invention are as follows: The present invention relates to a biological preparation for improving the quality of perch. The biological preparation includes an injection part, which mainly contains a single-stranded small RNA with the sequence: 5′-CAGUUUUGCAUGGGUUUGCAC-3′. The 5′ end of the single-stranded small RNA is modified with 2 phosphorothioate backbones, the 3′ end is modified with 4 phosphorothioate backbones and the 3′ end is cholesterol-modified, and the entire sequence is base-methylated. It may also include an edible additive part, including Clock core peptide 1 (LIATVRLAKPQFIKEMCT) and Clock core peptide 2 (LAQNTGPNTPAVATFAQ). The prominent advantages of the present invention affect the skeletal muscle development, liver lipid metabolism and the production of cellular inflammatory factors of perch through feed addition and injection, thereby improving the quality of perch; promoting intramuscular fat deposition, thus improving the taste and flavor of fish meat, and also enhancing the energy reserve of perch and shortening the breeding cycle. The increase in cellular inflammatory factors indicates an improvement in the disease resistance of perch, further increasing the survival rate during the breeding process and reducing the amount of antibiotics used. It will also avoid huge losses caused to farmers due to diseases.

[0023] A biological preparation for improving the quality of perch and its breeding method provided by the present invention start from the molecular mechanism of perch cultivation regulation, aiming to lay a foundation for further revealing the regulation mechanism of fish healthy development, and will also provide an important theoretical basis for improving the meat quality of fish in China and promoting the high-quality, efficient and healthy development of the aquaculture industry. The breeding method is simple, and it can provide theoretical support and practical application for the high-quality cultivation of artificially farmed fish, especially perch, and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the objectives, technical solutions and beneficial effects of the present invention clearer, the following drawings are provided for illustration:

[0025] Figure 1 It is a graph of the body length measurement of perch in the control group and the treatment group in Example 1.

[0026] Figure 2 It is the statistical result of the expression level of the MyoD gene in the muscle of perch in each group.

[0027] Figure 3 It is the statistical result of the expression level of the ApoC3 gene in the liver of perch in each group.

[0028] Figure 4 It is the statistical result of the expression levels of TNF-a, IL-1b and IL-8 genes in perch in each group. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] Reference will now be made in detail to various exemplary embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. The detailed description should not be construed as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] In this example, a total of 2 groups were set up. The first group was the control group fed with a normal diet (50% fish meal, 11.2% chicken meal, 15% flour, 9% soy protein, 4.5% squid paste, 8% fish oil and soybean oil, 2% monocalcium phosphate, 0.1% fish multivitamins, 0.1% mycotoxin adsorbent and multi-minerals, 0.1% compound probiotics); the second group was the treatment group (feed addition + injection group). When the sea bass was at 1 - 3 months old, in the normal diet, according to the weight of the normal diet, taurine was added at a ratio of 1 g / kg; Clock core peptide 1 (LIATVRLAKPQFIKEMCT) was added at 100 mg / kg; Clock core peptide 2 (LAQNTGPNTPAVATFAQ) was added at 100 mg / kg. Single-stranded small RNA (5 μl / g, injection volume / per sea bass body weight, the same hereinafter), Rev-erbα protein (5 μl / g) and Per1 protein (5 μl / g) were injected once every 15 days.

[0034] Stearic acid monoglyceride is used to nano-encapsulate chemically synthesized single-stranded small RNAs with special labels and chemical modifications. The sequence of the single-stranded small RNA is: 5′-CsAsGUUUUGCAUGGGUUUGsCsAsCs-Chol-3′. The 5′ end of this single-stranded small RNA is modified with 2 phosphorothioate backbones, the 3′ end is modified with 4 phosphorothioate backbones, and the 3′ end is cholesterol-modified, and the entire sequence is base-methylated modified.

[0035] The single-stranded small RNA in the injection part is formulated into a solution with a concentration of 5 mg / L.

[0036] Sea bass Rev-erbα and Per1 are obtained by the following method: Collect early-stage sea bass embryos and break them. Centrifuge the embryo lysate at 1200 - 1500 r / min for 10 - 15 min. After standing, take the supernatant and pass it through a chromatography column containing monoclonal antibodies against sea bass Rev-erbα or Per1 to obtain purified sea bass Rev-erbα and Per1 proteins respectively. The protein Rev-erbα solution and Per1 solution are configured into solutions with a concentration of 1 mg / ml. Recombinant proteins of Rev-erbα and Per1b can also be purchased commercially.

[0037] Feeding conditions: Raise in an outdoor circulating external water system. The aquaculture water body is 100 L. During the aquaculture period, the water temperature is 25 - 30 °C, pH is 6.5 - 7.2, dissolved oxygen > 6.5 mg / L, ammonia nitrogen < 0.2 mg / L, and nitrite nitrogen < 0.05 mg / L.

[0038] After two months of feeding, the specific results of the growth indicators are shown in Table 1. Compared with the control group, the weight gain and weight gain rate of sea bass in the treatment group increased, but there was no significant difference (P > 0.05). The body length measurement diagram of sea bass is as Figure 1 shown, and there is no significant difference between the two.

[0039] Table 1 Effects of dietary addition of Clock on the growth performance of sea bass (mean ± standard error) n = 5

[0040]

[0041] Example 2: Effects of a biological agent and method for improving the quality of sea bass on the expression of ApoC3 gene in the liver of sea bass

[0042] MicroRNA can participate in regulating the expression of Clock. We found through screening that miRNA-19d can selectively recognize sea bass Clock and inhibit its expression. The chemically synthesized small RNA molecules designed and used with labels and chemical modifications can specifically bind to miRNA-19d, reduce its expression, and then activate Clock to regulate the function of MyoD.

[0043] This example was divided into 3 groups in total. The first group was the control group fed with a normal diet (control group); in the second group, during the 1st - 3rd month of age of the sea bass, taurine was added to the normal diet at a ratio of 1 g / kg, Clock core peptide 1 (LIATVRLAKPQFIKEMCT) at 100 mg / kg, and Clock core peptide 2 (LAQNTGPNTPAVATFAQ) at 100 mg / kg based on the weight of the normal diet in the edible part (feed group); in the third group, during the 1st - 3rd month of age of the sea bass, the edible part was added to the normal diet at a ratio of taurine 1 g / kg, Clock core peptide 1 (LIATVRLAKPQFIKEMCT) 100 mg / kg, and Clock core peptide 2 (LAQNTGPNTPAVATFAQ) 100 mg / kg based on the weight of the normal diet, and at the same time, small RNA (5 μl / g), Rev-erbα (5 μl / g), and Per1b recombinant protein (5 μl / g) were injected once every 15 days (feed + injection group).

[0044] The sea bass was raised starting from 1 month old, and the feeding conditions were the same as before. After two months of feeding, the expression levels of the MyoD gene ( Figure 2 ) in the muscle and the ApoC3 gene ( Figure 3 ) in the liver of the sea bass in each group were detected by RT-qPCR method. (a, b indicate significant differences between groups, P < 0.05). The increase in the expression level of ApoC3 may promote the deposition of intramuscular fat (IMF), thereby improving the taste and flavor of the fish meat, optimizing the muscle fat content through lipid metabolism regulation, and meeting the needs of the consumer market. It may also affect the transport and storage of Omega-3 polyunsaturated fatty acids, thereby increasing the proportion of healthy fatty acids in the fish meat and improving the nutritional value of the sea bass.

[0045] Example 3: A method for improving the quality of sea bass and the effect of a biological agent on the expression of immune-related genes in sea bass

[0046] This example is divided into 3 groups. The first group is the control group fed with normal diet (control group); the second group is the feed group, in which taurine at a ratio of 1.5 g / kg, Clock core peptide 1 (LIATVRLAKPQFIKEMCT) at a ratio of 100 mg / kg, and Clock core peptide 2 (LAQNTGPNTPAVATFAQ) at a ratio of 100 mg / kg are added to the normal diet for sea bass at the age of 1 - 3 months; the third group is the feed + injection group, in which taurine at a ratio of 1.5 g / kg, Clock core peptide 1 (LIATVRLAKPQFIKEMCT) at a ratio of 100 mg / kg, and Clock core peptide 2 (LAQNTGPNTPAVATFAQ) at a ratio of 100 mg / kg are added to the normal diet for sea bass at the age of 1 - 3 months, and at the same time, small RNA (5 μl / g), Rev-erbα (5 μl / g), and Per1b recombinant protein (5 μl / g) are injected every 15 days.

[0047] The specific statistical results of the expression levels of immune-related genes in sea bass of each group are as Figure 4 shown (a and b respectively indicate significant differences between groups, P < 0.05). The results show that the mRNA expressions of TNF-a, IL-b, and IL-8 in the kidneys of sea bass in the feed + injection group are significantly increased compared with those in the control group. The increase in the expression of the inflammatory factor TNF-α indicates an enhanced disease resistance of sea bass.

[0048] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A biological agent for improving the quality of perch, characterized in that, The biological agent comprises an injection, and the injection comprises a chemically modified chemically synthesized single-stranded small RNA, Rev-erbα protein and Per1 protein; the sequence of the single-stranded small RNA is: 5′-CAGUUUUGCAUGGGUUUGCAC-3′.

2. The biological agent for improving the quality of perch according to claim 1, characterized in that, The biological agent further comprises an edible agent, and the edible agent is Clock core peptide 1, Clock core peptide 2 and taurine. The amino acid sequence of Clock core peptide 1 is: LIATVRLAKPQFIKEMCT; the amino acid sequence of Clock core peptide 2 is: LAQNTGPNTPAVATFAQ.

3. The biological agent for improving the quality of perch according to claim 1, wherein The 5′ end of the single-stranded small RNA is modified with 2 phosphorothioate backbones, the 3′ end is modified with 4 phosphorothioate backbones, the 3′ terminus is modified with cholesterol, and the entire sequence is modified with base methylation.

4. The preparation for improving the quality of perch according to claim 1, characterized in that, Collect early-stage embryos of sea bass and break them. Centrifuge the embryo lysate at 1200-1500 r / min for 10-15 min. After standing, take the supernatant and pass it through a chromatography column containing Rev-erbα or Per1 monoclonal antibody to obtain purified Rev-erbα and Per1 proteins respectively.

5. Use of the biological agent according to any one of claims 1 to 4 in fish feed or fish growth additives.

6. The application according to claim 5, characterized in that, The fish is sea bass.

7. A key method for improving the quality of perch, characterized in that, When the sea bass is 1-6 months old, inject the single-stranded small RNA, Rev-erbα protein and Per1 protein every 10-15 days.

8. The key method for improving the quality of perch according to claim 7, characterized in that, By weight, the injection dose ratio is 5-10 μl / g.

9. The key method for improving the quality of perch according to claim 8, characterized in that, In the premixed feed of the basic diet of sea bass, add Clock core peptide 1, Clock core peptide 2 and taurine. The amino acid sequence of Clock core peptide 1 is: LIATVRLAKPQFIKEMCT; the amino acid sequence of Clock core peptide 2 is: LAQNTGPNTPAVATFAQ.

10. The key method for improving the quality of bass according to claim 9, wherein The addition amount of Clock core peptide 1 is 100-200 mg / kg; the addition amount of Clock core peptide 2 is 100-200 mg / kg; the addition amount of taurine is 1-1.5 g / kg.

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