Application of neuropeptide Y in preparation of product for promoting growth of tilapia mossambica and improving environmental stress tolerance

Abstract

The invention discloses application of neuropeptide Y in preparation of a product for promoting growth of tilapia mossambica and improving environmental stress tolerance. A method for efficiently obtaining a large amount of neuropeptide Y is established by utilizing a bacillus subtilis recombinant expression system. The neuropeptide Y promotes ingestion of tilapia mossambica and relieves inflammatory response. The neuropeptide Y is added into the feed, so that the growth of the tilapia can be remarkably promoted, the survival rate, the antioxidant capacity and the tolerance to environmental stress can be improved, and the negative effects of oxidative stress and the like caused by reducing the content of soybean meal in the feed can be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology and bioengineering technology, and in particular to application of neuropeptide Y in preparing products capable of promoting tilapia growth and improving environmental stress tolerance. Background Art

[0002] Tilapia (Oreochromis sp.), a member of the genus Oreochromis in the order Perciformes and family Cichlidae, is widely farmed in over 130 countries for its rapid growth, short breeding cycles, and strong resistance to disease and stress. However, with increasing stocking density and scale, the aquaculture environment for tilapia is deteriorating, leading to problems such as decreased appetite, slow growth, and weakened immune function. Various environmental stressors, such as ammonia nitrogen and nitrite, are hindering the healthy development of the tilapia aquaculture industry.

[0003] Ammonia nitrogen and nitrite are common harmful substances in water, primarily derived from the decomposition of fish excrement and leftover feed. High concentrations of ammonia nitrogen and nitrite can suppress tilapia's immunity and metabolism, affecting its growth. Currently, the main approaches to prevent and alleviate the stress caused by ammonia nitrogen and nitrite on tilapia are optimizing water quality and feed management. However, for tilapia already under stress, there is a lack of effective regulators to quickly enhance their resilience and mitigate the damage caused by stress.

[0004] Neuropeptide Y (NPY) is widely distributed in the central and peripheral nervous systems of mammals and is an important neuropeptide endocrine factor. The NPY gene has a 300-bp open reading frame encoding a 99-amino acid NPY precursor. This NPY precursor consists of a 28-amino acid signal peptide, a 36-amino acid NPY mature peptide, and a 35-amino acid peptide segment of unknown function. The 36-amino acid mature peptide is the true biologically active NPY. Enzymatic cleavage of the NPY precursor yields the active NPY mature peptide, which then exerts its physiological functions. However, prior research and application of neuropeptide Y in alleviating ammonia and nitrite stress in tilapia aquaculture has not been thoroughly explored, and its potential for enhancing tilapia's resistance to environmental stress needs further exploration. Furthermore, NPY can be obtained through natural extraction and chemical synthesis, but these methods are expensive, limited in quantity, and complex, making them inadequate for the huge demand in aquaculture. Therefore, effective methods for obtaining large quantities of NPY are needed. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides the use of neuropeptide Y in preparing a product for promoting the growth of tilapia and improving environmental stress tolerance.

[0006] The first object of the present invention is to provide a use of neuropeptide Y in preparing a product for promoting the growth of tilapia and improving the tolerance of tilapia to environmental stress.

[0007] The second object of the present invention is to provide a use of neuropeptide Y in preparing a product for promoting the growth of tilapia under environmental stress.

[0008] The third object of the present invention is to provide a use of neuropeptide Y in preparing a product for improving the survival rate of tilapia under environmental stress.

[0009] The fourth object of the present invention is to provide a use of neuropeptide Y in preparing a product for improving the antioxidant capacity of tilapia under environmental stress.

[0010] A fifth object of the present invention is to provide a use of neuropeptide Y in preparing a product for reducing the soybean meal content in tilapia feed and promoting the growth of tilapia under environmental stress.

[0011] A sixth object of the present invention is to provide a use of neuropeptide Y in preparing a product for reducing the soybean meal content in tilapia feed and improving the survival rate of tilapia under environmental stress.

[0012] A seventh object of the present invention is to provide a use of neuropeptide Y in preparing a product for reducing the soybean meal content in tilapia feed and improving the antioxidant capacity of tilapia under environmental stress.

[0013] In order to achieve the above object, the present invention is implemented through the following scheme:

[0014] The present invention constructs an in vitro expression system for neuropeptide Y for the first time, obtaining recombinant neuropeptide Y and demonstrating its involvement in regulating tilapia feeding and inflammatory responses. In vitro, recombinant neuropeptide Y binds to NPY receptors in tilapia hypothalamic fragments, stimulating the expression of the feeding-promoting factor npy while simultaneously alleviating inflammatory responses. In vivo, intraperitoneal injection of recombinant neuropeptide Y more effectively regulates NPY receptor expression in the hypothalamus, stimulates tilapia appetite, promotes the restoration of inflammatory homeostasis in the telencephalon, hypothalamus, and liver, and alleviates inflammatory responses in the tilapia intestine. The present invention also demonstrates through feeding experiments that replacing 50% of soybean meal in the feed with cottonseed protein concentrate inhibits tilapia growth, antioxidant capacity, and reduces feed utilization efficiency. However, adding neuropeptide Y to the feed significantly improves tilapia growth performance, antioxidant capacity, and feed utilization efficiency. Further ammonia nitrogen stress and nitrite stress experiments confirmed that replacing 50% of soybean meal in the feed with cottonseed protein concentrate had a negative impact on the ability of tilapia to resist ammonia nitrogen and nitrite stress, and this negative impact could be reversed by rtNPY.

[0015] Therefore, the present invention seeks protection for the following:

[0016] Application of neuropeptide Y in preparing products for promoting the growth of tilapia and improving the tolerance of tilapia to environmental stress.

[0017] Application of neuropeptide Y in preparing a product for promoting tilapia growth under environmental stress.

[0018] Use of neuropeptide Y in preparing a product for improving the survival rate of tilapia under environmental stress.

[0019] Application of neuropeptide Y in preparing a product for improving the antioxidant capacity of tilapia under environmental stress.

[0020] Application of neuropeptide Y in preparing a product for reducing soybean meal content in tilapia feed and promoting tilapia growth under environmental stress.

[0021] Application of neuropeptide Y in preparing a product for reducing the soybean meal content in tilapia feed and improving the survival rate of tilapia under environmental stress.

[0022] Application of neuropeptide Y in preparing a product for reducing soybean meal content in tilapia feed and improving the antioxidant capacity of tilapia under environmental stress.

[0023] Preferably, the amino acid sequence of neuropeptide Y includes the sequence shown in SEQ ID NO.3.

[0024] Preferably, the neuropeptide Y is obtained by in vitro recombinant expression or chemical synthesis.

[0025] More preferably, the microorganism used for the in vitro recombinant expression includes Bacillus subtilis.

[0026] Further preferably, the Bacillus subtilis is Bacillus subtilis WB800N.

[0027] More preferably, the amino acid sequence of neuropeptide Y obtained by in vitro recombinant expression is shown as SEQ ID NO.4.

[0028] Further preferably, the nucleotide sequence of the nucleic acid molecule encoding the neuropeptide Y obtained by in vitro recombinant expression is as shown in SEQ ID NO. 5 or a completely complementary sequence to the sequence shown in SEQ ID NO. 5. The nucleotide sequence shown in SEQ ID NO. 5 matches the characteristics of the microorganism used for in vitro recombinant expression, which facilitates the correct expression of the target protein in the expression host and its ultimate secretion into the extracellular space.

[0029] The recombinant expression plasmid should be a double-stranded DNA plasmid, and its composition should be appropriate for the expression host, containing a mature promoter, multiple cloning site, resistance tag, and an appropriate signal peptide coding region. More preferably, the recombinant expression plasmid used for in vitro recombinant expression uses the pHT43 plasmid as its backbone. The recombinant plasmid is then chemically transformed into the expression host to obtain a recombinant microorganism containing the tilapia NPY gene.

[0030] Preferably, the environmental stress includes ammonia nitrogen stress and / or nitrite stress.

[0031] Preferably, the tilapia comprises Tilapia GIFT.

[0032] Preferably, the content of soybean meal in the tilapia feed is 35%wt to 50%wt.

[0033] More preferably, the content of soybean meal in the tilapia feed is 40%wt.

[0034] In some specific embodiments, the tilapia feed comprises: soybean meal, casein, fish oil, soybean oil, high-gluten flour, microcrystalline cellulose, vitamin and mineral premix, monocalcium phosphate, choline chloride, dimethyl-β-propionate and butylated hydroxytoluene.

[0035] In some specific embodiments, the tilapia feed comprises: 35% wt to 50% wt soybean meal, 8% wt to 15% wt casein, 1.0% wt to 2.0% wt fish oil, 3.0% wt to 5.5% wt soybean oil, 30% wt to 50% wt high-gluten flour, 5% wt to 12% wt microcrystalline cellulose, 0.7% wt to 1.3% wt vitamin and mineral premix, 1.0% wt to 2.0% wt calcium dihydrogen phosphate, 0.3% wt to 0.7% wt choline chloride, 0.1% wt to 0.3% wt dimethyl-β-propionate and 0.1% wt to 0.3% wt butylated hydroxytoluene.

[0036] In some specific embodiments, the tilapia feed is composed of the following ingredients: 40% wt soybean meal, 10% wt casein, 1.5% wt fish oil, 4.5% wt soybean oil, 35% wt high-gluten flour, 5.5% wt microcrystalline cellulose, 1% wt vitamin and mineral premix, 1.5% wt calcium dihydrogen phosphate, 0.5% wt choline chloride, 0.25% wt dimethyl-β-propionate and 0.25% wt butylated hydroxytoluene.

[0037] Preferably, the method for reducing the soybean meal content in tilapia feed comprises: replacing 30% wt to 75% wt soybean meal with cottonseed protein concentrate.

[0038] More preferably, the method for reducing the soybean meal content in tilapia feed comprises: replacing 50% wt soybean meal with cottonseed protein concentrate.

[0039] In some specific embodiments, the tilapia feed comprises: soybean meal, casein, cottonseed protein concentrate, fish oil, soybean oil, high-gluten flour, microcrystalline cellulose, vitamin and mineral premix, monocalcium phosphate, choline chloride, dimethyl-β-propionate and butylated hydroxytoluene.

[0040] In some specific embodiments, the tilapia feed includes: 15% wt to 35% wt soybean meal, 8% wt to 15% wt casein, 10% wt to 22.5% wt cottonseed protein concentrate, 1.0% wt to 2.0% wt fish oil, 3.0% wt to 5.5% wt soybean oil, 30% wt to 50% wt high-gluten flour, 5% wt to 12% wt microcrystalline cellulose, 0.7% wt to 1.3% wt vitamin and mineral premix, 1.0% wt to 2.0% wt calcium dihydrogen phosphate, 0.3% wt to 0.7% wt choline chloride, 0.1% wt to 0.3% wt dimethyl-β-propionate and 0.1% wt to 0.3% wt dibutyl hydroxytoluene.

[0041] In some specific embodiments, the tilapia feed is composed of the following ingredients: 20% wt soybean meal, 10% wt casein, 15% wt cottonseed protein concentrate, 1.5% wt fish oil, 4.5% wt soybean oil, 35% wt high-gluten flour, 10.5% wt microcrystalline cellulose, 1% wt vitamin and mineral premix, 1.5% wt calcium dihydrogen phosphate, 0.5% wt choline chloride, 0.25% wt dimethyl-β-propionate and 0.25% wt butylated hydroxytoluene.

[0042] Preferably, the crude protein content of the tilapia feed is 28.0% to 36.0%, and more preferably, the crude protein content of the tilapia feed is 32.20% to 32.25%.

[0043] Preferably, the crude fat content of the tilapia feed is 5% to 8%.

[0044] More preferably, the crude fat content of the tilapia feed is 6.19% to 6.31%.

[0045] Preferably, the tilapia feed is sprayed with neuropeptide Y.

[0046] More preferably, the tilapia feed is sprayed with the neuropeptide Y obtained by in vitro recombinant expression.

[0047] Further preferably, the mass ratio of the tilapia feed to the neuropeptide Y is 1 g: (0.1-1) μg.

[0048] More preferably, the mass ratio of the tilapia feed to the neuropeptide Y is 1 g:0.3 μg.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] This invention utilizes a Bacillus subtilis recombinant expression system to establish a method for efficiently obtaining large amounts of neuropeptide Y. Neuropeptide Y promotes tilapia feeding and alleviates inflammatory responses. Adding neuropeptide Y to tilapia feed significantly promotes growth, improves survival rate, antioxidant capacity, and tolerance to environmental stress. It also significantly mitigates negative effects such as oxidative stress caused by reducing the amount of soybean meal in the feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is the sequencing alignment result of the recombinant plasmid expressing tilapia NPY; NPY is the nucleotide sequence of the designed recombinant NPY; 1_D01.seq is the nucleotide sequence of the recombinant plasmid sequencing.

[0052] Figure 2The electrophoresis analysis results of the expression induced by the recombinant Bacillus subtilis bacteria are shown in FIG. 1 ; Lane M is a protein standard; Lanes 1 to 9 correspond to samples 1 to 9 in step 3 of Example 1, respectively.

[0053] Figure 3 Figure 5 is the mass spectrometry peak diagram of rtNPY, and Relative intensity is the relative intensity; A is the peak diagram of peptide YYSALR; B is the peak diagram of peptide HYINLITR.

[0054] Figure 4 The electrophoresis analysis results of the rtNPY purification effect; lane M is the protein standard; lanes 1 to 7 correspond to samples 1 to 7 in step 4 of Example 1, respectively.

[0055] Figure 5 The electrophoresis analysis results of the ultrafiltration concentration effect of rtNPY; lane M is the protein standard; lane 1 is the supernatant obtained by centrifugation after inducing WB800N / pHT43-NPY expression with 0.5 mM IPTG at 37°C for 12 h; lane 2 is the eluate with 250 mM imidazole; lane 3 is the concentrate after ultrafiltration; lane 4 is sNPY.

[0056] Figure 6 is the BCA standard curve.

[0057] Figure 7 The effect of rtNPY on NPY receptor-related genes in hypothalamic fragments of GIFT tilapia; A to E are the mRNA expression levels of y2-2, y4, y7, y8a and y8b, respectively; at the same time, *p<0.05, **p<0.01, ***p<0.001 vs. 0nM; at the same dose, #p<0.05, ##p<0.01 vs. 0h.

[0058] Figure 8 The effect of rtNPY on feeding-related genes in hypothalamic fragments of GIFT tilapia; A to F are the mRNA expression levels of npy, pyy, agrp, cart1, orexin and pomc, respectively; at the same time, *p<0.05, **p<0.01, ***p<0.001 vs. 0nM; at the same dose, #p<0.05, ##p<0.01 vs. 0h.

[0059] Figure 9The effect of rtNPY on inflammation-related genes in hypothalamic fragments of GIFT tilapia; A to F are the mRNA expression levels of il-1β, il-6, il-8, il-10, tnf-α and tgf-β1, respectively; at the same time, *p<0.05, **p<0.01, ***p<0.001 vs. 0nM; at the same dose, #p<0.05, ##p<0.01, ###p<0.001 vs. 0h.

[0060] Figure 10 Effects of intraperitoneal injection of sNPY and rtNPY on the expression levels of NPY receptors and feeding-related genes in the telencephalon and hypothalamus of GIFT tilapia; A is the mRNA expression level of NPY receptor-related genes in the telencephalon; B is the mRNA expression level of feeding-related genes in the telencephalon; C is the mRNA expression level of NPY receptor-related genes in the hypothalamus; D is the mRNA expression level of feeding-related genes in the hypothalamus; at the same time, *p<0.05, **p<0.01, ***p<0.001 vs. PBS.

[0061] Figure 11 Effects of intraperitoneal injection of sNPY and rtNPY on the expression levels of inflammation-related genes in the telencephalon, hypothalamus, liver and intestine of GIFT tilapia; A, telencephalon; B, hypothalamus; C, liver; D, foregut; E, midgut; F, hindgut; at the same time, *p<0.05, **p<0.01, ***p<0.001 vs. PBS.

[0062] Figure 12 The effects of intraperitoneal injection of sNPY and rtNPY on the expression levels of key genes in the GH / IGF-1 axis in the pituitary-liver of GIFT tilapia; A is the mRNA expression level of gh in the pituitary; B is the mRNA expression level of igf-1 in the liver.

[0063] Figure 13 are antioxidant-related indicators of tilapia serum; A is total antioxidant capacity; B is superoxide dismutase activity; C is glutathione peroxidase activity; D is catalase activity; and E is malondialdehyde content.

[0064] Figure 14 is the survival rate of four groups of tilapia after 96 hours of ammonia nitrogen and nitrite stress.

[0065] Figure 15 These are the antioxidant-related indicators of serum of four groups of tilapia after 96 h of ammonia nitrogen and nitrite stress; A is the total antioxidant capacity; B is the superoxide dismutase activity; C is the glutathione peroxidase activity; D is the catalase activity; and E is the malondialdehyde content. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0067] The culture media involved in the following examples are as follows: LB liquid medium: 1% W / V NaCl, 1% W / V peptone, 0.5% W / V yeast extract powder, and antibiotics at appropriate concentrations as needed when used; LB solid medium: 1% W / V NaCl, 1% W / V peptone, 0.5% W / V yeast extract powder, 2% W / V agar powder, and antibiotics at appropriate concentrations as needed when used; 10× minimum salt solution: 14 g K2HPO4, 6 g KH2PO4, 2 g (NH4)2SO4, 1 g Na3C6H5O7·2H2O, 0.1 g MgSO4, dissolved in sterile water in sequence and diluted to 100 mL; GMI solution: 5 mL 10× minimum salt solution, 0.05 g glucose, 0.01 g acid-hydrolyzed casein, and 0.05 g yeast extract, dissolved in sterile water in sequence, mixed before use, and diluted to 50 mL; GMII solution: 10 mL Dissolve 10× minimum salt solution, 0.5 g glucose, 0.004 g acid-hydrolyzed casein, 0.004 g yeast extract, 0.5 mL 0.5 M MgCl₂, and 0.5 mL 0.1 M CaCl₂ in sterile water, mix well, and adjust the volume to 100 mL. All components of the GMI and GMII solutions, except the 10× minimum salt solution, must be sterilized separately (autoclaving at 121°C for 20 minutes).

[0068] The raw materials of the tilapia feed involved in the following embodiments are as follows: soybean meal was purchased from Xiamen Jiakang Feed Co., Ltd.; casein was purchased from Xiamen Jiakang Feed Co., Ltd.; cottonseed protein concentrate was purchased from Zhaoyuan Wenji Food Co., Ltd.; fish oil was purchased from Xiamen Jiakang Feed Co., Ltd.; soybean oil was purchased from Guangzhou Pupu Network Technology Co., Ltd.; high-gluten flour was purchased from Taicang Jinjuhe Trading Co., Ltd.; microcrystalline cellulose was purchased from Henan Zuannuo Biotechnology Co., Ltd.; vitamin and mineral premixes were purchased from Xiamen Jiakang Feed Co., Ltd.; calcium dihydrogen phosphate was purchased from Xiamen Jiakang Feed Co., Ltd.; choline chloride was purchased from Xiamen Jiakang Feed Co., Ltd.; dimethyl-β-propionate was purchased from Shanghai Yantian Biotechnology Co., Ltd., with the product number MAYA-NZJ-293; and butylated hydroxytoluene was purchased from Shanghai Yantian Biotechnology Co., Ltd., with the product number MAYA-NZJ-810.

[0069] Example 1 Method for recombinant expression of tilapia neuropeptide Y using Bacillus subtilis

[0070] 1. Design and synthesis of recombinant genes expressing tilapia neuropeptide Y (NPY) and construction of recombinant plasmids

[0071] The NPY coding gene sequence (SEQ ID NO. 1) and the full-length amino acid sequence encoding the NPY precursor peptide (SEQ ID NO. 2) were obtained from a GIFT tilapia transcriptome library previously established in our laboratory. In this example, the amino acid sequence of the NPY mature peptide (SEQ ID NO. 3) was obtained from this full-length amino acid sequence.

[0072] The 6×His tag protein was inserted into the N-terminus of the NPY mature peptide to obtain the amino acid sequence of recombinant tilapia NPY (SEQ ID NO. 4). Codon optimization was then performed and the stop codon TGA was added to obtain the coding sequence of recombinant tilapia NPY (SEQ ID NO. 5). The recombinant tilapia NPY gene fragment was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the pHT43 plasmid to obtain a recombinant plasmid expressing tilapia NPY, which was designated pHT43-NPY.

[0073] After E. coli cloning and purification, plasmid extraction and sequencing identification, the sequencing results are as follows Figure 1 As shown, the constructed pHT43-NPY sequence was correct and stored at -80°C for future use.

[0074] 2. Construction of recombinant Bacillus subtilis expressing tilapia neuropeptide Y

[0075] The prior art has disclosed some methods for preparing recombinant neuropeptides based on genetic engineering technology, such as: Chinese patent application CN201010225765.7 relates to an Escherichia coli expression system for recombinant human neuropeptide Y; Chinese patent application CN200910214231.1 relates to a Pichia pastoris expression system for recombinant tilapia neuropeptide Y; Chinese patent application CN201810712076.5 relates to an Escherichia coli expression system for recombinant grouper neuropeptide Y; Chinese patent application CN201810680835.4 relates to a mammalian cell expression system for recombinant large yellow croaker neuropeptide Y; Chinese patent application CN201810681630.8 relates to a mammalian cell expression system for recombinant neuropeptide Y of cuttlefish. However, these existing technologies also have the following major problems: (1) The E. coli expression system easily forms inclusion bodies, making it difficult to obtain large amounts of soluble proteins with biological activity; (2) E. coli itself contains endotoxins and toxic proteins, which may be mixed in the final product; (3) The yeast expression system has low expression levels, long fermentation times, and is prone to incorrect protein glycosylation; (4) The culture medium cost of the mammalian cell expression system is extremely high, the culture cycle is long, the operating technology requirements are high, and sometimes it can lead to viral infection.

[0076] Bacillus subtilis is a strain that efficiently secretes proteins and has become an important biotechnology tool in the production of industrial enzymes and food and pharmaceutical proteins. The Bacillus subtilis expression system has the following characteristics: (1) it is non-pathogenic and does not produce substances such as endotoxins; (2) it has strong protein secretion capabilities; (3) it has no significant codon bias, and the product is not prone to forming inclusion bodies; and (4) it is simple and rapid to culture. Compared with existing technologies, this system has the following technical advantages, as shown in Table 1.

[0077] Table 1 Comparison of existing technologies and Bacillus subtilis expression systems for neuropeptide Y recombinant proteins

[0078]

[0079]

[0080] Bacillus subtilis WB800N (B. subtilis WB800N) is more conducive to the expression of exogenous proteins after inactivating eight extracellular proteases in the genome of B. subtilis 168 through gene mutation. Therefore, this example uses B. subtilis WB800N to achieve recombinant expression of NPY. The recombinant plasmid pHT43-NPY is transformed into B. subtilis WB800N competent cells to obtain positive transformants, namely recombinant B. subtilis expressing tilapia neuropeptide Y, denoted as WB800N / pHT43-NPY, establishing an effective method for obtaining large amounts of NPY. The specific steps are as follows:

[0081] Streak Bacillus subtilis WB800N on LB solid plate and incubate at 37℃ for 16h. Pick a single colony with a sterile inoculation loop and inoculate it into 5mL GMI solution. Incubate at 37℃ and 100rpm for 16h. Transfer 2mL of bacterial solution to 18mL GMI solution and incubate at 37℃ and 180rpm until OD 600 nm is 0.80; 10 mL of bacterial liquid was transferred to 90 mL of GMII solution and cultured at 37°C and 100 rpm for 90 min to obtain Bacillus subtilis WB800N competent cells; 200 ng of recombinant plasmid pHT43-NPY was gently mixed with 500 μL of Bacillus subtilis WB800N competent cells, placed in a 37°C water bath for 30 min, and then cultured at 37°C constant temperature shaker (180 rpm) for 60 min; the obtained culture was spread on an LB solid plate containing 5 μg / mL chloramphenicol and cultured at 37°C for 16 h; a single clone colony was picked and inoculated into LB liquid culture medium containing 5 μg / mL chloramphenicol, cultured at 37°C and 180 rpm for 24 h, the bacterial liquid was collected to extract the plasmid for sequencing identification, and the results showed that the bacteria corresponding to the plasmid with the correct sequence was the Bacillus subtilis recombinant bacteria (WB800N / pHT43-NPY) expressing tilapia neuropeptide Y.

[0082] According to the same method, the pHT43 plasmid was transformed into Bacillus subtilis WB800N competent cells to obtain a control Bacillus subtilis recombinant strain, which was recorded as WB800N / pHT43.

[0083] 3. Induced expression of recombinant Bacillus subtilis

[0084] WB800N / pHT43 and WB800N / pHT43-NPY were inoculated into 10 mL of LB liquid medium containing 5 μg / mL chloramphenicol, respectively, and cultured at 37°C and 180 rpm for 12 h. Four 500 mL conical flasks containing 100 mL of LB liquid medium containing 5 μg / mL chloramphenicol were taken and marked as bottles 1 to 4, and the WB800N / pHT43 bacterial solution was inoculated into bottles 1 and 2 at a volume ratio of 1:100, and the WB800N / pHT43-NPY bacterial solution was inoculated into bottles 3 and 4 at a volume ratio of 1:100. Bottles 1 to 4 were cultured at 37°C and 180 rpm until the OD 600 nm was 0.80, and 100 μL 1 M IPTG was added to bottle 2 and bottle 4, respectively, which were used as the WB800N / pHT43 induction group and WB800N / pHT43-NPY induction group, respectively. No inducer was added to bottle 1 and bottle 3, which were used as the WB800N / pHT43 negative control group and WB800N / pHT43-NPY negative control group, respectively. The shaking culture was continued for 4 h.

[0085] After the culture was completed, the bacterial suspensions from bottles 1 to 4 were collected and centrifuged at 8000 rpm for 10 min. The bacterial precipitates from bottles 1 to 4 were collected and washed with lysis buffer (200 mM NaCl, 50 mM Tris, pH = 8.0) and resuspended to obtain the bacterial precipitate sample of the WB800N / pHT43 negative control group (denoted as sample 1), the bacterial precipitate sample of the WB800N / pHT43 induced group (denoted as sample 2), the bacterial precipitate sample of the WB800N / pHT43-NPY negative control group (denoted as sample 3), and the bacterial precipitate sample of the WB800N / pHT43-NPY induced group (denoted as sample 4); the supernatants after centrifugation of bottles 1 to 4 were collected respectively, and concentrated by DOC-TCA-acetone precipitation method to obtain the supernatant sample of the WB800N / pHT43 negative control group (denoted as sample 5), the supernatant sample of the WB800N / pHT43 induced group (denoted as sample 6), the supernatant sample of the WB800N / pHT43-NPY negative control group (denoted as sample 7), and the supernatant sample of the WB800N / pHT43-NPY induced group (denoted as sample 8). In addition, the mature peptide of NPY (SEQ ID NO. 3), namely sNPY, was synthesized by chemical synthesis, and its protein sample was recorded as sample 9.

[0086] 2× protein loading buffer was added to each of samples 1 to 9, and the samples were boiled in a boiling water bath for 10 min to prepare protein electrophoresis samples. Tricine-SDS-PAGE gel electrophoresis was then performed, and Coomassie Brilliant Blue staining was performed after completion of the electrophoresis.

[0087] The electrophoresis results are as follows Figure 2As shown in the figure, in the culture supernatant of WB800N / pHT43-NPY, after the inducer IPTG was added, a clear protein band below 11 kDa was found (sample 8), which was the recombinant tilapia NPY (denoted as recombinanttilapia NPY, rtNPY). Since it contained 6 histidine-tagged proteins (about 0.84 kDa), the total molecular weight was slightly larger than that of chemically synthesized sNPY (SEQ ID NO. 3). However, the appearance of this protein band was not observed when the inducer IPTG was not added (sample 7).

[0088] Further mass spectrometry detection of rtNPY was performed, such as Figure 3 As shown in Figures A and B, two peptides unique to NPY in tilapia were found: YYSALR and HYINLITR. This indicates that rtNPY is recombinant NPY from GIFT tilapia and that rtNPY was successfully expressed in vitro.

[0089] 4. Purification and ultrafiltration concentration of rtNPY

[0090] The induction expression conditions were the same as in step 3, except that WB800N / pHT43-NPY was induced with 0.5 mM IPTG at 37°C for 12 h as the induced group, and WB800N / pHT43-NPY cultured without IPTG served as the control group. The bacterial cultures from the induced and control groups were collected and centrifuged at 8000 rpm for 10 min. The supernatant samples from the induced and control groups (referred to as sample 1) and sample 2 were collected.

[0091] The supernatant samples from the induced group were purified using HisTrap HP prepacked chromatography columns (Cytiva, USA). During the purification process, the flow-through after the Ni column (denoted as sample 3), the equilibration buffer (denoted as sample 4), the 50 mM imidazole eluate (denoted as sample 5), and the 250 mM imidazole eluate (denoted as sample 6) were collected. sNPY was designated as sample 7.

[0092] Samples 1 to 7 were tested by Tricine-SDS-PAGE to identify the purification effect. Figure 4 As shown, 50 mM imidazole eluent can completely elute the impurity proteins on the chromatography column, and 250 mM imidazole eluent can successfully elute the target protein rtNPY, showing an obvious and single target band (11 kDa), proving that the purification effect is good.

[0093] The purified protein solution was concentrated by ultrafiltration using a 3 kDa ultrafiltration tube, 250 mM imidazole was replaced with PBS, and the concentrated solution after ultrafiltration was collected for Coomassie Brilliant Blue staining. Figure 5As shown, the concentrate after ultrafiltration has an obvious and single target band (11 kDa) and is darker in color than the band of the 250 mM imidazole elution solution, indicating that the target protein rtNPY with high purity and high concentration is successfully obtained.

[0094] The protein concentration of the ultrafiltration concentrate was determined by BCA method, and the following Figure 6 As shown in the standard curve, 27.14 mg rtNPY was co-purified from 1 L WB800N / pHT43-NPY supernatant.

[0095] Example 2 Functional Study of rtNPY

[0096] The weight of the GIFT tilapia used in this example was 35±5 g, the culture water temperature was 25° C. to 28° C., and the fish were domesticated under 12 hours of light and 12 hours of darkness. The experiments were conducted after domestication for more than 2 weeks.

[0097] 1. Experiment on incubation of tilapia hypothalamic fragments with rtNPY

[0098] (1) Incubation of tilapia hypothalamic fragments

[0099] Healthy and uniform-sized GIFT tilapia were randomly selected and euthanized to collect the hypothalamus. Subsequently, the hypothalamus was washed three times with pre-cooled L15 medium and then cut into approximately 1 mm pieces using a tissue slicer. 3 The fragments were washed three times with pre-cooled L15 culture medium, and the hypothalamic fragments were resuspended in L15 culture medium at a concentration of 20 mg / mL to obtain a hypothalamic fragment resuspension.

[0100] The resuspended hypothalamic fragments were added to a 24-well plate at 1 mL / well and incubated at 28°C for 24 hours. The old medium was discarded and fresh L15 medium was added at 1 mL / well to starve the hypothalamic fragments for 1 hour. Subsequently, the hypothalamic fragments were incubated with L15 medium containing 0 nM, 10 nM, 100 nM, and 1000 nM rtNPY for 0, 3, 6, 9, and 12 hours, respectively.

[0101] (2) Detection of gene expression by fluorescence quantitative PCR (qPCR)

[0102] After the incubation, discard the L15 medium in the 24-well plate and use Hypothalamic fragments were resuspended in a 4% ethanol (Omega, Cat. No. L12XJ) reagent and transferred to enzyme-free EP tubes. Total RNA was extracted from the hypothalamic fragments and reverse transcribed to generate cDNA. Using cDNA as a template, qPCR was performed to measure the mRNA expression levels of neuropeptide (NPY) receptor genes (y2-2, y4, y7, y8a, and y8b), feeding-related genes (npy, pyy, agrp, cart1, orexin, and pomc), and inflammatory factors (il-1β, il-6, il-8, il-10, tnf-α, and tgf-β1). β-actin was used as an internal control. The primers used are shown in Table 2. A negative control was set up, and each sample was repeated twice.

[0103] The qPCR reaction system was as follows: template 1 μL, ddH2O 3.6 μL, upstream primer 0.2 μL, downstream primer 0.2 μL, 2×SYBR Green Pro Taq HS Premix II 5 μL. The qPCR reaction program was as follows: (1) 95°C, 1 min; (2) 95°C, 5 sec; 60°C, 30 sec; 72°C, 30 sec; 40 cycles; (3) 95°C, 15 sec; annealing at 65-95°C at an increment of 0.5°C / sec to generate a melting curve.

[0104] Table 2 qPCR primer sequences

[0105]

[0106]

[0107] Two-way ANOVA was used to analyze the significance of the effects of rtNPY incubation dose and incubation time on the RNA expression levels of each gene.

[0108] (3) Result analysis

[0109] like Figure 7 As shown in Figure A, after incubation with rtNPY, the mRNA expression level of y2-2 in tilapia hypothalamic fragments did not change significantly. The incubation dose of rtNPY had no significant effect on the mRNA expression of y2-2, while the incubation time had a significant effect, but the interaction was not significant. Figure 7As shown in Figure B, after incubation with rtNPY, at a concentration of 0 nM, the mRNA expression level of y4 in the tilapia hypothalamus fragments changed relatively steadily over time; at concentrations of 10 nM, 100 nM, and 1000 nM, the expression levels increased significantly after 12 h; and after incubation with rtNPY for 12 h, the mRNA expression levels of y4 in the 10 nM and 100 nM groups were significantly higher than those in the 0 nM group; the incubation dose, incubation time, and their interaction of rtNPY all had significant effects on the mRNA expression level of y4. Figure 7 As shown in Figure C, after incubation with rtNPY, at a concentration of 0 nM, the mRNA expression level of y7 in tilapia hypothalamus fragments changed relatively steadily over time; at concentrations of 10 nM and 100 nM, the mRNA expression level of y7 increased significantly after 12 h of incubation; and after 12 h of incubation with rtNPY, the mRNA expression level of y7 in the 10 nM and 100 nM groups was significantly higher than that in the 0 nM group; the incubation dose, incubation time and their interaction of rtNPY all had a significant effect on the mRNA expression level of y7. Figure 7 As shown in Figure D, after incubation with 10nM rtNPY for 9-12h and 100nM rtNPY for 12h, the mRNA expression level of y8a in tilapia hypothalamic fragments increased significantly; and after incubation with rtNPY for 12h, the mRNA expression level of y8a in the 10nM and 100nM groups was significantly higher than that in 0nM; the incubation dose, incubation time and their interaction of rtNPY had a significant effect on the mRNA expression level of y8a. Figure 7 As shown in Figure E, after incubation with 10nM and 100nM rtNPY for 9 to 12 hours, the mRNA expression level of y8b in tilapia hypothalamic fragments increased significantly, and both were significantly higher than 0nM; the incubation dose, incubation time and their interaction of rtNPY had a significant effect on the mRNA expression level of y8b.

[0110] like Figure 8 As shown in Figure A, after incubation with 10nM and 100nM rtNPY for 12h, the mRNA expression level of npy in tilapia hypothalamus fragments increased significantly, and both were significantly higher than 0nM; the incubation dose, incubation time and their interaction had a significant effect on the mRNA expression level of npy. Figure 8 As shown in Figures B to F, after incubation with rtNPY, the mRNA expression levels of pyy, agrp, cart1, orexin and pomc in tilapia hypothalamic fragments did not change significantly; the incubation dose, incubation time and their interaction of rtNPY had no significant effect on the mRNA expression of these five genes.

[0111] like Figure 9As shown in Figure A, after incubation with 0nM, 10nM and 100nM rtNPY for 12h, the mRNA expression level of il-1β in tilapia hypothalamus fragments increased significantly; and after incubation with rtNPY for 12h, the mRNA expression level of il-1β in the 100nM and 1000nM groups was significantly lower than that in 0nM; the incubation dose and incubation time of rtNPY had a significant effect on the mRNA expression of il-1β, but the interaction effect was not significant. Figure 9 As shown in Figure B, after incubation with rtNPY, the mRNA expression level of il-6 in tilapia hypothalamic fragments did not change significantly; the incubation dose, incubation time and their interaction on the mRNA expression of y2-2 were not significantly affected. Figure 9 As shown in Figure C, after incubation with 10 nM and 100 nM rtNPY, the mRNA expression level of il-8 in tilapia hypothalamus fragments showed a downward trend, and the mRNA expression level of il-8 after incubation for 6 and 12 hours was significantly lower than that at 0 hour; the incubation dose of rtNPY, incubation and their interaction all had a significant effect on the expression of il-8 gene. Figure 9 As shown in D, compared with 0h, the expression level of il-10 mRNA in tilapia hypothalamus fragments was significantly increased after incubation with 10nM and 100nM for 9-12h; and after incubation for 3-12h, the expression level of il-10 mRNA in tilapia hypothalamus fragments incubated with 10nM and 100nM rtNPY was significantly higher than that of 0nM; the incubation dose, incubation time and their interaction of rtNPY all had a significant effect on the expression of il-10 gene. Figure 9 As shown in Figure E, after 12 hours of incubation, the mRNA expression level of tnf-α in the tilapia hypothalamus fragments incubated with 100nM rtNPY was significantly lower than that with 0nM; the incubation dose, incubation time and their interaction of rtNPY all had a significant effect on the expression of tnf-α gene. Figure 9 As shown in Figure F, compared with 0h, the mRNA expression level of tgf-β1 in tilapia hypothalamic fragments was significantly increased after incubation with 0nM, 10nM, 100nM and 1000nM rtNPY for 3h, 9h and 12h; the incubation dose of rtNPY had no significant effect on the mRNA expression of tgf-β1, but the incubation time and its interaction had a significant effect.

[0112] 2. Effects of intraperitoneal injection of sNPY and rtNPY on feeding, inflammation-related genes, and the GH / IGF-1 axis in tilapia

[0113] (1) Incubation of tilapia hypothalamic fragments

[0114] Tilapia fry weighing 30±5 g were randomly divided into three groups, namely the PBS group, sNPY group, and rtNPY group, with 32 fry in each group. The fry were intraperitoneally injected with PBS (injection dose of 100 μL PBS), sNPY (injection dose of 100 μL NPY (1 μg / g body weight)), and rtNPY (injection dose of 100 μL NPY (1 μg / g body weight)), respectively. The PBS group served as the control.

[0115] (2) Fluorescence quantitative PCR detection of gene expression

[0116] The telencephalon and hypothalamus of tilapia in each group were collected 3, 6, 12 and 24 hours after injection, respectively. Total RNA was extracted from the tissue samples and reverse transcribed to obtain cDNA. Using cDNA as a template, qPCR was used to detect changes in neuropeptide receptor genes (y2-2, y4, y7, y8a and y8b) and feeding-related genes (npy, pyy, agrp, cart1, orexin and pomc).

[0117] The telencephalon, hypothalamus, liver and intestine of each group of tilapia were collected, and total RNA of tissue samples was extracted. cDNA was obtained by reverse transcription, and the changes of inflammation-related genes (il-1β, il-6, il-8, il-10, tnf-α, ifn-γ and tgf-β1) were detected by qPCR using cDNA as a template.

[0118] The pituitary and liver of tilapia in each group were collected, total RNA of tissue samples was extracted, and cDNA was obtained by reverse transcription. Using cDNA as a template, qPCR was used to detect the changes in key genes (gh and igf-1) in the GH / IGF-1 axis.

[0119] The qPCR detection method, reaction system, and reaction procedure were the same as those in step 1 of the present example, “rtNPY incubation of tilapia hypothalamus fragments experiment.” The primers used are shown in Tables 2 and 3.

[0120] Table 3 qPCR primer sequences

[0121]

[0122] (3) Result analysis

[0123] like Figure 10 As shown in Figure A, 3 hours after intraperitoneal injection of rtNPY, the expression levels of y2-2, y4, and y8a in the telencephalon were significantly upregulated. 6-12 hours after intraperitoneal injection of rtNPY, the expression level of y7 in the telencephalon was significantly upregulated; however, 24 hours after injection of rtNPY, the expression level of NPY receptors in the telencephalon returned to the level of the PBS group. Figure 10As shown in Figure B, 3 hours after intraperitoneal injection of rtNPY, the expression level of orexin in the telencephalon was significantly upregulated; 6 hours and 24 hours after intraperitoneal injection of rtNPY, the expression level of pyy in the telencephalon was significantly upregulated. Figure 10 As shown in Figure C, 3 hours after intraperitoneal injection of sNPY and rtNPY, the expression level of NPY receptors in the hypothalamus was significantly upregulated; as time passed after NPY injection, the high expression of NPY receptors in the hypothalamus gradually decreased and returned to the level of the PBS group; however, 24 hours after rtNPY injection, the expression levels of y2-2 and y8a in the hypothalamus were significantly higher than those in the PBS group. Figure 10 As shown in D, 3 hours after intraperitoneal injection of sNPY and rtNPY, the expression levels of npy, pyy, cart1 and agrp in the hypothalamus were significantly upregulated; with the passage of time after NPY injection (6 to 12 hours), the high expression of feeding-related genes in the hypothalamus gradually decreased and returned to the level of the PBS group; 24 hours after rtNPY injection, the expression levels of pyy, agrp and orexin in the hypothalamus were significantly higher than those in the PBS group.

[0124] The above results indicate that compared with sNPY, intraperitoneal injection of rtNPY can more effectively regulate the expression of NPY receptors in the hypothalamus and stimulate the appetite of tilapia.

[0125] like Figure 11 As shown in Figure A, 3 hours after intraperitoneal injection of rtNPY, the expression levels of il-1β, il-10 and ifn-γ in the telencephalon increased significantly; as time passed after rtNPY injection, the high expression of inflammation-related genes in the telencephalon gradually decreased and returned to the level of the PBS group; while intraperitoneal injection of sNPY had no significant effect on the expression of inflammation-related genes in the telencephalon. Figure 11 As shown in Figure B, 3 hours after intraperitoneal injection of sNPY and rtNPY, the expression levels of IL-8 and TGF-β1 in the hypothalamus were significantly reduced. 6-12 hours after rtNPY injection, the expression levels of IL-6 and IL-8 in the hypothalamus were significantly increased. However, 24 hours after injection of sNPY and rtNPY, the expression of inflammation-related genes in the hypothalamus returned to the level of the PBS group. Figure 11 As shown in Figure C, 3 hours after intraperitoneal injection of sNPY and rtNPY, the expression levels of inflammation-related genes in the liver were upregulated; 6 hours after rtNPY injection, the expression levels of IL-10, TNF-α and TGF-β1 in the liver were significantly higher than those in the PBS group; but 24 hours after injection of sNPY and rtNPY, the expression levels of inflammation-related genes in the liver returned to the levels of the PBS group. Figure 11As shown in Figure D, 3 hours after intraperitoneal injection of sNPY and rtNPY, the expression level of IL-8 in the foregut was significantly reduced; 12 hours after rtNPY injection, the expression level of IL-6 in the foregut was significantly higher than that in the PBS group; 24 hours after injection, the expression level of IFN-γ in the foregut of the sNPY group was significantly lower than that in the PBS group, while the expression levels of other inflammatory-related genes in the foregut were not significantly different from those in the PBS group. Figure 11 As shown in Figure E, 3 hours after intraperitoneal injection of sNPY and rtNPY, the expression level of il-8 in the midgut was significantly reduced, while the expression levels of il-1β and tgf-β1 were increased; 6 hours after injection of sNPY and rtNPY, the expression levels of il-1β, il-6, il-8, tnf-α and ifn-γ in the midgut were lower than those in the PBS group; 24 hours after injection, the expression levels of il-6 and tnf-α in the midgut of the sNPY and rtNPY groups were significantly lower than those in the PBS group, and the expression level of il-8 in the midgut of the rtNPY group was significantly lower than that in the PBS group. Figure 11 As shown in Figure F, 3 hours after intraperitoneal injection of sNPY and rtNPY, the expression level of IL-8 in the hindgut was significantly decreased; 12 hours after injection of sNPY and rtNPY, the expression levels of IL-1β, IL-6, IL-8, TNF-α and IFN-γ in the hindgut were significantly lower than those in the PBS group; 24 hours after injection of sNPY and rtNPY, the expression levels of IL-1β and IFN-γ in the hindgut were significantly lower than those in the PBS group, while the expression level of IL-10 was significantly higher than that in the PBS group.

[0126] The above results indicate that in order to quickly respond to the stimulation of sNPY and rtNPY, tilapia will activate the body's inflammatory response in a short period of time, but over time, the inflammatory response in the telencephalon, hypothalamus and liver will return to steady state, and intraperitoneal injection of sNPY and rtNPY can alleviate the inflammatory response in the tilapia intestine.

[0127] like Figure 12 As shown in Figure A, intraperitoneal injection of sNPY and rtNPY had no significant effect on the expression level of gh in the pituitary. Figure 12 As shown in Figure 5B, intraperitoneal injection of sNPY and rtNPY had no significant effect on the expression level of IGF-1 in the liver.

[0128] The above results indicate that rtNPY is involved in regulating the feeding and inflammatory response of tilapia. It can bind to the NPY receptor in the hypothalamic fragments of tilapia and stimulate the expression of the feeding-promoting factor npy, while alleviating the systemic inflammatory response.

[0129] Example 3 Effects of cottonseed protein concentrate replacing soybean meal and rtNPY addition on growth performance, antioxidant capacity and environmental stress resistance of tilapia

[0130] 1. Effects of cottonseed protein concentrate replacing soybean meal and rtNPY addition on growth performance and antioxidant capacity of tilapia

[0131] (1) Feed preparation and spraying

[0132] Based on the national standard GB / T 22919.10-2024 for tilapia feed, which stipulates a crude protein content of 28.0% to 36.0% and a crude fat content of ≥5.0%, a soybean meal feed (containing 32.25% crude protein and 6.31% crude fat) was prepared as shown in Table 4. Subsequently, the formula of the soybean meal feed was adjusted to produce a cottonseed protein concentrate feed as shown in Table 4. The specific differences between the two feeds are: compared with the soybean meal feed, the cottonseed protein concentrate feed contains 20.0% wt less soybean meal and 15% wt more cottonseed protein concentrate; in addition, to ensure that the crude protein and crude fat contents of the two feeds are basically the same, the cottonseed protein concentrate feed also contains 5% wt of microcrystalline cellulose.

[0133] Table 4 Composition and nutritional content of tilapia feed (dry matter %)

[0134]

[0135] Soybean meal feed and cottonseed protein concentrate feed were prepared sequentially according to the feed formula shown in Table 4. The rtNPY prepared in Example 1 was sprayed onto the soybean meal feed at a dose of 0.3 μg rtNPY / g feed, resulting in a soybean meal + rtNPY feed. The rtNPY prepared in Example 1 was sprayed onto the cottonseed protein concentrate feed at a dose of 0.3 μg rtNPY / g feed, resulting in a cottonseed protein concentrate + rtNPY feed. Finally, four different feed groups were prepared: soybean meal feed (denoted as SBM), soybean meal + rtNPY feed (denoted as SBM + rtNPY), cottonseed protein concentrate feed (denoted as CPC), and cottonseed protein concentrate + rtNPY feed (denoted as CPC + rtNPY).

[0136] (2) Breeding test

[0137] GIFF tilapia (30 fish / group, average body weight 3.99 ± 0.03 g) were randomly divided into four groups and fed a soybean meal diet, a soybean meal + rtNPY diet, a cottonseed protein concentrate diet, and a cottonseed protein concentrate + rtNPY diet, respectively. These groups were designated as the SBM group, the SBM + rtNPY group, the CPC group, and the CPC + rtNPY group, respectively. Tilapias were fed twice daily at full capacity with ad libitum access for 56 days.

[0138] (3) Growth performance evaluation

[0139] After the breeding experiment of step (2) is finished, the body weight (g) of each group of tilapia is weighed, and the growth performance related index and survival rate of tilapia are counted. The calculation formula is as follows: weight gain rate (%) = (final body weight - initial body weight) / initial body weight × 100%; specific growth rate (%) = (ln final body weight - ln initial body weight) / number of days × 100%; feeding rate (%) = food intake / [(initial body weight + final body weight) / 2] / number of days × 100%; feed coefficient = food intake / (final body weight - initial body weight); fatness (%) = body weight / body length 3 × 100%; organ-to-body ratio (%) = organ weight / final body weight × 100%; liver-to-body ratio (%) = liver weight / final body weight × 100%; survival rate (%) = final number / initial number × 100%.

[0140] Table 5 Statistical results of growth performance related indicators and survival rate of tilapia in each group

[0141]

[0142] As shown in Table 5, the final average weight and weight gain rate of the SBM+rtNPY group were significantly higher than those of the other three groups, indicating that the addition of rtNPY to soybean meal feed promoted the weight gain of tilapia; the specific growth rate of the SBM+rtNPY group was also significantly higher than that of the other three groups, indicating that the addition of rtNPY to soybean meal feed accelerated the growth rate of tilapia; in terms of feeding rate and feed conversion ratio, the feeding rate and feed conversion ratio of the SBM group and the SBM+rtNPY group were lower among the four groups of tilapia, and the feeding rate of the CPC group was the highest, but the feed conversion ratio was also The results were the largest, indicating that replacing 50% soybean meal with cottonseed protein concentrate in the diet reduced feed utilization efficiency in tilapia. The CPC+rtNPY group had higher feed intake and feed conversion rates than the SBM and SBM+rtNPY groups, but lower than the CPC group, indicating that rtNPY supplementation improved feed utilization efficiency in tilapia. There were no significant differences in fatness, organ-to-body ratio, and liver-to-body ratio among the four groups of tilapia, and all had 100% survival rates, indicating that tilapia survived well during the experiment and that different feed compositions had no significant effect on fish survival. Overall, the SBM+rtNPY group performed best in terms of growth performance, while the CPC group performed relatively poorly. However, the addition of rtNPY had a positive effect on tilapia growth.

[0143] (4) Evaluation of antioxidant capacity

[0144] After the breeding experiment in step (2) was completed, the serum of each group of tilapia was collected and the total antioxidant capacity (T-AOC, product number: A015-2-1), superoxide dismutase (SOD, product number: A001-1-2) activity, glutathione peroxidase (GSH-Px, product number: A005-1-2) activity, catalase (CAT, product number: A007-1-1) activity and malondialdehyde (MDA, product number: A003-1-2) content were determined using a commercial kit produced by Nanjing Jiancheng Bioengineering Research Institute.

[0145] The changes of antioxidant related indicators in the serum of tilapia Figure 13 As shown in Figures A to E, compared with the SBM group, the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity and catalase activity of tilapia serum in the SBM+rtNPY group increased to a certain extent, while the malondialdehyde (MDA) content decreased, indicating that the addition of rtNPY to soybean meal feed improved the antioxidant capacity of tilapia; compared with the SBM group, the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity and catalase activity of tilapia serum in the CPC group decreased significantly, while the MDA content increased significantly, indicating that cottonseed feed can improve the antioxidant capacity of tilapia. Cottonseed protein concentrate weakened the antioxidant capacity of tilapia by inhibiting antioxidant enzyme activity and aggravating lipid peroxidation. After replacing 50% soybean meal in the feed with cottonseed protein concentrate, the antioxidant capacity of tilapia was reduced and oxidative stress was promoted. Compared with the CPC group, the total antioxidant capacity, superoxide dismutase activity, and glutathione peroxidase activity of tilapia serum in the CPC+rtNPY group were significantly increased, while the malondialdehyde content was significantly decreased, indicating that adding rtNPY to feed with low soybean meal content can effectively improve the antioxidant capacity of tilapia and reverse the oxidative stress caused by the reduction of soybean meal.

[0146] 2. Effects of ammonia nitrogen and nitrite stress on the survival rate and antioxidant capacity of tilapia

[0147] (1) Stress treatment

[0148] After the aquaculture experiment in step (2) of the previous part was completed, the four groups of tilapia were subjected to salt stress treatment with ammonia nitrogen at a concentration of 32.80 mg / L and nitrite at a concentration of 60 mg / L for a total of 96 hours.

[0149] (2) Survival rate determination

[0150] After the stress was over, the survival rate of tilapia in each group was counted. Figure 14As shown in the results, after 96 h of ammonia stress, the survival rates of tilapia in the SM, SM+rtNPY, CPC, and CPC+rtNPY groups were 56.67%, 76.67%, 36.67%, and 50%, respectively. After 96 h of nitrite stress, the survival rates of tilapia in the SM, SM+rtNPY, CPC, and CPC+rtNPY groups were 53.33%, 60.00%, 43.33%, and 56.67%, respectively. These results indicate that replacing 50% soybean meal with cottonseed protein concentrate in the diet reduces the ability of tilapia to resist ammonia and nitrite stress, while adding rtNPY to the diet improves the tolerance of tilapia to ammonia and nitrite stress, especially reversing the negative effects of soybean meal reduction on tilapia intolerance to ammonia and nitrite stress, thereby improving the survival of tilapia under ammonia and nitrite stress.

[0151] (3) Evaluation of antioxidant capacity

[0152] The serum of the remaining surviving tilapia in each group was collected, and the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, catalase activity and malondialdehyde content were determined according to the method of this example.

[0153] After 96 h of ammonia nitrogen stress, the changes in antioxidant-related indicators in the serum of tilapia in each group are as follows: Figure 15 As shown in Figures A to E, compared with the SBM group, the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, and catalase activity of tilapia serum in the SBM+rtNPY group were increased, while the malondialdehyde content decreased; compared with the SBM group, the total antioxidant capacity, superoxide dismutase activity, and catalase activity of tilapia serum in the CPC group were significantly decreased, and the glutathione peroxidase activity was significantly decreased, while the malondialdehyde content increased; compared with the CPC group, the total antioxidant capacity, superoxide dismutase activity, and glutathione peroxidase activity of tilapia serum in the CPC+rtNPY group were increased, while the malondialdehyde content decreased.

[0154] After 96 hours of nitrite stress, the changes in antioxidant-related indicators in the serum of tilapia in each group are as follows: Figure 15As shown in Figures A to E, there were no significant differences in the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity, catalase activity and malondialdehyde content of tilapia serum between the SBM group and the SBM+rtNPY group; compared with the SBM group, the total antioxidant capacity and catalase activity of tilapia serum in the CPC group were significantly decreased, the superoxide dismutase activity and GSH peroxidase activity were somewhat decreased, and the malondialdehyde content was increased; compared with the CPC group, the total antioxidant capacity, superoxide dismutase activity, glutathione peroxidase activity and catalase activity of tilapia serum in the CPC+rtNPY group were significantly increased, while the malondialdehyde content was decreased.

[0155] The above results indicate that the addition of rtNPY to the feed can improve the ability of tilapia to resist ammonia nitrogen stress and nitrite stress, especially for the feed with cottonseed protein concentrate replacing 50% soybean meal. The addition of rtNPY can reverse the negative effects of tilapia intolerance to ammonia nitrogen stress and nitrite stress caused by the reduction of soybean meal.

[0156] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. Application of neuropeptide Y in the preparation of products for promoting the growth of tilapia and improving the tolerance of tilapia to environmental stress.

2. Application of neuropeptide Y in the preparation of products for promoting tilapia growth under environmental stress.

3. Application of neuropeptide Y in the preparation of a product for improving the survival rate of tilapia under environmental stress.

4. Application of neuropeptide Y in the preparation of a product for improving the antioxidant capacity of tilapia under environmental stress.

5. Application of neuropeptide Y in the preparation of products for reducing soybean meal content in tilapia feed and promoting tilapia growth under environmental stress.

6. Use of neuropeptide Y in the preparation of a product for reducing the soybean meal content in tilapia feed and improving the survival rate of tilapia under environmental stress.

7. Use of neuropeptide Y in the preparation of a product for reducing the soybean meal content in tilapia feed and improving the antioxidant capacity of tilapia under environmental stress.

8. The use according to any one of claims 1 to 7, characterized in that: The amino acid sequence of neuropeptide Y includes the sequence shown in SEQ ID NO.

3.

9. The use according to any one of claims 1 to 7, characterized in that: The neuropeptide Y is obtained by in vitro recombinant expression or chemical synthesis.

10. The use according to claim 9, characterized in that The microorganism used for the in vitro recombinant expression includes Bacillus subtilis.

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