Composition for improving feed utilization rate by targeting leptin gene of micropterus salmoides as well as preparation method and application of composition

Abstract

The invention belongs to the technical field of aquaculture, and discloses a composition targeting a leptin gene of micropterus salmoides and capable of improving the feed utilization rate as well as a preparation method and application of the composition. The composition is prepared from the following components in parts by weight: 1 to 30 parts of wax gourd polysaccharide, 5 to 35 parts of gamma-aminobutyric acid and 1 to 20 parts of siRNA. Through complementary preparation of wax gourd polysaccharide, gamma-aminobutyric acid and siRNA, triple effects of gene silencing, appetite promotion and metabolism enhancement are realized. According to the invention, a wax gourd polysaccharide water extraction and alcohol precipitation process, a siRNA chemical synthesis process and a double-phase embedding-gradient composite process are adopted, so that the stability and bioavailability of active ingredients are improved. By down-regulating leptin expression, appetite and feed utilization rate of the micropterus salmoides fed with high-sugar feed are enhanced, blood sugar and blood fat levels are reduced, growth of fish bodies is promoted, and the micropterus salmoides culture medium can be applied to the field of micropterus salmoides culture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of aquaculture, and particularly relates to a composition for improving feed utilization rate by targeting leptin gene of Micropterus salmoides, a preparation method thereof and application thereof in Micropterus salmoides culture. BACKGROUND

[0002] Micropterus salmoides is an important freshwater cultured precious fish in China, which has the advantages of fast growth, delicious meat and strong disease resistance, and the culture scale is expanding year by year. However, Micropterus salmoides is a typical carnivorous fish, and the utilization rate of carbohydrates is low. Factors such as feeding high-sugar feed can easily lead to problems such as reduced appetite, metabolic disorder, decreased feeding rate, low feed utilization rate, liver fat deposition and slow growth, which seriously restricts the improvement of culture benefit.

[0003] Leptin is a key hormone for negative regulation of feeding, and its expression level is positively correlated with fat accumulation. Silencing leptin by RNAi technology can relieve the inhibition of hypothalamic feeding center and activate lipolysis pathway, but naked siRNA is easily degraded in aquatic animals and has low cell uptake rate. Winter melon polysaccharide (WBP) has been proved to have anti-diabetic function and can significantly improve the digestive enzyme activity of aquatic animals; gamma-aminobutyric acid (GABA) is a naturally occurring inhibitory neurotransmitter in aquatic animals, which can quickly stimulate taste receptors and improve feeding. There is no report on the trinity and synergistic targeting of leptin gene by "plant polysaccharide + functional amino acid + gene silencing element" for improving appetite and metabolism of Micropterus salmoides. SUMMARY

[0004] In order to solve the above problems existing in the prior art, the primary purpose of the present application is to provide a composition for improving feed utilization rate by targeting leptin gene of Micropterus salmoides.

[0005] Another purpose of the present application is to provide a preparation method of the composition for improving feed utilization rate, which adopts a winter melon polysaccharide high-purity extraction process suitable for industrial amplification, a synthesis process of high-efficiency and low-off-target siRNA double-strand for leptin mRNA of Micropterus salmoides, and a processing process of WBP-GABA-siRNA composition.

[0006] Still another purpose of the present application is to provide the application of the composition for improving feed utilization rate.

[0007] The purpose of the present application is realized by the following technical solutions: A composition for improving feed utilization rate by targeting leptin gene of Micropterus salmoides is composed of winter melon polysaccharide 1-30 parts by weight, gamma-aminobutyric acid 5-35 parts by weight and siRNA 1-20 parts by weight.

[0008] Preferably, the composition consists of 5-25 parts of wax gourd polysaccharide, 10-30 parts of gamma-aminobutyric acid and 5-15 parts of siRNA.

[0009] More preferably, the composition consists of 10-20 parts of wax gourd polysaccharide, 15-20 parts of gamma-aminobutyric acid and 10-12 parts of siRNA.

[0010] The preparation method of the composition for improving feed utilization rate by targeting the leptin gene of Micropterus salmoides, comprising the following steps: S1. Mix fresh wax gourd peel and wax gourd meat, dry at 60-100℃, crush and sieve, add deionized water, soak at 90-100℃, centrifuge at 2500-4000 rpm to collect the supernatant, add anhydrous ethanol to the supernatant and stand, centrifuge at 2000-3000 rpm to collect the precipitate, dissolve the precipitate in a mixed solution of chloroform and n-butanol, centrifuge at 3000-4000 rpm at 35-50℃, and vacuum freeze-dry to obtain wax gourd polysaccharide, abbreviated as WBP; S2. Design siRNA sense strand 5'-GGUUUGUACUAUUACACUATT-3' and antisense strand 5'-UAGUGUAAUAGUACAAACCTT-3' with the leptin gene (GenBank accession number: MN887534.1) coding region nucleotide as the target; react the phosphoramidite monomer with a concentration of 0.1-0.9 mol / L and the activated agent tetrazole with a concentration of 0.2-0.5 mol / L at 25-30℃ to synthesize siRNA double strands, then purify by high performance liquid chromatography, and vacuum freeze-dry to obtain leptin-siRNA; S3. Add GABA solution to WBP solution, stand at 25-35℃ to form WBP-GABA pre-complex; then add leptin-siRNA solution, vortex and incubate at 25℃ to obtain WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano; S4. Mix WGR-Nano and hydroxypropyl methylcellulose solution by stirring, vacuum dry at 50-80℃ to obtain the composition for improving feed utilization rate by targeting the leptin gene of Micropterus salmoides.

[0011] Preferably, in step S1, the mass ratio of winter melon peel to winter melon flesh is (3~15):(7~20), the total mass of winter melon peel and winter melon flesh to the volume ratio of deionized water is (1~5) g:(20~30) / mL; the volume ratio of chloroform to n-butanol in the mixed solution is (4~10):(1~5), the sieve aperture is 40~80 mesh, the drying time is 1~2 h, the soaking time is 2~3 h, the centrifugation time at 2500~4000 rpm is 15~30 min, the volume ratio of supernatant to anhydrous ethanol is 1:(3~5), the standing time is 12~24 h, the centrifugation time at 2000~3000 rpm is 10~30 min, and the centrifugation time at 3000~4000 rpm is 30~60 min.

[0012] Preferably, in step S2, the high-performance liquid chromatography uses a C18 reversed-phase column, and the mobile phase is a 0.1 wt% trifluoroacetic acid-acetonitrile aqueous solution, with gradient elution using a trifluoroacetic acid-acetonitrile aqueous solution containing 5-40% acetonitrile by volume; the reaction time is 1.5-2 h.

[0013] Preferably, the volume ratio of GABA solution to WBP solution in step S3 is (1~30):(5~35), the molar ratio of N atoms of WBP amino groups in WBP solution to P atoms of siRNA phosphate groups in leptin-siRNA solution is (8~16):(1~20); the concentration of WBP solution is 5~10 mg / mL, pH= 6.8~7.5; the concentration of GABA solution is 20~40 mg / mL, the concentration of leptin-siRNA solution is 50~70 μmol / L; the vortexing time is 10~30 s, the incubation time is 20~30 min, and the standing time is 15~30 min.

[0014] Preferably, in step S4, the mass ratio of hydroxypropyl methylcellulose to water in the hydroxypropyl methylcellulose solution is 1:10, and the mass ratio of WGR-Nano to the hydroxypropyl methylcellulose solution is (3~15):(5~20); the stirring speed is 1000~3000 rpm, the stirring time is 15~30 min, and the vacuum drying time is 5~10 h.

[0015] A largemouth bass feed includes the aforementioned composition; the composition is added to the largemouth bass feed at an amount of 0.5~1.2 wt%.

[0016] The application of the composition targeting the leptin gene of largemouth bass to improve feed utilization in aquaculture.

[0017] Compared with the prior art, the present application has the following beneficial effects: 1. The present application first integrates the "RNAi gene silencing-plant polysaccharide-functional amino acid" in one, breaks through the traditional single appetite stimulation or single lipid-lowering idea, WBP as a natural cationic polysaccharide can encapsulate / protect siRNA, and can form a mucus protective layer in the gastrointestinal tract to improve the stability and absorption of GABA and siRNA; GABA rapidly stimulates taste buds to improve the food intake rate, WBP continuously improves intestinal health, and leptin silencing long-acting removes the "satiety signal", the three complement each other to realize "appetite stimulation-lipid-lowering" simultaneously. 2. The composition of the present application can greatly reduce the feed cost and improve the economic benefit of aquaculture as a high-efficiency environmentally friendly fish feed additive. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 To study the effect of siRNA technology interfering leptin on the expression of leptin gene in the liver of largemouth bass.

[0019] Figure 2 To study the effect of siRNA technology interfering leptin on the food intake of largemouth bass.

[0020] Figure 3 To study the effect of the control feed and high-sugar feed of Example 7 on the expression of leptin gene in the brain and liver of largemouth bass.

[0021] Figure 4 To study the effect of the control feed and high-sugar feed of Example 7 on the food intake of largemouth bass. DETAILED DESCRIPTION

[0022] The content of the present application will be further illustrated below in combination with specific examples, but should not be understood as a limitation of the present application. If not specifically indicated, the technical means used in the examples are conventional means familiar to those skilled in the art. Unless specifically indicated, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0023] The gamma-aminobutyric acid and hydroxypropyl methyl cellulose used in the examples of the present application are from Suzhou Biotechnology Co., Ltd.

[0024] Example 1 1. Mix winter melon peel and winter melon flesh in a mass ratio of 3:7, dry at 60℃ for 1 hour, and then pulverize through a 40-mesh sieve to obtain winter melon powder. Then, add the above winter melon powder in a material-to-liquid ratio of 1g:20mL to deionized water, soak at 90℃ for 2 hours, and centrifuge at 2500 rpm for 15 minutes to collect the supernatant. Add 3 times the volume of anhydrous ethanol to the supernatant, let stand at 5℃ for 12 hours, and centrifuge at 2000 rpm for 10 minutes to collect the precipitate. Dissolve the precipitate in a mixed solution of chloroform and n-butanol (chloroform to n-butanol volume ratio of 4:1), centrifuge at 3000 rpm for 30 minutes at 35℃, repeat 3 times, and vacuum freeze at -5℃ to obtain winter melon polysaccharide, abbreviated as WBP.

[0025] 2. Targeting the coding region of the largemouth bass leptin gene (GenBank accession number: MN887534.1), siRNA was designed with a sense strand of 5'-GGUUUGUACUAUUACACUATT-3' and an antisense strand of 5'-UAGUGUAAUAGUACAAACCTT-3'. Both strands were modified with two thymine (TT) nucleotides at their 3' ends to enhance stability. Subsequently, a solid-phase phosphorus amide reaction was used to synthesize double-stranded siRNA by reacting 0.1 mol / L phosphorus amide monomer and 0.2 mol / L tetrazolium activator at 25 °C for 1.5 h. The siRNA was then purified by high-performance liquid chromatography (HPLC) using a C18 reversed-phase column (250 mm × 4.6 mm) and a mobile phase of 0.1 wt% trifluoroacetic acid-acetonitrile aqueous solution at a flow rate of 1.0 h. The largemouth bass leptin-siRNA was prepared by gradient elution with trifluoroacetic acid-acetonitrile aqueous solution containing 5% acetonitrile at a flow rate of mL / min and vacuum freezing at -5℃.

[0026] 3. A γ-aminobutyric acid (GABA) solution (20 mg / mL) at a volume ratio of 1:5 was slowly added dropwise to a WBP solution (5 mg / mL, pH = 6.8), and the mixture was allowed to stand at 25°C for 15 min to form a WBP-GABA pre-complex. Subsequently, a leptin-siRNA solution at a concentration of 50 μmol / L was added dropwise (the molar ratio of N atoms in the amino group of WBP in the WBP solution to P atoms in the phosphate group of siRNA in the leptin-siRNA solution was 8:1), the mixture was vortexed for 10 s, and incubated at 25°C for 20 min to obtain the WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano.

[0027] 4. Mix WGR-Nano (mass ratio 3:5) with hydroxypropyl methylcellulose solution (mass ratio 1:10 of hydroxypropyl methylcellulose and water), stir at 1000 rpm for 15 min, and dry under vacuum at 50°C for 5 h to obtain composition 1.

[0028] Example 2 1. Fresh winter melon peel and flesh were mixed in a mass ratio of 5:10, dried at 80℃ for 2 h, and then pulverized through an 80-mesh sieve. Subsequently, deionized water was added at a material-to-liquid ratio of 1:25 (g / mL), and the mixture was soaked at 100℃ for 2.5 h. The supernatant was collected by centrifugation at 3000 rpm for 20 min. 3.5 volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 5℃ for 24 h. The precipitate was collected by centrifugation at 3000 rpm for 20 min. The precipitate was dissolved in a mixed solution of chloroform and n-butanol (chloroform to n-butanol volume ratio of 5:2), centrifuged at 3500 rpm for 40 min at 40℃, and repeated three times. The mixture was then vacuum-frozen at -5℃ to obtain WBP.

[0029] 2. Targeting the coding region of the largemouth bass leptin gene (GenBank accession number: MN887534.1), siRNA was designed with a sense strand of 5'-GGUUUGUACUAUUACACUATT-3' and an antisense strand of 5'-UAGUGUAAUAGUACAAACCTT-3'. Both strands were modified with two thymine (TT) nucleotides at their 3' ends to enhance stability. Subsequently, a solid-phase phosphorus amide reaction was used to synthesize double-stranded siRNA by reacting 0.1 mol / L phosphorus amide monomer and 0.2 mol / L tetrazolium activator at 30℃ for 2 h. The siRNA was then purified by high-performance liquid chromatography (HPLC) using a C18 reversed-phase column (250 mm × 4.6 mm) and a mobile phase of 0.1 wt% trifluoroacetic acid-acetonitrile aqueous solution at a flow rate of 1.0 h. The leptin-siRNA was prepared by gradient elution with trifluoroacetic acid-acetonitrile aqueous solution containing 10% acetonitrile at a flow rate of mL / min and vacuum freezing at -5℃.

[0030] 3. A GABA solution (20 mg / mL) with a volume ratio of 3:7 was slowly added dropwise to a WBP solution (5 mg / mL, pH = 6.8), and the mixture was allowed to stand at 30°C for 20 min to form a WBP-GABA pre-complex. Subsequently, a leptin-siRNA solution with a concentration of 50 μmol / L was added dropwise (the molar ratio of N atoms of the WBP amino group in the WBP solution to P atoms of the siRNA phosphate group in the leptin-siRNA solution was 10:2), the mixture was vortexed for 30 s, and incubated at 25°C for 25 min to obtain the WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano.

[0031] 4. Mix WGR-Nano and hydroxypropyl methylcellulose solution (hydroxypropyl methylcellulose and water are prepared at a mass ratio of 1:10) in a mass ratio of 5:6, stir at 2000 rpm for 25 min, and dry under vacuum at 50℃ for 5 h to obtain composition 2.

[0032] Example 3 1. Fresh winter melon peel and flesh were mixed in a mass ratio of 8:11, dried at 80℃ for 2 h, and then pulverized through an 80-mesh sieve. Subsequently, deionized water was added at a material-to-liquid ratio of 3:22 (g / mL), and the mixture was soaked at 100℃ for 3 h. The supernatant was collected by centrifugation at 3000 rpm for 30 min. Five volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 5℃ for 24 h. The precipitate was collected by centrifugation at 2500 rpm for 30 min. The precipitate was dissolved in a mixed solution of chloroform and n-butanol (chloroform to n-butanol volume ratio = 6:3), centrifuged at 50℃ at 4000 rpm for 45 min, and repeated three times. The mixture was then vacuum-frozen at -5℃ to obtain WBP.

[0033] 2. Targeting the coding region of the largemouth bass leptin gene (GenBank accession number: MN887534.1), siRNA was designed with a sense strand of 5'-GGUUUGUACUAUUACACUATT-3' and an antisense strand of 5'-UAGUGUAAUAGUACAAACCTT-3'. Both strands were modified with two thymine (TT) nucleotides at their 3' ends to enhance stability. Subsequently, a solid-phase phosphorus amide reaction was used to synthesize double-stranded siRNA by reacting 0.1 mol / L phosphorus amide monomer and 0.2 mol / L tetrazolium activator at 30 °C for 1.5 h. The siRNA was then purified by high-performance liquid chromatography (HPLC) using a C18 reversed-phase column (250 mm × 4.6 mm) and a mobile phase of 0.1 wt% trifluoroacetic acid-acetonitrile aqueous solution at a flow rate of 1.0 h. The leptin-siRNA was prepared by gradient elution with trifluoroacetic acid-acetonitrile aqueous solution containing 15% acetonitrile at a flow rate of mL / min, followed by vacuum freezing at -5°C.

[0034] 3. A GABA solution (20 mg / mL) with a volume ratio of 24:32 was slowly added dropwise to a WBP solution (5 mg / mL, pH = 6.8), and the mixture was allowed to stand at 35°C for 30 min to form a WBP-GABA pre-complex. Subsequently, a leptin-siRNA solution with a concentration of 50 μmol / L was added dropwise (the molar ratio of N atoms of the WBP amino group in the WBP solution to P atoms of the siRNA phosphate group in the leptin-siRNA solution was 9:16), the mixture was vortexed for 30 s, and incubated at 25°C for 30 min to obtain the WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano.

[0035] 4. Mix WGR-Nano and hydroxypropyl methylcellulose solution (hydroxypropyl methylcellulose and water in a mass ratio of 1:10) at a mass ratio of 5:8, stir at 2000 rpm for 30 min, and dry under vacuum at 50°C for 5 h to obtain composition 3.

[0036] Example 4 1. Fresh winter melon peel and flesh were mixed in a mass ratio of 7:10, dried at 100℃ for 2 h, and then pulverized through an 80-mesh sieve. Subsequently, deionized water was added at a material-to-liquid ratio of 3:25 g / mL, and the mixture was soaked at 100℃ for 3 h. The supernatant was collected by centrifugation at 4000 rpm for 30 min. Five volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 5℃ for 24 h. The precipitate was collected by centrifugation at 3000 rpm for 30 min. The precipitate was dissolved in a mixed solution of chloroform and n-butanol (chloroform to n-butanol volume ratio = 7:4), centrifuged at 50℃ for 4000 rpm for 50 min, and repeated three times. The mixture was then vacuum-frozen at -5℃ to obtain WBP.

[0037] 2. Targeting the coding region of the largemouth bass leptin gene (GenBank accession number: MN887534.1), siRNA was designed with a sense strand of 5'-GGUUUGUACUAUUACACUATT-3' and an antisense strand of 5'-UAGUGUAAUAGUACAAACCTT-3'. Both strands were modified with two thymine (TT) nucleotides at their 3' ends to enhance stability. Subsequently, a solid-phase phosphorus amide reaction was used to synthesize the siRNA duplex at 30℃ for 2 h, involving 0.1 mol / L phosphorus amide monomer and 0.2 mol / L tetrazolium as the activator. The siRNA was then purified by high-performance liquid chromatography (HPLC) using a C18 reversed-phase column (250 mm × 4.6 mm) and a mobile phase of 0.1% trifluoroacetic acid-acetonitrile aqueous solution at a flow rate of 1.0 h. The leptin-siRNA was prepared by gradient elution with trifluoroacetic acid-acetonitrile aqueous solution containing 20% ​​acetonitrile at a flow rate of mL / min, followed by vacuum freezing at -5°C.

[0038] 3. A GABA solution (25 mg / mL, volume ratio 25:33) was slowly added dropwise to a WBP solution (8 mg / mL, pH = 6.8), and the mixture was allowed to stand at 35°C for 30 min to form a WBP-GABA pre-complex. Subsequently, a 50 μmol / L leptin-siRNA solution was added dropwise (the molar ratio of N atoms of the WBP amino group in the WBP solution to P atoms of the siRNA phosphate group in the leptin-siRNA solution was 10:19), the mixture was vortexed for 30 s, and incubated at 25°C for 30 min to obtain the WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano.

[0039] 4. Mix WGR-Nano and hydroxypropyl methylcellulose solution (hydroxypropyl methylcellulose and water in a mass ratio of 1:10) at a mass ratio of 6:13, stir at 3000 rpm for 25 min, and dry under vacuum at 50°C for 5 h to obtain composition 4.

[0040] Example 5 1. Fresh winter melon peel and flesh were mixed in a mass ratio of 12:17, dried at 100℃ for 2 h, and then pulverized through an 80-mesh sieve. Subsequently, deionized water was added at a material-to-liquid ratio of 5:23 (g / mL), and the mixture was soaked at 100℃ for 3 h. The supernatant was collected by centrifugation at 4000 rpm for 25 min. Five volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 5℃ for 24 h. The precipitate was collected by centrifugation at 3000 rpm for 20 min. The precipitate was dissolved in a mixed solution of chloroform and n-butanol (chloroform to n-butanol volume ratio of 7:4), centrifuged at 4000 rpm for 60 min at 45℃, and repeated three times. The mixture was then vacuum-frozen at -5℃ to obtain WBP.

[0041] 2. Targeting the coding region of the largemouth bass leptin gene (GenBank accession number: MN887534.1), siRNA was designed with a sense strand of 5'-GGUUUGUACUAUUACACUATT-3' and an antisense strand of 5'-UAGUGUAAUAGUACAAACCTT-3'. Both strands were modified with two thymine (TT) nucleotides at their 3' ends to enhance stability. Subsequently, a solid-phase phosphorus amide reaction was used to synthesize double-stranded siRNA by reacting 0.1 mol / L phosphorus amide monomer and 0.2 mol / L tetrazolium activator at 30℃ for 2 h. The siRNA was then purified by high-performance liquid chromatography (HPLC) using a C18 reversed-phase column (250 mm × 4.6 mm) and a mobile phase of 0.1 wt% trifluoroacetic acid-acetonitrile aqueous solution at a flow rate of 1.0 h. The leptin-siRNA was prepared by gradient elution with a trifluoroacetic acid-acetonitrile aqueous solution containing 30% acetonitrile at a flow rate of mL / min and vacuum freezing at -5℃.

[0042] 3. A GABA solution (35 mg / mL) with a volume ratio of 7:12 was slowly added dropwise to a WBP solution (15 mg / mL, pH = 6.8), and the mixture was allowed to stand at 35°C for 30 min to form a WBP-GABA pre-complex. Subsequently, a 50 μmol / L leptin-siRNA solution was added dropwise (the molar ratio of N atoms of the WBP amino group in the WBP solution to P atoms of the siRNA phosphate group in the leptin-siRNA solution was 14:5), the mixture was vortexed for 30 s, and incubated at 25°C for 30 min to obtain the WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano.

[0043] 4. Mix WGR-Nano and hydroxypropyl methylcellulose solution (hydroxypropyl methylcellulose and water in a mass ratio of 13:18) at a mass ratio of 1:10, stir at 3000 rpm for 30 min, and dry under vacuum at 50°C for 5 h to obtain composition 5.

[0044] Example 6 1. Fresh winter melon peel and flesh were mixed in a mass ratio of 15:20, dried at 100℃ for 2 h, and then pulverized through an 80-mesh sieve. Subsequently, deionized water was added at a material-to-liquid ratio of 5:30 (g / mL), and the mixture was soaked at 100℃ for 3 h. The supernatant was then collected by centrifugation at 4000 rpm for 30 min. Five volumes of anhydrous ethanol were added to the supernatant, and the mixture was allowed to stand at 5℃ for 24 h. The precipitate was collected by centrifugation at 3000 rpm for 30 min. The precipitate was dissolved in a mixed solution of chloroform and n-butanol (chloroform to n-butanol volume ratio = 10:5), centrifuged at 50℃ for 4000 rpm for 60 min, and repeated three times. The mixture was then vacuum-frozen at -5℃ to obtain WBP.

[0045] 2. Targeting the coding region of the largemouth bass leptin gene (GenBank accession number: MN887534.1), siRNA was designed with a sense strand of 5'-GGUUUGUACUAUUACACUATT-3' and an antisense strand of 5'-UAGUGUAAUAGUACAAACCTT-3'. Both strands were modified with two thymine (TT) nucleotides at their 3' ends to enhance stability. Subsequently, a solid-phase phosphorus amide reaction was used to synthesize the siRNA duplex at 30℃ for 2 h, reacting 0.1 mol / L phosphorus amide monomer and 0.2 mol / L tetrazolium as the activator. The siRNA was then purified by high-performance liquid chromatography (HPLC) using a C18 reversed-phase column (250 mm × 4.6 mm) and a mobile phase of 0.1% trifluoroacetic acid-acetonitrile aqueous solution at a flow rate of 1.0 h. The leptin-siRNA was prepared by gradient elution with a trifluoroacetic acid-acetonitrile aqueous solution containing 40% acetonitrile at a flow rate of mL / min, followed by vacuum freeze-drying at -5°C.

[0046] 3. A GABA solution (concentration 40 mg / mL) with a volume ratio of 30:25 was slowly added dropwise to a WBP solution (concentration 20 mg / mL, pH = 6.8), and the mixture was allowed to stand at 35°C for 30 min to form a WBP-GABA pre-complex. Subsequently, a leptin-siRNA solution with a concentration of 50 μmol / L was added dropwise (the molar ratio of N atoms of the WBP amino group in the WBP solution to P atoms of the siRNA phosphate group in the leptin-siRNA solution = 16:20), the mixture was vortexed for 30 s, and incubated at 25°C for 30 min to obtain the WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano.

[0047] 4. Mix WGR-Nano and hydroxypropyl methylcellulose solution (hydroxypropyl methylcellulose and water in a mass ratio of 15:20) at 3000 rpm for 30 min, and dry under vacuum at 50°C for 5 h to obtain composition 6.

[0048] Example 7 Leptin-siRNA injection experiment: 160 healthy juvenile largemouth bass (weight 30±2g) were randomly divided into 4 groups (control group, negative control group, siRNA experiment group 1 (10μmol / L siRNA) and siRNA experiment group 2 (20μmol / L siRNA), with 4 replicates per group and 10 fish per group. They were temporarily housed in a 100L recirculating aquaculture tank at a water temperature of 26±1℃ and dissolved oxygen ≥5mg / L for 3 days to adapt to the environment. Subsequently, the groups were injected intraperitoneally, namely (1) siRNA experiment group 1: the leptin-siRNA prepared in Example 1 was diluted to 10μmol / L with sterile physiological saline; (2) siRNA experiment group 2: the leptin-siRNA prepared in Example 1 was diluted to 20μmol / L with sterile physiological saline; (3) negative control group: negative siRNA (sequence 5'-UUCUCCGAAC) with no homology to the leptin gene was selected. (4) Control group: only sterile saline was used. The injection volume of each fish was 100 μL / 10g body weight, that is, each fish in siRNA experiment group 1 and siRNA experiment group 2 was injected with leptin-siRNA, the negative control group was injected with an equal amount of negative siRNA, and the control group was injected with an equal amount of sterile saline. Subsequently, at 24h, 48h and 72h after injection, the feed intake of each group of fish was counted, and 4 fish in each group were randomly selected for rapid dissection to obtain liver tissue, which was placed in RNA preservation solution and frozen at -80℃.

[0049] RNA was extracted from liver tissue according to the Takara® Plus RNA Purification Kit instructions. cDNA was reverse transcribed into cDNA using the Prime Script® RTreageat Kit as a template, and EF1α was used as an internal control gene for quantitative real-time PCR of the leptin gene. The 10 μL reaction mixture contained 1 μL cDNA dilution buffer, 5 μL SYBR® Premix Ex Taq™ II, 0.3 μL of upstream primer, and 3.4 μL of downstream primer (see Table 1) in double-distilled water. The Ct values ​​of the target gene were normalized using EF1α, and 2... -△△Ct The method is used to calculate the relative expression level of genes.

[0050] Figure 1 To investigate the effect of siRNA technology on leptin gene expression in the liver of largemouth bass. Figure 2 The effect of siRNA technology on leptin-induced feeding behavior in largemouth bass. Figure 1 and2 The results showed that leptin expression was significantly decreased in group 2 of the siRNA experiment, but the food intake of largemouth bass was significantly increased. These results indicate that the leptin gene plays a key role in regulating the feeding function of largemouth bass.

[0051] Table 1. Primer sequences required for the experiment

[0052] Note: EF1α is an extension factor 1α.

[0053] (1) High sugar nutrition experiment: 160 healthy juvenile fish (average weight 57.69±0.2g) were randomly selected for a 12-week breeding experiment. The experiment was divided into two groups, with each group repeated 4 times. During the period, the control group was fed with feed (10% sugar level) and the high sugar group was fed with feed (20% sugar level) twice a day (08:00 and 17:00). The control group's feed composition was 40wt% fishmeal, 26wt% soybean meal, 5wt% fish oil, 5wt% soybean oil, 10wt% corn starch, 10wt% wheat bran, 2wt% calcium dihydrogen phosphate, and 2wt% premix. The high-sugar group's feed composition was 40wt% fishmeal, 26wt% soybean meal, 5wt% fish oil, 5wt% soybean oil, 20wt% corn starch, 2wt% calcium dihydrogen phosphate, and 2wt% premix (the premix provided 314.0g CaCO3, 469.3g KH2PO4, 147.4g MgSO4·7H2O, 49.8g NaCl, 10.9g FeSO4·7H2O, 0.62g CuSO4·5H2O, 4.67g ZnSO4·7H2O, 3.12g MnSO4·7H2O, and KI per kilogram of feed). 0.16g, CoCl2·6H2O 0.08g, (NH4)6Mo7O 24 • 4H₂O 0.06g, NaSeO₃ 0.02g, VA 500000IU, VC 10000mg, VD₃ 50000IU, VE 2500mg, VK₃ 1000mg, VB₁ 5000mg, VB₂ 5000mg, VB₆ 5000mg, VB₁₂ 5mg, inositol 25000mg, pantothenic acid 10000mg, choline 100000mg, niacin 25000mg, folic acid 1000mg, biotin 250mg). All fish were starved for 24 hours after the rearing period. Six fish from each treatment were randomly selected and rapidly anesthetized with 100mg / L MS-222. Brain and liver tissues were rapidly frozen in liquid nitrogen and stored at -80℃. Feed intake (g feed / number of fish / days) = total intake / (total number of fish × number of rearing days).

[0054] (2) qRT-PCR: RNA was extracted from liver tissue according to the instructions of the Takara® Plus RNA Purification Kit. cDNA was reverse transcribed into the leptin gene as a template using the Prime Script® RTreageat Kit instructions, and EF1α was used as an internal control gene for quantitative real-time PCR. The 10 μL reaction mixture contained 1 μL of cDNA dilution buffer, 5 μL of SYBR® Premix Ex Taq™ II, 0.3 μL of upstream primer, and 3.4 μL of double-distilled water for downstream primers (see Table 1). The Ct value of the target gene was normalized using EF1α, and 2... -△△Ct The method is used to calculate the relative expression level of genes. Figure 3 The effects of the control group's diet and the high-sugar group's diet in Example 7 on the expression of leptin genes in the brain and liver of largemouth bass. Figure 4 This describes the effect of the control group's diet and the high-sugar group's diet in Example 7 on the feed intake of largemouth bass. Each data point represents four replicates, and different lowercase superscripts indicate significant differences between experimental groups. Figure 3 and 4 The study showed that after 12 weeks of rearing, the expression levels of leptin in the brain and liver of largemouth bass fed a high-sugar diet (HC) were significantly increased, but their food intake was significantly reduced. This indicates that leptin is involved in the regulation of blood glucose in largemouth bass, and that a high-sugar diet promotes its expression and reduces the food intake of largemouth bass.

[0055] Application Example 1 (1) Culture experiment of juvenile largemouth bass (approximately 15 g): 720 uniformly sized juvenile fish (initial weight: 15.29 ± 0.03 g) were randomly placed into 24 net cages (size: 2.0 m × 1.0 m × 1.0 m), with 30 fish in each cage. The net cages were divided into 6 groups, with each group repeated 4 times. The fish were cultured for 12 weeks according to the feed formula shown in Table 2. During this period, the feeding and growth of the juvenile fish were observed. The basic feed consisted of 40 wt% fish meal, 26 wt% soybean meal, 5 wt% fish oil, 5 wt% soybean oil, 20 wt% corn starch, 2 wt% calcium dihydrogen phosphate, and 2 wt% premix. The feed composition of each group is shown below: ①Basic Group: fed only basic feed; ②Basic + WBP group: Feed the basic diet with 1wt% WBP added; ③Basic + GABA group: Feed the basic diet with 1wt% GABA added; ④ Basic + WBP-GABA group: Feed the basic diet with 1wt% WBP-GABA precomplex added; ⑤ Basic + Composition 1 Group: Feed the basic feed with 1 wt% of Composition 1 from Example 1; ⑥ Basic + Composition 2 Group: Feed the basic feed with 1 wt% of Composition 2 of Example 2.

[0056] WBP is the product prepared in step 1 of Example 1. The WBP-GABA precomplex is the product prepared in step 3 of Example 1.

[0057] Table 2 Feed formulations for different treatment groups

[0058] Premix: Each kilogram of premix contains the following minerals (g / kg) and vitamins (IU or mg / kg): CuSO4·5H2O 2.0g; FeSO4·7H2O 25g; ZnSO4·7H2O 22g; MnSO4·4H2O 7g; Na2SeO3 0.04g; KI 0.026g; CoCl2·6H2O 0.1g; VA 900,000 IU; VD 200,000 IU; VE 4500mg; VK 3220mg; VB1 320mg; VB2 1090mg; niacin 2800mg; VB5 2000 mg; VB6 500 mg; VB 12 1.6 mg; Vitamin C 5000 mg; Pantothenic acid 1000 mg; Folic acid 165 mg; Choline 60000 mg; Inositol 15000 mg.

[0059] (2) After the rearing period, the following analyses were performed: weight gain rate (%), specific growth rate, serum glucose, insulin and triglyceride levels (mmol / L), feed intake, and feed utilization rate. The method for analyzing leptin expression in brain tissue was the same as in Example 7, and the methods for analyzing serum glucose, insulin, and triglyceride levels were the same as those used in the kits produced by Nanjing Jiancheng Institute. The calculation methods for weight gain rate, specific growth rate, and feed utilization rate are as follows: Weight gain rate = (final weight - initial weight) / initial weight × 100%; Specific growth rate = (LnW) / ... t -LnW0) / t×100%, feed utilization rate = total feed consumption / (final weight - initial weight); where W0 and W t The values ​​are the initial body mass and the body mass (g) on ​​day t of the experiment, respectively, where t is the experiment time (d).

[0060] Table 3 shows the effects of different treatment groups' diets on the growth and physiological and biochemical indicators of largemouth bass. As shown in Table 3, the basal + composition 2 group significantly increased the weight gain rate, specific growth rate, feed intake, feed utilization rate, and plasma insulin level of largemouth bass, but significantly decreased leptin gene expression, serum glucose, and triglyceride levels. This indicates that the basal + composition 2 diet can reduce blood glucose and blood lipid levels in largemouth bass fed a high-sugar diet by inhibiting leptin expression, thereby increasing feed intake and feed utilization rate, and ultimately promoting the growth of largemouth bass.

[0061] Table 3. Effects of different treatment groups on growth and physiological and biochemical parameters of largemouth bass.

[0062] Note: Items marked with the same lowercase letter in the same row indicate that the differences are not significant. Application Example 2 (1) Largemouth bass juvenile (about 50g) culture experiment: 600 juvenile fish of uniform size (initial weight: 56.15±0.04g) were randomly placed into 24 net cages (size: 2.0m×1.0m×1.0m), with 30 fish in each net cage. The net cages were divided into 5 groups, and each group was repeated 4 times. The fish were cultured for 12 weeks, during which the feeding and growth of the juvenile fish were observed. The basic feed consists of 40wt% fishmeal, 26wt% soybean meal, 5wt% fish oil, 5wt% soybean oil, 20wt% corn starch, 2wt% calcium dihydrogen phosphate, and 2wt% premix (each kilogram of premix contains the following minerals (g / kg) and vitamins (IU or mg / kg): CuSO4·5H2O 2.0 g; FeSO4·7H2O 25 g; ZnSO4·7H2O 22 g; MnSO4·4H2O 7 g; Na2SeO3 0.04 g; KI 0.026 g; CoCl2·6H2O 0.1 g; VA 900,000 IU; VD 200,000 IU; VE 4500 mg; VK 3220 mg; VB1 320 mg; VB2 1090 mg; niacin 2800 mg; VB5 2000 mg; VB6 500 mg; VB 12 The diets for each group consisted of: 1.6 mg of vitamin C, 5000 mg of vitamin C, 1000 mg of pantothenic acid, 165 mg of folic acid, 60000 mg of choline, and 15000 mg of inositol. The composition of each diet is shown below. ① Basic + Composition 2 Group: Feed the basic feed with 1 wt% of Composition 2 from Example 2; ② Basic + Composition 3 Group: Feed the basic feed with 1 wt% of Composition 3 of Example 3; ③ Basic + Composition 4 Group: Feed the basic feed with 1 wt% of Composition 4 from Example 4; ④ Basic + Composition 5 Group: Feed the basic feed with 1 wt% of Composition 5 of Example 5; ⑤ Basic + Composition 6 Group: Feed the basic feed with 1 wt% of Composition 6 of Example 6.

[0063] (2) After the aquaculture is completed, follow-up indicator analysis is carried out with reference to Application Example 1.

[0064] Table 4 shows the effects of different treatment groups' diets on the growth and physiological and biochemical indicators of largemouth bass. Each data point represents four replicates, and different superscript letters indicate significant differences between experimental groups. Table 4 shows that the basal + combined diet group 6 significantly increased the weight gain rate and specific growth rate of largemouth bass, and significantly increased plasma insulin levels, feed intake, and feed utilization. However, it significantly decreased leptin gene expression, serum glucose, and triglyceride levels. This indicates that the basal + combined diet group 6 can promote the growth of largemouth bass by inhibiting leptin expression, reducing blood glucose and lipid levels in largemouth bass fed a high-sugar diet, increasing feed intake and feed utilization.

[0065] Table 4. Effects of different treatment groups on growth and physiological and biochemical parameters of largemouth bass.

[0066] Note: Items marked with the same lowercase letter in the same row indicate that the differences are not significant. Based on the growth and physicochemical results of relevant aquaculture experiments, this invention confirms that the regulatory composition of this invention can increase the feed intake and serum insulin content of largemouth bass by downregulating leptin expression, and reduce the blood glucose and blood lipid levels of largemouth bass that consume leptin, thereby improving the feed utilization and growth performance of largemouth bass.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A composition for improving feed utilization by targeting the leptin gene in largemouth bass, characterized in that, The composition, by weight, consists of 1-30 parts of winter melon polysaccharide, 5-35 parts of γ-aminobutyric acid, and 1-20 parts of siRNA.

2. The composition for improving feed utilization by targeting the leptin gene in largemouth bass according to claim 1, characterized in that, The composition, by weight, consists of 5-25 parts of winter melon polysaccharide, 10-30 parts of γ-aminobutyric acid, and 5-15 parts of siRNA.

3. The composition for improving feed utilization by targeting the leptin gene in largemouth bass according to claim 2, characterized in that, The composition, by weight, consists of 10-20 parts of winter melon polysaccharide, 15-20 parts of γ-aminobutyric acid, and 10-12 parts of siRNA.

4. A method for preparing the composition for improving feed utilization by targeting the leptin gene of largemouth bass according to any one of claims 1-3, characterized in that, It includes the following steps: S1. Fresh winter melon peel and flesh are mixed, dried at 60-100℃, pulverized and sieved, deionized water is added, soaked at 90-100℃, and then centrifuged at 2500-4000 rpm to collect the supernatant. The supernatant is added to anhydrous ethanol and allowed to stand. The precipitate is collected by centrifugation at 2000-3000 rpm. The precipitate is dissolved in a mixed solution of chloroform and n-butanol, centrifuged at 35-50℃ at 3000-4000 rpm, and then freeze-dried under vacuum to obtain winter melon polysaccharide, abbreviated as WBP. S2. Using the coding region nucleotides of the largemouth bass leptin gene (GenBank accession number: MN887534.1) as the target, siRNA was designed with a sense strand of 5'-GGUUUGUACUAUUACACUATT-3' and an antisense strand of 5'-UAGUGUAAUAGUACAAACCTT-3'. The siRNA double strand was synthesized by reacting phosphoramide monomer at a concentration of 0.1~0.9 mol / L and tetrazolium activator at a concentration of 0.2~0.5 mol / L at 25~30℃ using the solid-phase phosphoramide method. The siRNA was then purified by high performance liquid chromatography and freeze-dried under vacuum to obtain leptin-siRNA. S3. Add the GABA solution dropwise to the WBP solution and let it stand at 25~35℃ to form the WBP-GABA precomplex; After adding leptin-siRNA solution and vortexing, the mixture was incubated at 25°C to obtain the WBP-GABA-siRNA ternary complex, abbreviated as WGR-Nano. S4. Mix WGR-Nano and hydroxypropyl methylcellulose solution and vacuum dry at 50-80℃ to obtain a composition that targets the leptin gene of largemouth bass and improves feed utilization.

5. The method for preparing the composition for improving feed utilization by targeting the leptin gene of largemouth bass according to claim 4, characterized in that, In step S1, the mass ratio of winter melon peel to winter melon flesh is (3~15):(7~20), and the total mass of winter melon peel and winter melon flesh to the volume ratio of deionized water is (1~5) g:(20~30) / mL; the volume ratio of chloroform to n-butanol in the mixed solution is (4~10):(1~5); the sieve aperture is 40~80 mesh; the drying time is 1~2 h; the soaking time is 2~3 h; the centrifugation time at 2500~4000 rpm is 15~30 min; the volume ratio of supernatant to anhydrous ethanol is 1:(3~5); the standing time is 12~24 h; the centrifugation time at 2000~3000 rpm is 10~30 min; and the centrifugation time at 3000~4000 rpm is 30~60 min.

6. The method for preparing the composition for improving feed utilization by targeting the leptin gene of largemouth bass according to claim 4, characterized in that, In step S2, the high-performance liquid chromatography uses a C18 reversed-phase column and a mobile phase of 0.1 wt% trifluoroacetic acid-acetonitrile aqueous solution, with gradient elution using a trifluoroacetic acid-acetonitrile aqueous solution containing 5-40% acetonitrile by volume; the reaction time is 1.5-2 h.

7. The method for preparing the composition for improving feed utilization by targeting the leptin gene of largemouth bass according to claim 4, characterized in that, In step S3, the volume ratio of GABA solution to WBP solution is (1~30):(5~35), and the molar ratio of N atoms of the amino group in WBP solution to P atoms of phosphate group in leptin-siRNA solution is (8~16):(1~20); the concentration of WBP solution is 5~10 mg / mL, pH= 6.8~7.5; the concentration of GABA solution is 20~40 mg / mL, and the concentration of leptin-siRNA solution is 50~70 μmol / L; the vortexing time is 10~30 s, the incubation time is 20~30 min, and the standing time is 15~30 min.

8. The method for preparing the composition for improving feed utilization by targeting the leptin gene of largemouth bass according to claim 4, characterized in that, In step S4, the mass ratio of hydroxypropyl methylcellulose to water in the hydroxypropyl methylcellulose solution is 1:10, and the mass ratio of WGR-Nano to hydroxypropyl methylcellulose solution is (3~15):(5~20); the stirring speed is 1000~3000 rpm, the stirring time is 15~30 min, and the vacuum drying time is 5~10 h.

9. A feed for largemouth bass, characterized in that, The largemouth bass feed includes the composition according to any one of claims 1-3; the composition is added to the largemouth bass feed at an amount of 0.5-1.2 wt%.

10. The use of the composition for improving feed utilization by targeting the leptin gene of largemouth bass according to any one of claims 1-3 in aquaculture.

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