阅读说明：本技术 一种缓解罗氏沼虾脂质过氧化的Dorsal基因干扰方法 (Dorsal gene interference method for relieving lipid peroxidation of macrobrachium rosenbergii ) 是由 孙存鑫 刘波 钱妤 周群兰 戈贤平 于 2019-11-28 设计创作，主要内容包括：本发明公开了一种缓解罗氏沼虾脂质过氧化的Dorsal基因干扰方法,包括以下步骤：(1)根据罗氏沼虾Dorsal基因的核苷酸序列合成序列特异性的dsRNA；(2)调整dsMsDorsal的浓度,按4μg/g虾的剂量注射,控制注射体积在30μL以内,(3)检测dsMsDorsal对组织损伤、细胞凋亡和基因表达的影响。本发明针对罗氏沼虾Dorsal基因设计的dsMsDorsal能快捷、高效、特异性地抑制罗氏沼虾摄食饲料脂肪酸不平衡、脂肪酸氧化等原因导致的脂质过氧化,实现罗氏沼虾健康、高效养殖。(The invention discloses a Dorsal gene interference method for relieving lipid peroxidation of macrobrachium rosenbergii, which comprises the following steps: (1) synthesizing sequence-specific dsRNA (double-stranded ribonucleic acid) according to the nucleotide sequence of the Macrobrachium rosenbergii Dorsal gene; (2) adjusting the concentration of dsMsDorsal, injecting at a dose of 4 μ g/g shrimp, controlling the injection volume within 30 μ L, and (3) detecting the effect of dsMsDorsal on tissue damage, apoptosis and gene expression. The dsMsDorsal designed aiming at the Dorsal gene of the Macrobrachium rosenbergii can quickly, efficiently and specifically inhibit lipid peroxidation caused by imbalance of fatty acid in ingested feed of the Macrobrachium rosenbergii, oxidation of fatty acid and the like, and realize healthy and efficient culture of the Macrobrachium rosenbergii.)

1. A Dorsal gene interference method for relieving lipid peroxidation of Macrobrachium rosenbergii is characterized by comprising the following steps:

① synthesizing a sequence-specific dsMsDorsal according to the nucleotide sequence of the Macrobrachium rosenbergii Dorsal gene, which comprises the following steps:

(1) preparation of cDNA

Taking the hepatopancreas of the macrobrachium rosenbergii, extracting Total RNA by using a Trizol method, detecting the RNA integrity of the extracted Total RNA by using 1.2% agarose gel electrophoresis, determining the RNA concentration by using a Nano Drop ultraviolet spectrophotometer, carrying out reverse transcription on the RNA with the concentration reaching the standard by using a kit to obtain cDNA, and storing the cDNA at the temperature of minus 20 ℃ for later use;

(2) design of primers

pL webpage end design dsRNA primers are designed by using http:// www.flyrnai.org/cgi-bin/RNAi _ find _ primers, and a T7 promoter sequence is added to the 5' end of each primer pair during primer design, so that primer sequences are obtained as follows:

sense strand

TAATACGACTCACTATAGGGCAAGTGTTCCTCGAAGGCTC，

Antisense strand

TAATACGACTCACTATAGGGAACTTCACCAATTTGTCCGC；

(3) Amplification of cDNA

Amplifying by using a kit and taking cDNA as a template, wherein the amplification step is 94 ℃ for 5min → [94 ℃ for 30s → 53 ℃ for 45s → 72 ℃ for 1min ] × 35cycle → 72 ℃ for 10min, detecting the quality of an amplification product by using 1.2% agarose gel electrophoresis, determining that a band is clear and has no impurity band as qualified, and cutting, recovering and purifying the qualified target band;

(4) transcription and synthesis of dsRNA

Synthesizing dsRNA by transcription according to the instruction of a transcription kit, wherein TranscriptAId Enzyme Mix and nucleotide are placed on ice, 5 multiplied TranscriptAId Reaction Buffer is placed at room temperature, Reaction liquid is slowly and uniformly mixed, transcription is carried out on a PCR instrument, the Reaction condition is 37 ℃ for 2 h, and the obtained transcription product is subsequently purified;

(5) purification of dsRNA

a) Adding 2 μ L of DNase I and RNase free into the reaction solution, mixing well, and standing at 37 deg.C for 15 min;

b) adding 2 μ L of 0.5M EDTA to adjust the pH of the reaction solution to 8.0; standing at 65 deg.C for 10 min;

c) adding 150 mu L of DEPC water and 15 mu L of 3M sodium acetate into the reaction solution, and fully and uniformly mixing, wherein the pH value of the sodium acetate solution is 5.2;

d) adding 150 μ L of the mixture in a ratio of 1: 1, fully and uniformly mixing the phenol/chloroform mixture, centrifuging, and transferring the upper aqueous phase into a new centrifugal tube;

e) adding 150 mu L of chloroform into a new centrifugal tube, fully and uniformly mixing, centrifuging, and transferring the upper-layer water phase into the new centrifugal tube;

f) adding ethanol with a volume twice that of the new centrifuge tube, cooling at-20 ℃ for at least 30 min, centrifuging at 4 ℃ for 20 min, and removing supernatant;

g) adding 500 μ L of 70% cold ethanol into the precipitate, rinsing, centrifuging at 13000 rpm at 4 deg.C for 5min, discarding supernatant, and drying, wherein the 70% cold ethanol is prepared with DEPC water;

h) adding 20 mu LDEPC water to dissolve the precipitate to prepare a dsMsDorsal solution, and storing at-20 ℃ for later use;

② adjusting the concentration of the synthesized dsMsDorsal, and injecting into the pericardial cavity according to the dose of 2-4 mug/g shrimp;

③ the results of dsMsDorsal testing the effect of anti-oxidase activity, tissue damage, apoptosis and gene expression show that the Dorsal gene interference method for relieving lipid peroxidation of Macrobrachium rosenbergii can effectively relieve the growth inhibition and liver pancreas damage of Macrobrachium rosenbergii caused by lipid peroxidation in production.

2. The Dorsal gene interference method for alleviating lipid peroxidation of Macrobrachium rosenbergii of claim 1, wherein the RNA concentration standard in step (1) is 200 ng/μ L or more, i.e. the RNA concentration is above 200 ng/μ L, and the reverse transcription kit is Takara Primer Script II 1st Strand cDNA Synthesis kit.

3. The Dorsal gene interference method for alleviating lipid peroxidation of Macrobrachium rosenbergii as claimed in claim 1, wherein the T7 promoter sequence in step (2) is TAATACGACTCACTATAGGG.

4. The Dorsal gene interference method for alleviating lipid peroxidation of Macrobrachium rosenbergii as claimed in claim 1, wherein the Kit model used for cDNA amplification in step (3) is Takara PrimeSTAR HS DNAPlloytase Kit, Code number R010A, and the Kit model used for Gel cutting purification is Takara MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0 Kit, Code number 9762.

5. The Dorsal gene interference method for alleviating lipid peroxidation of Macrobrachium rosenbergii as claimed in claim 1, wherein the dsRNA Transcription kit model in step (4) is Thermo Scientific Transcript A ID T7High Yield Transcription kit # K0441, and the reaction time of PCR instrument is prolonged to 4-8 h for the transcript less than or equal to 100 nt.

6. The Dorsal gene interference method for alleviating lipid peroxidation of Macrobrachium rosenbergii as claimed in claim 1, wherein the injection volume of dsMsDorsal in step ② is controlled within 30 μ L, and the injection is performed every 1-2 weeks according to the cultivation period.

7. The Dorsal gene interference method for alleviating lipid peroxidation of Macrobrachium rosenbergii as claimed in claim 1, wherein the nucleotide sequence of the Dorsal gene is GenBank: KX 219631.1.

8. The Dorsal gene interference method for alleviating lipid peroxidation in Macrobrachium rosenbergii as claimed in claim 1, wherein the injection concentration of dsMsDorsal is 4 μ g/g.

The technical field is as follows:

the invention belongs to the field of physiological regulation of aquatic animals, and particularly relates to a method for knocking and slowing down fat peroxidation of macrobrachium rosenbergii Dorsal gene by using an RNA interference technology.

Background art:

macrobrachium rosenbergii has been widely cultivated in China east China and south China since the introduction of Macrobrachium rosenbergii in the last 70 th century, and the annual yield reaches 13.2 million tons. In the culture process, the macrobrachium rosenbergii generally has the problem of lacking in lipid nutrition research, and due to inappropriate fat level and fatty acid composition in the feed and the oxidation of the fatty acid generated in the storage process of the feed, the growth of the macrobrachium rosenbergii is inhibited, the fat metabolism disorder of the macrobrachium rosenbergii is also caused, and the prawn body is oxidized and damaged to a certain extent. Therefore, the regulation and control of the fat metabolism of the macrobrachium rosenbergii have important significance for the healthy and efficient culture of the macrobrachium rosenbergii.

The Dorsal gene is a homologous subtype of NF- κ B in invertebrates, first found in drosophila. Research shows that Dorsal has a close relationship with inflammatory response, cell proliferation and apoptosis. In recent years, in the research of shrimps, Dorsal can activate the downstream antibacterial peptide gene expression in the Toll signal pathway and participate in antibacterial and antiviral reactions. The regulation of Dorsal in shrimp fat metabolism has been rarely reported.

RNA interference (RNAi) technology, which refers to a highly conserved, dsRNA-induced, highly efficient and specific degradation process of homologous mrnas during evolution, has been widely used in the fields of exploring gene functions and the treatment of infectious diseases and malignant tumors. In recent years, RNAi has also been increasingly used in gene function verification and antiviral studies in invertebrates. Because aquatic invertebrates such as shrimps and the like do not have stable passage cell lines, related RNAi research is mainly focused on the in-vivo level, and different individuals, different primers and different dsRNA introduction modes have larger influence differences on experimental results, so that a relatively stable RNA interference system is established for the Dorsal gene, which is essential for physiological research of fat of Macrobrachium rosenbergii and healthy and efficient culture.

The invention content is as follows:

the invention aims to solve the technical problem of providing a Dorsal gene interference method capable of effectively relieving lipid peroxidation of macrobrachium rosenbergii.

The invention discloses a Dorsal gene interference method for relieving lipid peroxidation of macrobrachium rosenbergii, which comprises the following steps:

① synthesizing a sequence-specific dsMsDorsal according to the nucleotide sequence of the Macrobrachium rosenbergii Dorsal gene, which comprises the following steps:

(1) preparation of cDNA

Taking the hepatopancreas of the macrobrachium rosenbergii, extracting Total RNA by using a Trizol method, detecting the RNA integrity of the extracted Total RNA by using 1.2% agarose gel electrophoresis, determining the RNA concentration by using a Nano Drop ultraviolet spectrophotometer, carrying out reverse transcription on the RNA with the concentration reaching the standard by using a kit to obtain cDNA, and storing the cDNA at the temperature of minus 20 ℃ for later use;

(2) design of primers

pL webpage end design dsRNA primers are designed by using http:// www.flyrnai.org/cgi-bin/RNAi _ find _ primers, and a T7 promoter sequence is added to the 5' end of each primer pair during primer design, so that primer sequences are obtained as follows:

sense strand

TAATACGACTCACTATAGGGCAAGTGTTCCTCGAAGGCTC，

Antisense strand

TAATACGACTCACTATAGGGAACTTCACCAATTTGTCCGC；

(3) Amplification of cDNA

Amplifying by using a kit and taking cDNA as a template, wherein the amplification step is 94 ℃ for 5min → [94 ℃ for 30s → 53 ℃ for 45s → 72 ℃ for 1min ] × 35cycle → 72 ℃ for 10min, detecting the quality of an amplification product by using 1.2% agarose gel electrophoresis, cutting a target band, recovering and purifying;

(4) transcription and synthesis of dsRNA

Synthesizing dsRNA by transcription according to the instruction of a transcription kit, wherein TranscriptAId Enzyme Mix and nucleotide are placed on ice, 5 multiplied TranscriptAId Reaction Buffer is placed at room temperature, Reaction liquid is slowly and uniformly mixed, transcription is carried out on a PCR instrument, the Reaction condition is 37 ℃ for 2 h, and the obtained transcription product is subsequently purified;

(5) purification of dsRNA

a) Adding 2 μ L of DNase I and RNase free into the reaction solution, mixing well, and standing at 37 deg.C for 15 min;

b) adding 2 μ L of 0.5M EDTA to adjust the pH of the reaction solution to 8.0; standing at 65 deg.C for 10 min;

c) adding 150 mu L of DEPC water and 15 mu L of 3M sodium acetate into the reaction solution, and fully and uniformly mixing, wherein the pH value of the sodium acetate solution is 5.2;

d) adding 150 μ L of the mixture in a ratio of 1: 1, fully and uniformly mixing the phenol/chloroform mixture, centrifuging, and transferring the upper aqueous phase into a new centrifugal tube;

e) adding 150 mu L of chloroform into a new centrifugal tube, fully and uniformly mixing, centrifuging, and transferring the upper-layer water phase into the new centrifugal tube;

f) adding ethanol with a volume twice that of the new centrifuge tube, cooling at-20 ℃ for at least 30 min, centrifuging at 4 ℃ for 20 min, and removing supernatant;

g) adding 500 μ L of 70% cold ethanol into the precipitate, rinsing, centrifuging at 13000 rpm at 4 deg.C for 5min, discarding supernatant, and drying, wherein the 70% cold ethanol is prepared with DEPC water;

h) adding 20 mu LDEPC water to dissolve the precipitate to prepare a dsMsDorsal solution, and storing at-20 ℃ for later use;

② performing pericardial cavity injection on the synthesized dsMsDorsal according to the shrimp dose of 2-4 mug/g;

③ the results of the detection of the effect of dsMsDorsal on tissue damage, apoptosis and gene expression show that the Dorsal gene interference method for relieving lipid peroxidation of Macrobrachium rosenbergii can effectively relieve the growth inhibition of Macrobrachium rosenbergii and liver pancreatic injury caused by lipid peroxidation in production.

Preferably, the standard of RNA concentration in the step (1) reaches the standard of being more than or equal to 200 ng/. mu.L, namely the RNA concentration can be used at the concentration of more than 200 ng/. mu.L, and the reverse transcription kit model is a Takara Primer Script II 1st Strand cDNAsSynthesis kit.

Preferably, the promoter sequence of T7 in step (2) is TAATACGACTCACTATAGG.

Preferably, the Kit model used for cDNA amplification in the step (3) is Takara PrimeSTAR HS DNAPlloyetase Kit, Code number R010A, and the Kit model used for Gel cutting purification is Takara MiniBESTAgare Gel DNA Extraction Kit Ver.4.0 Kit, Code number 9762.

Preferably, the dsRNA Transcription kit model in the step (4) is Thermo scientific Transcription kit T7High Yield Transcription kit # K0441, and the reaction time of the PCR instrument is prolonged to 4-8 h for the transcript less than or equal to 100 nt.

Preferably, the injection volume of dsMsDorsal in the step ② is controlled within 30 μ L, and the injection is performed every 1-2 weeks according to the cultivation period.

Preferably, the nucleotide sequence number of the Dorsal gene is GenBank: KX 219631.1.

Preferably, the injection concentration of dsMsDorsal is 4 μ g/g.

Drawings

FIG. 1 is a graph showing the effect of different injection sites of the present invention on the silencing effect of Dorsal.

FIG. 2a is a graph showing the effect of a dsMsDorsal injection concentration of 1. mu.g/g on silencing effect according to the invention.

FIG. 2b is a graph showing the effect of the dsMsDorsal injection concentration of 2. mu.g/g on silencing effect of the present invention.

FIG. 2c is a graph showing the effect of the dsMsDorsal injection concentration of 4. mu.g/g on silencing effect of the present invention.

FIG. 3 is a graph showing the effect of different fat sources of the present invention on the hepatopancreas tissue structure of Macrobrachium rosenbergii.

FIG. 4 is a graph showing the effect of different fat sources of the present invention on apoptosis of the hepatopancreas cells of Macrobrachium rosenbergii.

FIG. 5 is a graph showing the effect of different fat sources of the present invention on the expression of the Toll-Dorsal gene of Macrobrachium rosenbergii.

FIG. 6 is a graph of the effect of dsMsDorsal injection on Macrobrachium rosenbergii hepatopancreas Toll-Dorsal pathway expression in accordance with the present invention.

FIG. 7 is a graph of the effect of dsMsDorsal injection on the hepatopancreas tissue structure of Macrobrachium rosenbergii.

The specific implementation mode is as follows:

the substantive content of the present invention is further illustrated by the following examples, but the description of the examples of the present invention is only for illustrating the present invention and should not be construed as limiting the invention as detailed in the claims. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the examples, the methods are conventional methods unless otherwise specified, and the reagents used are conventional reagents sold or formulated according to conventional methods unless otherwise specified.

A Dorsal gene interference method capable of effectively relieving lipid peroxidation of Macrobrachium rosenbergii comprises the following steps:

① synthesizing a sequence-specific dsMsDorsal according to the nucleotide sequence of the Macrobrachium rosenbergii Dorsal gene;

② performing pericardial cavity injection on the synthesized dsMsDorsal according to the shrimp dose of 2-4 mug/g;

③ the results of dsMsDorsal testing the effect of anti-oxidase activity, tissue damage, apoptosis and gene expression show that the Dorsal gene interference method for relieving lipid peroxidation of Macrobrachium rosenbergii can effectively relieve the growth inhibition and liver pancreas damage of Macrobrachium rosenbergii caused by lipid peroxidation in production.