Dsrna for shrimp sglt1 gene and application thereof

Abstract

The present application relates to a kind of dsRNA of prawn SGLT1 gene and its application.The target sequence of the dsRNA is derived from the coding sequence corresponding to the transmembrane region outside loop, intracellular loop or extracellular loop of sodium-dependent glucose cotransporter SGLT1 gene coding protein.The dsRNA of prawn SGLT1 gene provides a new molecular means for preventing and controlling acute hepatopancreas necrosis disease.The dsRNA molecule can be synthesized by in vitro transcription or prokaryotic / eukaryotic expression system, and can be further constructed to contain the sequence expression cassette, recombinant vector and recombinant bacteria.The dsRNA molecule of the present application is small in molecular weight, biodegradable, and will not cause environmental pollution or drug residue, with the advantages of safety, environmental protection, high efficiency and scale production, etc., and can be used as a new biological agent or feed additive for preventing and controlling Vibrio parahaemolyticus AHPND infection.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a dsRNA of the shrimp SGLT1 gene and its application. Background Technology

[0002] Litopenaeus vannamei ( Penaeus vannamei Litopenaeus vannamei, also known as the whiteleg shrimp, has become the world's most produced economically important shrimp species due to its rapid growth, low feeding costs, strong adaptability, and high meat yield. In recent years, with the development of high-density intensive aquaculture, the frequent occurrence of various diseases has severely restricted the stable and sustainable development of the Litopenaeus vannamei aquaculture industry. Among them, Acute Hepatopancreatic Necrosis Disease (AHPND) is currently one of the most serious bacterial diseases causing large-scale mortality and economic losses in Litopenaeus vannamei.

[0003] The primary pathogen of AHPND is *Vibrio parahaemolyticus* (Vp) carrying the pVA1 plasmid. The PirA / PirB two-component toxin encoded by this plasmid is widely considered the main pathogenic factor of AHPND. The PirB protein, in particular, possesses structural features similar to typical pore-forming toxin-like virulence factors and can produce significant toxic effects on host cells. Previous studies have shown that PirAB toxin can disrupt the structural integrity of shrimp hepatopancreatic epithelial cells, inducing apoptosis and tissue degenerative necrosis, thereby leading to hepatopancreatic dysfunction and acute death in shrimp.

[0004] Currently, the prevention and control of AHPND mainly relies on traditional methods such as aquaculture environment control, antibiotic feeding, and the addition of traditional Chinese medicine to feed. These methods can improve the aquatic environment, inhibit bacterial growth, or enhance shrimp immunity to a certain extent in the short term, but their control effects are generally unstable. Long-term or improper use of antibiotics can not only easily lead to the emergence and spread of drug-resistant strains, but also cause drug residues and environmental pollution, posing food safety risks. Although traditional Chinese medicine immune enhancers are considered relatively safe and environmentally friendly, their effective ingredients are complex, their mechanisms of action are unclear, their production processes are cumbersome, and their batch stability is poor, making it difficult to achieve standardized and large-scale application.

[0005] In summary, current technologies still lack effective control measures that can block the effects of pathogenic bacteria toxins at the molecular level. Summary of the Invention

[0006] The present invention aims to disclose a dsRNA of the shrimp SGLT1 gene and its application, in order to solve one or more technical problems existing in the existing methods and provide at least one beneficial option or create conditions. By providing a novel prevention and control technology that can weaken the pathogenic effect of pathogenic toxins at the host level and enhance the disease resistance of shrimp, efficient, safe and sustainable prevention and control of AHPND can be achieved.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention is to provide a dsRNA of the shrimp SGLT1 gene. The target sequence of the dsRNA is derived from the coding sequence corresponding to the outer loop, intracellular loop, or extracellular loop of the transmembrane region of the sodium-dependent glucose cotransporter 1 (SGLT1) gene. SGLT1 is a typical membrane protein, widely distributed on the cell membrane, and its function is to mediate the transmembrane transport of glucose and sodium ions. The shrimp SGLT1 gene is... Pv SGLT1 gene. Pv SGLT1, as a membrane protein, also possesses an extracellular loop, a transmembrane region, and an intracellular loop. The dsRNA targets the outer loop, intracellular loop, or extracellular loop of the transmembrane region, thereby improving gene silencing efficiency and reducing off-target effects.

[0008] In a further embodiment of the first aspect of the present invention, the nucleotide sequence of the dsRNA is shown in SEQ ID No:1. The prepared dsRNA was experimentally verified to significantly reduce the transcription and translation levels of the SGLT1 gene. Subsequently, based on successful interference, shrimp were injected with PirB protein or Vibrio parahaemolyticus AHPND strain.

[0009] A second aspect of the present invention is to provide a dsRNA expression cassette. The dsRNA expression cassette contains the dsRNA described in the first aspect of the present invention.

[0010] A third aspect of the present invention is to provide a recombinant vector. The recombinant vector contains the dsRNA described in the first aspect of the present invention or the dsRNA expression cassette described in the second aspect of the present invention.

[0011] A fourth aspect of the present invention is to provide a recombinant bacterium. The recombinant bacterium contains the dsRNA described in the first aspect of the present invention, the dsRNA expression cassette described in the second aspect of the present invention, or the recombinant vector described in the third aspect of the present invention.

[0012] The fifth aspect of the present invention is to provide an application direction, including at least one of the dsRNA described in the first aspect of the present invention, the dsRNA expression cassette described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, or the recombinant bacteria described in the fourth aspect of the present invention for preparing a formulation, the purpose of which is to prevent, inhibit, or treat acute hepatopancreatic necrosis disease in shrimp.

[0013] In a further embodiment of the fifth aspect of the present invention, the preparation prevents, inhibits or treats acute hepatopancreatic necrosis disease in shrimp by reducing the membrane binding amount and internalization rate of the pathogenic factor PirB.

[0014] A sixth aspect of the present invention is to provide a formulation. The formulation contains one or more components selected from the dsRNA of the first aspect of the present invention, the dsRNA expression cassette of the second aspect of the present invention, the recombinant vector of the third aspect of the present invention, or the recombinant bacteria of the fourth aspect of the present invention.

[0015] In a further embodiment of the sixth aspect of the invention, the formulation is an injection, a spray, or a feed additive.

[0016] In a further embodiment of the sixth aspect of the present invention, the dsRNA preparation is suitable for targeting the SGLT1 gene mRNA sequence of Litopenaeus vannamei, Litopenaeus japonicus, Litopenaeus chinensis, or Litopenaeus sinensis.

[0017] The dsRNA of the shrimp SGLT1 gene of this invention provides a novel molecular approach for the prevention and control of acute hepatopancreatic necrosis disease. The dsRNA molecule can be synthesized through in vitro transcription or a prokaryotic / eukaryotic expression system, and expression cassettes, recombinant vectors, and recombinant bacteria containing this sequence can be further constructed. The dsRNA of this invention has a small molecular weight, is biodegradable, and will not cause environmental pollution or drug residues. It has advantages such as safety, environmental friendliness, high efficiency, and scalability, and can be used as a novel biological agent or feed additive for the prevention and control of Vibrio parahaemolyticus AHPND infection.

[0018] Compared with existing technologies, this invention is the first to verify the mechanism of action of shrimp SGLT1 in AHPND pathogenesis at the host molecular level using RNA interference technology. After interfering with SGLT1, the membrane binding of PirB decreased significantly, damage to hemocytes and hepatopancreas was significantly reduced, and the disease resistance of shrimp was improved. The results indicate that downregulation of SGLT1 can effectively block the membrane action and pore-forming process of PirB, thereby significantly alleviating the pathological symptoms of AHPND. The dsRNA of the shrimp SGLT1 gene described in this invention can serve as a safe, environmentally friendly, and efficient control method, and has significant application value and promising prospects in the prevention and control of AHPND. Attached Figure Description

[0019] Figure 1 This is a graph showing the effect of dsRNA on the amount of PirB cells bound to the cell membrane, as described in Example 1. Figure 2 This is a diagram illustrating the effect of dsRNA on pathological changes in shrimp hepatopancreatic tissue as described in Example 2. Figure 3This is a graph showing the effect of dsRNA on the challenge experiment described in Example 3; Figure 4 This is a graph showing the detection and analysis of PirB-induced hematopoietic cell necrosis by dsRNA as described in Example 4. Detailed Implementation

[0020] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0021] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0022] Example 1, the dsRNA interference Pv Analysis of the effect of SGLT1 on PirB cell membrane binding amount (1) Primers for SGLT1 were designed and synthesized based on the nucleic acid sequence of Litopenaeus vannamei SGLT1 (GenBank ID: 113828104) in the NCBI database. The control gene was EGFP (GenBank ID: 20473140). The primer sequences are as follows: The upstream primer for the dsRNA is: 5'-GTTCATTGCTGTTTGAAGTATACAACCG-3' (SEQ ID No:2); The downstream primer for the dsRNA is: 5'-GGACATATGTGGAGCCTCTGTCATAG-3' (SEQ ID No:3); The upstream primer of the dsRNA expression cassette with T7 promoter: 5'-GGATCCTAATACGACTCACTATAGGGTTCATTGCTGTTTGAAGTATACAACCG-3' (SEQ ID No:4); The downstream primer of the dsRNA expression cassette with the T7 promoter is: 5'-GGATCCTAATACGACTCACTATAGGGGACATATGTGGAGCCTCTGTCATAG-3' (SEQ ID No: 5). dsEGFP upstream primer: 5-CGTAAACGGCCACAAGTT-3' (SEQ ID No: 6); dsEGFP downstream primer: 5-TTCACCTTGATGCCGTTC-3' (SEQ ID No:7); Upstream primer of dsEGFP expression cassette with T7 promoter: 5-GGATCCTAATACGACTCACTATAGGCGTAAACGGCCACAAGTT-3' (SEQ ID No: 8); Downstream primer of the dsEGFP expression cassette with T7 promoter: 5-GGATCCTAATACGACTCACTATAGGTTCACCTTGATGCCGTTC-3' (SEQ ID No: 9).

[0023] (2) PCR amplification was performed using Litopenaeus vannamei blood cell cDNA and pEGFP-N1 plasmid as templates. The resulting products were purified and used as in vitro transcription templates. T7 RNA polymerase was used for in vitro transcription to obtain the dsRNA and dsEGFP.

[0024] (3) The synthesized dsRNA and dsEGFP were diluted with DEPC water to 100 ng / µL. Twenty healthy shrimp were taken from each group, and 100 µL of the dsRNA or 100 µL of dsEGFP was injected into the abdominal base of each shrimp.

[0025] (4) 48 h after injection, each shrimp in the experimental group and the control group was injected with 100 µL of purified 4 µmol / L PirB protein or an equal amount of EGFP. 2 h after PirB protein injection, blood cells from 3 shrimp in each group were randomly selected for total RNA and total protein extraction.

[0026] (5) Interference effect detection: qPCR was used to detect the expression changes of SGLT1 at the mRNA level, and the relative expression level was calculated with β-actin as the internal reference gene.

[0027] (6) PirB binding detection: Immunofluorescence observation: After fixing the isolated blood cells, immunofluorescence staining was performed using anti-PirB antibody (1:1000). The secondary antibody was Alexa Fluor 5P4-labeled goat anti-rabbit IgG (1:400). The binding fluorescence signal of PirB on the blood cell membrane was observed using a ZEISS LSM 800 confocal microscope, and the average fluorescence intensity was quantitatively analyzed using software.

[0028] Western blot validation: Blood cell membrane proteins were extracted, and the content of PirB was detected using anti-PirB antibody. β-actin was used as an internal reference protein. After ECL staining, the differences between the two groups were compared by gray value analysis.

[0029] The results are as follows Figure 1As shown, compared with the control group (dsEGFP), the expression level of SGLT1 at the mRNA level in the interference group (dsRNA) was significantly decreased (interference efficiency >80%), indicating that RNA interference was successful (see...). Figure 1 (At point A in the middle). Immunofluorescence results showed that the binding signal of PirB on the hemocyte membrane was significantly weakened after SGLT1 interference, which was consistent with the results of Western blot analysis. The content of PirB in the protein components of shrimp hemocytes was significantly reduced, with the average gray value decreasing by about 50% (see...). Figure 1 (from position B to position D). The results in summary indicate that downregulating SGLT1 expression via dsRNA interference significantly reduces the membrane-binding capacity of PirB.

[0030] Example 2: Pathological changes in shrimp hepatopancreas tissue after dsRNA interference with PvSGLT1 and the action of PirB. (1) The dsRNA and dsEGFP were prepared according to the method provided in Example 1 and injected into the abdominal base muscle of healthy Litopenaeus vannamei at a concentration of 100 ng / µL, with 20 shrimp in each group. 48 h after injection, the experimental group (dsRNA group) and the control group (dsEGFP group) were injected with 100 µL of purified 4 µmol / L PirB protein or an equal amount of EGFP, respectively.

[0031] (2) 24 h after PirB injection, three shrimp were randomly selected from each group, and the hepatopancreas tissue was quickly taken and fixed in 4% paraformaldehyde solution overnight. After dehydration with graded ethanol, clearing with xylene and embedding in paraffin, the tissue was sectioned (5 µm thick), and the structural changes of the hepatopancreas tissue were observed by hematoxylin-eosin (H&E) staining.

[0032] The results are as follows Figure 2 As shown, H&E staining revealed that in the control group (dsEGFP+EGFP), the hepatopancreatic acinar structure was intact, the luminal cells were tightly arranged, and the tissue morphology was normal. After PirB challenge (dsEGFP+PirB group), the hepatopancreatic acinar structure was significantly swollen, the luminal cells were loosely arranged, some basement membranes detached, the tubules collapsed, and local vacuolar degeneration and necrosis foci appeared (indicated by red arrows). In contrast, in the experimental group where SGLT1 was interfered with (dsRNA+PirB), the hepatopancreatic acinar structure remained largely intact, the luminal cells were tightly arranged, the basement membrane was continuous, and no obvious detachment or necrosis was observed. This indicates that interfering with SGLT1 can significantly reduce PirB-induced hepatopancreatic tissue damage.

[0033] Example 3: Survival analysis of shrimp after challenge with PirB protein or infection with VpAHPND (1) The dsRNA and dsEGFP were prepared according to the method provided in Example 1 and injected into the abdominal base muscle of healthy Litopenaeus vannamei at a concentration of 100 ng / µL, with 30 shrimp in each group. PirB protein challenge experiment or VpAHPND infection experiment was performed 48 h after injection.

[0034] (2) PirB challenge experiment: The experimental group (dsRNA) and the control group (dsEGFP) were injected with 100 µL of purified PirB protein at a concentration of 4 µmol / L or an equal amount of EGFP as a negative control. The number of surviving individuals was recorded every 12 h from 0 to 96 after injection, and the cumulative survival rate was calculated.

[0035] (3) VpAHPND infection experiment: The experimental group and the control group were injected with 100 µL containing 1×10 5 VpAHPND bacterial suspension at CFU / mL or PBS (negative control) were used to count the number of survivors during the 0–96 h post-infection period.

[0036] The results showed that the control group (dsEGFP+PirB) shrimp showed significant mortality within 48 hours, and the survival rate dropped to about 30% after 72 hours, while the survival rate of the experimental group remained at around 70%. Figure 3 As shown at point A in the diagram), the difference was statistically significant (p<0.01). In the VpAHPND live bacteria infection experiment ( Figure 3 As shown in section B), all members of the control group died within 48 hours, while approximately 35% of the experimental group survived at 48 hours (p<0.01). This indicates that interfering with SGLT1 can significantly improve the survival rate of Litopenaeus vannamei under PirB challenge and VpAHPND infection.

[0037] Example 4: Analysis of the effect of the dsRNA on PirB-induced hematopoietic cell necrosis (1) RNA interference experiments were performed according to the method provided in Example 1. The dsRNA (experimental group) and dsEGFP (control group) were injected into healthy Litopenaeus vannamei, respectively. Blood cells were collected for in vitro culture after 48 h. The uninjected sample was set as the blank control group (Untreated).

[0038] (2) PirB induction experiment: each group of blood cells was inoculated at 1×10 6The cells were seeded onto glass slides at a density of 1 μmol / mL, and incubated with 1 µmol / L purified PirB protein for 2 h. After incubation, the cells were washed with PBS and then subjected to PI / Hoechst double staining. The final concentration of PI (propidium iodide) working solution was 5 µmol / L, and the cells were incubated at room temperature in the dark for 10 min; Hoechst 33342 was incubated in the dark for 15 min. After washing with PBS, images of the red (PI) and blue (Hoechst) channels were captured using a ZEISSLSM 800 confocal microscope, and the proportion of necrotic cells was statistically analyzed using software.

[0039] The results are as follows Figure 4 As shown, the untreated group showed no obvious PI staining signal in blood cells; the control group (dsEGFP+PirB) showed a large number of red fluorescent positive cells, indicating increased cell membrane permeability and necrosis; while the experimental group (dsRNA+PirB) showed a significant reduction in red signal and a significant decrease in the proportion of necrotic cells. Figure 4 (As shown at point A in the middle). Statistical results showed that the proportion of necrotic cells in the control group was 50%, while that in the experimental group was only about 20% (p<0.001). Figure 4 (As shown at point B in the middle). This indicates that PirB can significantly induce necrosis of shrimp hemocytes, while the dsRNA can significantly reduce the proportion of necrotic cells. Downregulating the expression of host SGLT1 can effectively alleviate the toxic effects of PirB, providing a new molecular protection strategy for the prevention and control of AHPND.

[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A dsRNA of the shrimp SGLT1 gene, characterized in that, The target sequence originates from the coding sequence corresponding to the transmembrane region of the transcellular loop, intracellular loop, or extracellular loop of the sodium-dependent glucose cotransporter SGLT1 gene.

2. The dsRNA of the shrimp SGLT1 gene according to claim 1, characterized in that, The nucleotide sequence of the dsRNA is shown in SEQ ID No:

1.

3. A dsRNA expression cassette, characterized in that, Contains the dsRNA as described in claim 1 or 2.

4. A recombinant vector, characterized in that, Contains the dsRNA as described in any one of claims 1 to 2 or the dsRNA expression cassette as described in claim 3.

5. A recombinant bacterium, characterized in that, It contains the dsRNA of any one of claims 1 to 2, the dsRNA expression cassette of claim 3, or the recombinant vector of claim 4.

6. The use of the dsRNA of any one of claims 1 to 2, the dsRNA expression cassette of claim 3, the recombinant vector of claim 4, or the recombinant bacteria of claim 5 in the preparation of a formulation for the prevention, inhibition, or treatment of acute hepatopancreatic necrosis in shrimp.

7. The application according to claim 6, characterized in that, The formulation reduces the membrane binding and internalization rate of the pathogenic factor PirB.

8. A formulation, characterized in that, It contains one or more components of the dsRNA of any one of claims 1 to 2, the dsRNA expression cassette of claim 3, the recombinant vector of claim 4, and the recombinant bacteria of claim 5.

9. The formulation according to claim 8, characterized in that, The preparation is an injection, spray, or feed additive.

10. The formulation according to claim 9, characterized in that, The formulation is used for Litopenaeus vannamei, Litopenaeus japonicus, Litopenaeus chinensis, or Litopenaeus sinensis.

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