A gene target related to white shrimp wssv infection resistance and growth performance maintenance and application thereof

Abstract

The present application belongs to the technical field of bioengineering, and particularly relates to a gene target point related to WSSV infection resistance and growth performance maintenance of Litopenaeus vannamei and application thereof. The present application obtains a gene LvCaspase-znf related to WSSV infection mortality of Litopenaeus vannamei, the encoded protein of which contains a caspase family conserved domain and a zinc finger domain, the protein has nucleic acid binding capacity, can bind WSSV-derived nucleic acid fragments, and has self-interaction capacity or oligomerization capacity. The present application also provides dsRNA targeting the above gene, after knocking down the expression of the above gene through the dsRNA, the mortality of Litopenaeus vannamei after WSSV infection is reduced, the dsRNA can be used for preparing related products for reducing the mortality of Litopenaeus vannamei after WSSV infection and improving the WSSV infection resistance of Litopenaeus vannamei, and provides a new technical scheme for the prevention and control of aquatic breeding viral diseases and the development of WSSV-resistant products.

Description

Technical Field

[0001] This invention belongs to the technical field of bioengineering, specifically relating to a gene target related to the resistance to WSSV infection and the maintenance of growth performance in Litopenaeus vannamei and its application. Background Technology

[0002] White spot syndrome virus (WSSV) is a significant viral pathogen affecting Litopenaeus vannamei aquaculture, causing acute mortality and severe economic losses. Current WSSV control measures are insufficient in terms of specificity, stability, and scalability. Therefore, screening key host factors and establishing molecular control strategies based on host targets are of significant practical value.

[0003] Caspase family proteins are commonly involved in processes such as cell death, apoptosis regulation, and immune responses. Zinc finger domain proteins are often associated with nucleic acid recognition, transcriptional regulation, or protein-protein interactions. Previous studies have suggested that crustacean caspase-related proteins may be involved in viral infection processes, but the functions of different species, different family members, and different combinations of domains vary.

[0004] Currently, the role of the LvCaspase-znf gene from Litopenaeus vannamei in the WSSV infection mortality process has not been fully elucidated, and in particular, there is a lack of systematic application plans for the effects of gene knockdown on WSSV challenge mortality, protein-nucleic acid binding capacity, and protein oligomerization capacity.

[0005] WSSV infection not only causes acute death but can also lead to abnormalities in nutrient digestion and absorption, energy supply, nucleotide synthesis, protein processing, tissue structure maintenance, and molting-related metabolic processes, thereby affecting the physiological homeostasis and aquaculture performance of shrimp during the infection period. If anti-WSSV molecular interventions can reduce mortality while avoiding significant adverse effects on the normal growth-related metabolism of uninfected individuals and mitigating abnormalities in growth-related pathways under infection conditions, they would be more beneficial for practical aquaculture applications.

[0006] Therefore, identifying and utilizing LvCaspase-znf as a host molecular target for Litopenaeus vannamei to resist WSSV infection, developing interfering nucleic acid molecules and related products targeting this gene, and evaluating their effects on growth-related molecular states such as nutrient metabolism, energy metabolism, protein synthesis, molting, and tissue homeostasis can provide a new technical approach for the prevention and control of viral diseases in aquaculture, the development of antiviral products, and the maintenance of aquaculture production performance. Summary of the Invention

[0007] To address the above problems, the present invention aims to provide a gene target related to WSSV infection resistance and growth performance maintenance in Litopenaeus vannamei and its application.

[0008] The technical content of this invention is as follows: This invention provides an application of LvCaspase-znf gene / protein as a target in screening candidate substances for reducing mortality, reducing WSSV viral load, or inhibiting WSSV viral replication in Litopenaeus vannamei. The candidate substances target LvCaspase-znf and inhibit the expression of LvCaspase-znf gene / protein. The selected candidate substances were used to enhance the resistance or tolerance of Litopenaeus vannamei to WSSV infection; The candidate substances include small molecule compounds, peptides, nucleic acids, feed additives, or gene editing elements; The LvCaspase-znf gene is derived from Litopenaeus vannamei, and its nucleotide sequence is shown in SEQ ID NO.3, encoding a protein containing a conserved caspase family domain and a zinc finger domain. The LvCaspase-znf protein is a protein encoded by the LvCaspase-znf gene, and its amino acid sequence is shown in SEQ ID NO.4. The protein has nucleic acid binding ability, can bind nucleic acid fragments derived from WSSV, and has self-interacting ability or oligomerization ability. The screening process further includes: detecting the effects of candidate substances on the nucleic acid binding capacity, self-interaction capacity, and / or oligomerization capacity of the LvCaspase-znf protein, and detecting changes in shrimp mortality, viral load, and / or expression levels of virus-related genes after white spot syndrome virus infection; wherein, candidate substances that can inhibit the nucleic acid binding, self-interaction, or oligomerization of the LvCaspase-znf protein and reduce mortality or viral load are substances with potential for anti-WSSV virus infection.

[0009] The present invention also provides a dsRNA that inhibits the expression of the LvCaspase-znf gene, which targets a continuous 520 nucleotide fragment in the LvCaspase-znf gene, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0010] The present invention also provides an LvCaspase-znf gene / protein expression inhibitor, wherein the LvCaspase-znf gene expression inhibitor includes the above-mentioned dsRNA that inhibits the expression level of LvCaspase-znf gene, thereby inhibiting LvCaspase-znf gene expression and thus inhibiting LvCaspase-znf protein expression; The LvCaspase-znf gene expression inhibitor also includes siRNA, shRNA, or antisense nucleic acid fragments that target the LvCaspase-znf gene.

[0011] The present invention also provides a composition for reducing mortality, reducing WSSV viral load, or inhibiting WSSV replication in Litopenaeus vannamei, the composition comprising the above-mentioned LvCaspase-znf gene / protein expression inhibitor, and at least one of an aquaculture-acceptable vector, excipient, or diluent.

[0012] This invention also provides the application of an LvCaspase-znf gene / protein expression inhibitor or a composition thereof in the preparation of a formulation for reducing mortality of Litopenaeus vannamei after WSSV infection, maintaining growth-related metabolic status and / or aquaculture performance of Litopenaeus vannamei under WSSV infection conditions; The formulation includes feed additives, injectable formulations, or liquid formulations for soaking. The injectable formulation contains the above-mentioned dsRNA and an aquaculture-acceptable injectable carrier or diluent. The growth-related metabolic states include nutrient digestion and absorption, carbohydrate metabolism, lipid metabolism, amino acid metabolism, nucleotide metabolism, protein synthesis or processing, molting and chitin metabolism, muscle structure, cytoskeleton, extracellular matrix, cell junctions, mineral absorption, and one or more of the PI3K-Akt, Ras, or MAPK signaling pathways. The formulation includes methods to alleviate WSSV infection-induced nutrient metabolism inhibition, energy metabolism disorder, tissue damage, and / or abnormal growth-related molecular states.

[0013] This invention also provides a kit for assessing the mortality risk or resistance to WSSV infection in Litopenaeus vannamei, the kit comprising one or more of the following: (a) Primer pairs for specific detection of LvCaspase-znf gene expression levels; The nucleotide sequences of the primer pairs are shown in SEQ ID NO.10 and SEQ ID NO.11; (b) Antibodies that specifically bind to LvCaspase-znf protein; (c) The above-mentioned dsRNA molecules used as positive or negative controls; (d) A double-stranded DNA probe for detecting the nucleic acid binding activity of LvCaspase-znf protein, the double-stranded DNA probe being formed by annealing the oligonucleotides shown in SEQ ID NO.12 and SEQ ID NO.13.

[0014] This invention also provides a method for screening or breeding shrimp strains with resistance to WSSV virus infection, characterized by comprising the following steps: 1) Determine the expression level of LvCaspase-znf gene / protein in the candidate shrimp population; 2) Based on the results of step 1), select individuals with LvCaspase-znf gene / protein expression levels lower than the population average as parents for family breeding.

[0015] The beneficial effects of this invention are as follows: This invention relates to gene targets related to WSSV infection resistance and growth performance maintenance in Litopenaeus vannamei and their applications. The invention obtains a host gene, LvCaspase-znf, associated with WSSV infection mortality in Litopenaeus vannamei. The encoded protein contains conserved caspase family domains and zinc finger domains. LvCaspase-znf protein possesses nucleic acid binding capacity, capable of binding WSSV-derived nucleic acid fragments, and exhibits self-interaction or oligomerization capabilities, providing a functional basis for its involvement in the WSSV infection process. This invention also provides a dsRNA targeting the LvCaspase-znf gene. Knocking down LvCaspase-znf gene expression using this dsRNA reduces mortality in Litopenaeus vannamei after WSSV infection, indicating that LvCaspase-znf can serve as a host molecular target for WSSV infection control in Litopenaeus vannamei. Furthermore, it can be used to prepare products that reduce mortality after WSSV infection and improve the shrimp's resistance to WSSV infection, providing a new technical solution for the prevention and control of viral diseases in aquaculture and the development of anti-WSSV products.

[0016] Based on transcriptome analysis of the uninfected group and the WSSV-infected group at 24 h and 48 h, this invention demonstrates that dsLvCaspase-znf treatment did not significantly disrupt the normal growth-related molecular states of Litopenaeus vannamei under uninfected conditions. Furthermore, it can alleviate abnormalities in pathways related to nutrient metabolism, energy metabolism, protein processing, cytoskeleton, cell junctions, and tissue repair under WSSV infection conditions, helping to maintain growth-related metabolic states and aquaculture performance during the infection period. This provides a new technical solution for the prevention and control of WSSV disease in Litopenaeus vannamei, the screening of antiviral targets, and the reduction of aquaculture losses. Attached Figure Description

[0017] Figure 1 Diagram showing the analysis of the open reading frame of the LvCaspase-znf gene and the domains of its encoded protein; Figure 2 Multiple sequence alignment results for the LvCaspase-znf encoded protein 1; Figure 3Multiple sequence alignment results for the LvCaspase-znf encoded protein 2; Figure 4 The results show the expression changes of the LvCaspase-znf gene in Litopenaeus vannamei after WSSV infection; Figure 5 The results show the knockdown efficiency of dsLvCaspase-znf on the LvCaspase-znf gene. Figure 6 The results show the survival rate of Litopenaeus vannamei after WSSV challenge following LvCaspase-znf knockdown. Figure 7 The results of WSSV virus load detection in Litopenaeus vannamei after LvCaspase-znf knockdown; Figure 8 The results of pull-down detection of the binding of LvCaspase-znf encoded protein to WSSV-derived nucleic acid fragments; Figure 9 Results of detection of the self-interaction or oligomerization ability of LvCaspase-znf encoded protein; Figure 10 KEGG enrichment analysis of differentially expressed genes between the dsGFP group and the dsLvCaspase-znf group at 0 h, 24 h and 48 h; Figure 11 GO enrichment analysis of differentially expressed genes between the dsGFP group and the dsLvCaspase-znf group at 0 h, 24 h and 48 h; Figure 12 Cluster heatmap analysis of differential genes between the dsGFP group and the dsLvCaspase-znf group at 0 h, 24 h and 48 h. Detailed Implementation

[0018] The present invention will be further described in detail below through specific implementation examples and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. After reading the present invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope of the appended claims.

[0019] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0020] The Litopenaeus vannamei used in this invention is sourced from Yudeli Aquatic Products Co., Ltd., Zhuhai City, Guangdong Province.

[0021] Example 1 Screening for targets to reduce mortality after WSSV infection in Litopenaeus vannamei and their applications 1. Obtaining and structurally analyzing the target gene Total RNA was extracted from the gills, hepatopancreas, intestine, stomach, hemocytocytes, muscle, subcarpal epidermis, nerve tissue, eyestalks, heart, antennae, and appendages of Litopenaeus vannamei, and cDNA was obtained by reverse transcription. Using the shrimp cDNA as a template, specific primers were designed based on the ORF sequence information of LvCaspase-znf provided by transcriptome / genome data. The nucleotide sequences of the primer pairs are shown in SEQ ID NO. 1 and SEQ ID NO. 2. The open reading frame sequence of the target gene LvCaspase-znf was amplified, as shown in... Figure 1 As shown.

[0022] The PCR amplification products were recovered and purified after detection by agarose gel electrophoresis, and then ligated into prokaryotic and eukaryotic expression vectors. The recombinant expression vectors were transformed into *E. coli* DH5α competent cells to obtain recombinant engineered bacteria. Positive clones were selected for sequencing verification. Sequencing results are as follows: Figure 1 The LvCaspase-znf gene has an open reading frame length of 1887 bp, encoding 629 amino acids. Its nucleotide sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4, which is 628 aa. The 629th position is the stop codon Ter.

[0023] InterPro was used for domain prediction. The results are as follows: Figure 1 As shown, the LvCaspase-znf encoded protein contains a caspase family conserved domain and a zinc finger domain. The zinc finger domain is located at amino acids 1-371, and the caspase family conserved domain is located at amino acids 432-614. Multiple sequence alignment analysis was performed using MEGA, and the results are as follows: Figure 2 , Figure 3 As shown, LvCaspase-znf has some homology with other crustacean homologous caspase family proteins.

[0024] 2. Changes in target gene expression after WSSV infection Healthy Litopenaeus vannamei shrimp were randomly divided into a WSSV infection group and a PBS control group (n=30 per group, with 3 replicates). The WSSV infection group was injected with WSSV virus solution, while the control group was injected with an equal volume of PBS. Gills, hepatopancreas, hemolymph, and intestinal tissues were collected at 0, 4, 8, 12, 24, and 48 h post-infection to extract RNA, which was then detected by qRT-PCR using EF1α as an internal reference gene.

[0025] like Figure 4As shown in the results, compared with the control group, the expression level of LvCaspase-znf in gills, hepatopancreas, hemolymph and intestinal tissues was significantly upregulated after WSSV infection, reaching a peak at 8 h, with a fold change of 3-30 times, and the P value was less than 0.05.

[0026] 3. Design, synthesis, and knockdown efficiency detection of dsRNA RNAi targeting fragments were designed based on the nucleotide sequence of LvCaspase-znf, and a 520 bp fragment was amplified. dsLvCaspase-znf was synthesized using in vitro transcription. Simultaneously, dsGFP was synthesized using GFP or other unrelated sequences as a negative control.

[0027] Based on the LvCaspase-znf gene sequence shown in SEQ ID NO.3, a continuous 520-nucleotide fragment was selected as the RNAi targeting fragment, and the nucleotide sequence of the targeting fragment is shown in SEQ ID NO.5.

[0028] Primers were designed for amplifying the template for dsRNA synthesis. The nucleotide sequences of the LvCaspase-znf gene-specific primers dsLvCaspase-znf-F and dsLvCaspase-znf-R are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0029] Furthermore, to obtain PCR products carrying T7 promoter sequences at both ends, T7 promoter sequences were ligated to the 5′ ends of dsLvCaspase-znf-F and dsLvCaspase-znf-R, respectively, to obtain full-length upstream primer T7-dsLvCaspase-znf-F and full-length downstream primer T7-dsLvCaspase-znf-R for in vitro transcription template amplification, with nucleotide sequences as shown in SEQ ID NO.8 and SEQ ID NO.9, respectively.

[0030] Using a plasmid containing the LvCaspase-znf gene fragment (SEQ ID NO.5) as a template, PCR amplification was performed using primer pairs T7-dsLvCaspase-znf-F and T7-dsLvCaspase-znf-R to obtain a dsRNA synthesis template carrying T7 promoter sequences at both ends.

[0031] The PCR amplification system consisted of: 100 ng template DNA, 50 μL 10×PCR Buffer, 10 μL dNTPs, 3 μL upstream primer, 3 μL downstream primer, 2 μL DNA polymerase, and ddH2O to a final volume of 100 μL. The PCR program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55-65℃ touch-down annealing for 30 s, 72℃ extension for 150 s, for a total of 35 cycles; and a final extension at 72℃ for 10 min.

[0032] After the PCR product showed a target band of approximately 520 bp during agarose gel electrophoresis, the gel was recovered or the PCR product was purified to obtain the dsRNA synthesis template.

[0033] dsRNA was synthesized in vitro using an in vitro transcription kit. The specific reaction system was performed according to the kit instructions.

[0034] After the reaction, DNase I was added to digest the DNA template, and then dsRNA was purified using a kit. The concentration of dsRNA was measured using a Nanodrop nucleic acid concentration analyzer, and the integrity of dsRNA was detected by agarose gel electrophoresis. The dsRNA was named dsLvCaspase-znf. dsGFP or dsRNA with irrelevant sequences was used as a negative control.

[0035] Healthy Litopenaeus vannamei were randomly divided into a dsLvCaspase-znf group, a dsGFP negative control group, and a PBS control group. Each Litopenaeus vannamei was injected with 1 μg dsRNA / g according to its body weight. Gill and intestinal tissues were collected 48 h after injection, and the expression level of LvCaspase-znf was detected by qRT-PCR. The nucleotide sequences of the primer pairs used are shown in SEQ ID NO.10 and SEQ ID NO.11.

[0036] The results are as follows Figure 5 As shown, compared with the dsGFP group, the expression level of LvCaspase-znf in the dsLvCaspase-znf group was significantly reduced, with a knockdown efficiency of 60.1-61.0% and P values ​​less than 0.05.

[0037] 4. Detection of WSSV challenge mortality rate after knockdown of the target gene Forty-eight hours after dsRNA injection, Litopenaeus vannamei in each group was challenged with WSSV. The shrimp were observed for eight consecutive days post-challenge, and the number of dead shrimp in each group was recorded at 0h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h, and the cumulative survival rate was calculated.

[0038] The results are as follows Figure 6As shown, compared with the dsGFP+WSSV negative control group, the cumulative mortality rate of the dsLvCaspase-znf+WSSV group was significantly reduced. Specifically, on day 8 post-challenge, the cumulative mortality rate of the dsLvCaspase-znf+WSSV group was approximately 40%, while that of the dsGFP+WSSV group was approximately 80%; the PBS control group showed virtually no mortality, with a cumulative mortality rate of approximately 0%. These results indicate that knocking down the LvCaspase-znf gene can reduce the mortality rate of Litopenaeus vannamei after WSSV infection.

[0039] The above results indicate that knocking down LvCaspase-znf can reduce the mortality rate of Litopenaeus vannamei after WSSV infection and improve the tolerance or resistance of Litopenaeus vannamei to WSSV infection.

[0040] 5. Detection of WSSV viral load and viral gene expression like Figure 7 As shown, compared with the dsGFP negative control group, the WSSV virus copy number in the hepatopancreas, intestine, and gill tissues of Litopenaeus vannamei in the dsLvCaspase-znf group was significantly reduced. Among them, the viral load in the dsGFP group was around 10... 6 The viral load was on the order of 0.1 μg DNA, while in the dsLvCaspase-znf group it was reduced to 10. 3- 10 4 The copy number was on the order of 0.1 μg DNA. These results indicate that knocking down the LvCaspase-znf gene significantly inhibited the accumulation of WSSV in Litopenaeus vannamei.

[0041] The above results indicate that the LvCaspase-znf gene can be used as a target to screen for candidate substances that can effectively control the WSSV infection process, reduce viral proliferation, and improve post-infection survival.

[0042] 6. Detection of the nucleic acid binding capacity of the target protein To verify the nucleic acid binding capacity of the LvCaspase-znf protein in cellular states, the LvCaspase-znf gene constructed in the vector pcDNA3.1-N-HA was transfected into 293T cells, with an empty vector plasmid HA used as a negative control. Biotin-labeled nucleic acid pull-down and competitive pull-down experiments were employed to detect their binding capacity to DNA or RNA. Biotin-labeled nucleic acid pull-down experiment: Biotin-labeled ISD double-stranded DNA (formed by annealing oligonucleotides shown in SEQ ID NO.12 and SEQ ID NO.13, with a biotin-labeled 5' end), biotin-labeled poly(I:C), and biotin-labeled WSSV-derived DNA-like nucleic acid probes (their upstream and downstream sequences are shown in SEQ ID NO.14 and SEQ ID NO.15, respectively, with a biotin-labeled 5' end) were incubated with streptavidin magnetic beads to form nucleic acid-magnetic bead complexes. Cell lysate or purified protein is added to a nucleic acid-magnetic bead complex to capture the binding protein; after washing to remove non-specific binding proteins, the binding protein is eluted, and the target protein is detected by Western blot.

[0043] Competitive pull-down assay: To determine the specificity of binding, an excess of unlabeled competing double-stranded nucleic acids was added to the pull-down system of the biotin-labeled nucleic acid probe. The unlabeled ISD competing double-stranded nucleic acid used the same nucleotide sequence as SEQ ID NO.12 and SEQ ID NO.13 (without biotin labeling); the unlabeled WSSV competing double-stranded nucleic acid used the same nucleotide sequence as SEQ ID NO.14 and SEQ ID NO.15 (without biotin labeling). Western blot analysis showed that if the unlabeled homologous nucleic acid significantly reduced the amount of target protein pulled down, the binding was considered specific.

[0044] like Figure 8 The results showed that biotin-labeled WSSV-derived dsDNA could bind to LvCaspase-znf-HA protein. After adding 10 times the amount of unlabeled WSSV-derived dsDNA, the binding signal was significantly weakened, indicating that LvCaspase-znf can specifically or relatively specifically bind to WSSV-derived nucleic acid fragments. Furthermore, domain analysis showed that the zinc finger domain fragment could bind to nucleic acids, while the absence of the zinc finger domain significantly weakened or almost eliminated the nucleic acid binding signal of LvCaspase-znf, indicating that the zinc finger domain is an important domain mediating its nucleic acid binding.

[0045] The above results indicate that the LvCaspase-znf protein possesses nucleic acid binding capabilities, specifically binding to WSSV-derived dsDNA fragments, immunostimulatory DNA (ISD), and RNA (Poly(I:C)). These results suggest that the LvCaspase-znf protein may participate in the immune response or pathological process of Litopenaeus vannamei to WSSV infection by recognizing WSSV-derived nucleic acid molecules.

[0046] 7. Detection of self-interactions or oligomerization of the target protein To investigate whether the LvCaspase-znf encoded protein possesses self-interacting or oligomerizing capabilities, the cysteine ​​(C) at position 536 of the LvCaspase-znf protein was replaced with alanine (A) to express and purify the MBP-LvCaspase-znf (C536A) fusion protein, with MBP protein serving as a negative control. Validation was performed using chemical cross-linking combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and non-denaturing polyacrylamide gel electrophoresis. Using the purified MBP-LvCaspase-znf (C536A) protein as material, protein migration was assessed under conditions of no nucleic acid, ISD, Poly(I:C), or n-dodecyl-β-D-maltose treatment.

[0047] like Figure 9 As shown, chemical cross-linking combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiment: without the addition of cross-linking agent, the main detected band was the MBP-LvCaspase-znf (C536A) monomer band; after the addition of cross-linking agent BS3 or DSS, under the condition of ISD or Poly(I:C), obvious MBP-LvCaspase-znf (C536A) oligomer band appeared in the high molecular weight region, and obvious oligomer or polymer complex signal could be detected, indicating that LvCaspase-znf has oligomerization ability under nucleic acid binding conditions.

[0048] Non-denaturing polyacrylamide gel electrophoresis: After the addition of dodecyl-β-D-maltose, the results showed that MBP-LvCaspase-znf (C536A) protein could form high-molecular-weight migration bands or diffuse complex signals after stimulation with nucleic acid ISD or Poly(I:C). These results indicate that MBP-LvCaspase-znf (C536A) protein possesses self-interacting capabilities after incubation with ISD or Poly(I:C) nucleic acids and can form oligomers or high-molecular-weight complexes. Combined with the reduced WSSV infection mortality rate after RNAi knockdown, this suggests that LvCaspase-znf may participate in the lethal process of WSSV infection through nucleic acid recognition and protein oligomerization.

[0049] 8. Target gene-based detection kits This embodiment provides a kit for detecting LvCaspase-znf expression levels, including qRT-PCR primers, reverse transcription reagents, quantitative real-time PCR reagents, and a positive control; The primers include upstream primer SEQ ID NO.10 and downstream primer SEQ ID NO.11. This kit can be used to detect changes in LvCaspase-znf expression in Litopenaeus vannamei during WSSV infection, thereby aiding in the evaluation of the WSSV infection response status of Litopenaeus vannamei or the efficacy of candidate antiviral regulators.

[0050] Transcriptome analysis of growth-related metabolic states after dsLvCaspase-znf treatment To evaluate the effects of dsLvCaspase-znf treatment on growth-related molecular states in Litopenaeus vannamei, four groups were established: dsGFP untreated group, dsLvCaspase-znf untreated group, dsGFP+WSSV challenged for 24 h group, dsLvCaspase-znf+WSSV challenged for 24 h group, dsGFP+WSSV challenged for 48 h group, and dsLvCaspase-znf+WSSV challenged for 48 h group. Gill tissue was collected from each group, total RNA was extracted, libraries were constructed, and transcriptome sequencing was performed.

[0051] Differential expression analysis, GO enrichment analysis, KEGG enrichment analysis, and GSEA multi-pathway enrichment analysis were performed on the sequencing results, focusing on nutrient digestion and absorption, carbohydrate metabolism, lipid metabolism, amino acid metabolism, nucleotide metabolism, protein synthesis or processing, molting and chitin metabolism, muscle structure, cytoskeleton, extracellular matrix, cell junctions, mineral absorption, and PI3K-Akt, Ras, and MAPK signaling pathways.

[0052] The results are as follows Figure 10 , Figure 11 , Figure 12As shown, this study investigated the effect of dsRNA injection of the LvCaspase-znf gene on growth-related molecular states in Litopenaeus vannamei. In this embodiment, the dsGFP treatment group served as a control. GO enrichment analysis, KEGG enrichment analysis, and differential gene clustering heatmap analysis were performed on differentially expressed genes between the dsGFP and dsLvCaspase-znf groups at 0 h, 24 h, and 48 h. The results showed that at 0 h, KEGG enrichment analysis revealed that differentially expressed genes were mainly enriched in endoplasmic reticulum protein processing pathways, protein export pathways, protein digestion and absorption pathways, mineral absorption pathways, extracellular matrix-receptor interaction pathways, PI3K-Akt signaling pathways, PPAR signaling pathways, cholesterol metabolism pathways, and glycerophospholipid metabolism pathways. GO functional analysis showed that differentially expressed genes were involved in endopeptidase inhibition activity, endopeptidase regulatory activity, metal ion transmembrane transport activity, calcium / sodium ion antitransport activity, threonine metabolism, α-amino acid catabolism, and negative regulation of proteolysis. The above results indicate that the dsRNA described in this invention can regulate protein processing and transport, amino acid metabolism, mineral ion transport, lipid metabolism, extracellular matrix maintenance, and cell survival-related signaling processes in Litopenaeus vannamei under basal conditions, which is beneficial for maintaining protein homeostasis, tissue structure stability, and mineral balance required for normal shrimp growth.

[0053] Further analysis revealed that, at 24 h, KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in fructose and mannose metabolic pathways, pentose and glucuronic acid interconversion pathways, glycolysis / gluconeogenesis pathways, galactose metabolic pathways, glycerol lipid metabolic pathways, linoleic acid metabolic pathways, arachidonic acid metabolic pathways, purine metabolic pathways, folic acid biosynthesis pathways, vitamin B6 metabolic pathways, amino sugar and nucleotide sugar metabolic pathways, fat digestion and absorption pathways, and carbohydrate digestion and absorption pathways. GO functional analysis showed that differentially expressed genes were involved in carbohydrate metabolism, small molecule metabolism, de novo biosynthesis of pyrimidine nucleobases, hexosaminease activity, β-N-acetylhexosaminease activity, hydrolases acting on carbon-nitrogen bonds (excluding peptide bonds), pyridoxal phosphate binding, and vitamin B6 binding. The above results indicate that the dsRNA described in this invention can regulate carbohydrate metabolism, lipid metabolism, nucleotide synthesis, vitamin cofactor metabolism, glycan metabolism, and nutrient digestion and absorption in Litopenaeus vannamei. These processes are closely related to shrimp energy supply, nutrient utilization, lipid reserves, nucleic acid synthesis, cell proliferation, glycan remodeling, chitin-related metabolism, molting, and exoskeleton renewal. This suggests that the dsRNA described in this invention helps improve the growth-related metabolic state of shrimp under pathogen stress.

[0054] By 48 h, KEGG enrichment analysis showed that the differentially expressed genes were further enriched in cytochrome P450-mediated exogenous substance metabolism pathways, drug metabolism-cytochrome P450 pathways, steroid hormone biosynthesis pathways, retinol metabolism pathways, ascorbic acid and aldonic acid metabolism pathways, folic acid biosynthesis pathways, folic acid-mediated one-carbon metabolism pathways, sphingolipid metabolism pathways, lysosomal pathways, peroxisome pathways, pancreatic secretion pathways, and mineral uptake pathways. GO functional analysis showed that the differentially expressed genes were involved in catalytic activity, oxidoreductase activity, hydrolase activity, monooxygenase activity, peptidase activity, glycosidic hydrolase activity, carbohydrate metabolism, lipid metabolism, organic acid metabolism, and carboxylic acid metabolism. These results indicate that the dsRNA described in this invention can regulate detoxification metabolism, redox reactions, glycan degradation, lipid remodeling, lysosomal function, peroxisome function, and mineral homeostasis in Litopenaeus vannamei during the later stages of infection or stress, thereby helping to mitigate the adverse effects of pathogen stress on tissue structure and growth metabolism.

[0055] Further analysis of the differential gene clustering heatmap revealed differences in gene expression patterns between the dsGFP control group and the dsLvCaspase-znf treatment group at different time points. The differentially expressed genes could be clustered into multiple expression modules, with related gene clusters primarily involving endoplasmic reticulum protein processing pathways, protein export pathways, mineral uptake pathways, pyrimidine metabolism pathways, lysine degradation pathways, pentose and glucuronide interconversion pathways, peroxisome pathways, lysosome pathways, pancreatic secretion pathways, cytochrome P450-mediated exogenous substance metabolism pathways, steroid hormone biosynthesis pathways, extracellular matrix-receptor interaction pathways, tight junction pathways, actin cytoskeleton regulation pathways, PI3K-Akt signaling pathways, Ras signaling pathways, and the Drosophila MAPK signaling pathway. These pathways are closely related to growth-related processes such as protein synthesis and processing, nutrient absorption, carbohydrate metabolism, lipid metabolism, amino acid metabolism, nucleotide metabolism, mineral uptake, extracellular matrix maintenance, cell junctions, cytoskeleton regulation, cell proliferation, tissue repair, molting, and exoskeleton formation. Therefore, the dsRNA described in this invention can regulate the molecular networks related to nutrient metabolism, energy metabolism, protein processing, glycan metabolism, mineral absorption, and tissue structure maintenance in Litopenaeus vannamei under WSSV stress by targeting and silencing the LvCaspase-znf gene. This reduces the adverse effects of pathogen infection on growth-related metabolic processes in shrimp, thereby helping to maintain normal growth, tissue homeostasis, and aquaculture adaptability of Litopenaeus vannamei under infection stress.

[0056] The above results indicate that dsLvCaspase-znf treatment can not only reduce the mortality rate after WSSV infection, but also mitigate the adverse effects of virus infection on the growth-related metabolic state and tissue homeostasis of Litopenaeus vannamei under WSSV infection conditions, thus providing a molecular basis for maintaining aquaculture production performance and reducing aquaculture losses during the infection period.

Claims

1. The application of an LvCaspase-znf gene / protein expression inhibitor in the preparation of a formulation for improving the resistance of Litopenaeus vannamei to WSSV infection or reducing the mortality rate of Litopenaeus vannamei after WSSV infection, characterized in that, The inhibitor is a dsRNA that targets the nucleotide fragment shown in SEQ ID NO.5; The nucleotide sequence of the LvCaspase-znf gene is shown in SEQ ID NO.

3.

2. The application according to claim 1, characterized in that, The formulation includes feed additives, injectable formulations, or liquid formulations for soaking.

3. The application according to claim 2, characterized in that, The injectable formulation comprises the dsRNA of claim 1 and an aquaculture-acceptable injectable carrier or diluent.

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