Application of insect mitochondrial protein COX5B in insect-borne virus infection resistance

Abstract

The invention discloses application of insect COX5B protein or related biological materials thereof in preparation of products for resisting black-streaked dwarf virus infection and products for controlling insects to spread black-streaked dwarf viruses. The invention also discloses an application of the specific antibody of the insect COX5B protein in detection of black-streaked dwarf virus in an insect body. The invention also discloses a method for controlling transmission of southern rice black-streaked dwarf virus by sogatella furcifera. It is found that the sogatella furcifera COX5B protein can inhibit acquisition and copying of SRBSDV, so that accumulation of the SRBSDV is inhibited, host survival is enhanced, the COX5B protein can inhibit propagation of the SRBSDV in sogatella furcifera, and the COX5B protein can be used for preparing products for resisting black-streaked dwarf virus infection and products for controlling propagation of the black-streaked dwarf virus by insects so as to improve the black-streaked dwarf virus resistance of crops; the kit can also be used for detecting black-streaked dwarf viruses in insect bodies.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of an insect mitochondrial protein, COX5B, in combating insect-borne virus infections. Background Technology

[0002] Cytochrome c oxidase subunit 5B (COX5B) is a terminal enzyme in the respiratory chain, located on the inner mitochondrial membrane. Cytochrome c oxidase is the terminal oxidase of the mitochondrial respiratory chain, catalyzing the transfer of electrons from cytochrome c cells to oxygen molecules, producing water and ATP. The main function of COX5B protein is to stabilize the structure of cytochrome c oxidase and participate in the catalytic reaction. Numerous studies have been conducted on COX5B protein, but most have focused on mammals. Due to evolutionary differences between vertebrates and invertebrates, our understanding of the role of COX5B protein in insect vectors remains limited.

[0003] Rice viral diseases severely restrict the yield and quality of crops worldwide. Southern rice black-streaked dwarf virus (SRBSDV), causing large-scale outbreaks, can lead to yield reductions of up to 90% or even total crop failure, making it the most significant viral disease affecting rice production in southern my country and the Indochina Peninsula over the past decade. SRBSDV is transmitted by the migratory planthopper *Sogatella frucifera* via a persistent, proliferative pathway. Once inside the planthopper, the virus interacts with various proteins within the vector, overcoming transmission barriers. Therefore, studying the interactions between virus-encoded proteins and insect vector proteins not only holds promise for elucidating the molecular mechanisms of viral infection, replication, and spread within the vector insect but will also provide important target molecules for effective virus control. Summary of the Invention

[0004] Based on the technical problems existing in the background art, this invention proposes the application of insect mitochondrial protein COX5B in resistance to insect-borne virus infection. This invention discovers that the COX5B protein of the white-backed planthopper can inhibit the acquisition and replication of SRBSDV, thereby inhibiting the accumulation of SRBSDV and enhancing host survival. Furthermore, the COX5B protein can inhibit the spread of SRBSDV in the white-backed planthopper. It can be used to prepare products for resistance to black-streaked dwarf virus infection, products for controlling the spread of black-streaked dwarf virus by insects, thereby improving the resistance of crops to black-streaked dwarf virus; it can also be used to detect black-streaked dwarf virus in insects.

[0005] This invention proposes the application of an insect COX5B protein or related biomaterials in the preparation of products resistant to black-streaked dwarf virus infection and products for controlling the spread of black-streaked dwarf virus by insects;

[0006] Among them, insect COX5B protein-related biological materials include at least one of the following: insect COX5B protein agonists, expression cassettes containing insect COX5B protein encoding genes, recombinant vectors containing insect COX5B protein encoding genes, transgenic cell lines containing insect COX5B protein encoding genes, transgenic plants containing insect COX5B protein encoding genes, transgenic microorganisms containing insect COX5B protein encoding genes, and recombinant proteins containing insect COX5B protein encoding genes.

[0007] The aforementioned products for combating Black-streaked Dwarf Virus infection refer to products that resist or inhibit Black-streaked Dwarf Virus infection.

[0008] The aforementioned insect COX5B protein encoding gene can be an insect COX5B protein encoding gene or its active fragment, or a encoding gene with ≥80% homology and possessing the function and activity of insect COX5B protein.

[0009] Their homology can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0010] The aforementioned recombinant vectors can be plasmids, rods, etc.

[0011] The aforementioned transgenic cell lines can be transgenic insect cell lines, etc.

[0012] The aforementioned genetically modified plants can include genetically modified rice, etc.

[0013] The aforementioned genetically modified microorganisms can be genetically modified Escherichia coli, yeast, etc.

[0014] The aforementioned biomaterials can be prepared using conventional methods in this field.

[0015] Preferably, the insect is the white-backed planthopper.

[0016] The inventors performed gene sequencing on the COX5B protein of the white-backed planthopper and determined that the amino acid sequence of the COX5B protein of the white-backed planthopper is shown in SEQ ID NO: 1, and the DNA sequence of the COX5B protein of the white-backed planthopper is shown in SEQ ID NO: 2.

[0017] Preferably, the black-streaked dwarf virus is the Southern Rice Black-Streaked Dwarf Virus.

[0018] Southern rice black-streaked dwarf virus (SRBSDV) belongs to the Fijivirus genus. Its genome contains 10 double-stranded RNA segments, and 13 ORFs have been annotated so far. It is speculated that at least 6 structural proteins (P1, P2, P3, P4, P8, P10) and 5 non-structural proteins (P6, P7-1, P7-2, P9-1, P9-2) are included. The functions of most proteins are known, but the functions of some proteins are still unknown. For example, the S5 segment of SRBSDV encodes two proteins, P5-1 and P5-2. P5-1 is a major component of the fibrillary region of the viral protoplasm and interacts with the P6 protein encoded by S6 in the assembly process of viral particles. The function of the P5-2 protein has not yet been reported.

[0019] This invention also proposes the application of a specific antibody against insect COX5B protein in the detection of black-streaked dwarf virus in insects.

[0020] Specific antibodies against the aforementioned insect COX5B protein can be labeled and detected, for example, by labeling with detectable markers or enzymes that produce detectable markers. These detectable markers include: radioactive isotopes for detection, luminescent substances, colored substances, colloidal gold markers, polyethylene glycol, streptavidin, or biotin, etc.; the luminescent substances can be fluorescent agents (such as FITC), etc.

[0021] Specific antibodies against insect COX5B protein can be prepared using conventional methods in this field.

[0022] Preferably, the insect is the white-backed planthopper.

[0023] Preferably, the black-streaked dwarf virus is the Southern Rice Black-Streaked Dwarf Virus.

[0024] This invention also proposes a method for controlling the transmission of Southern Rice Black-Streaked Dwarf Virus by the white-backed planthopper. This method enhances the antiviral ability of the white-backed planthopper by spraying or injecting it with the COX5B protein or related biological materials, thereby blocking the transmission chain of Southern Rice Black-Streaked Dwarf Virus.

[0025] Among them, insect COX5B protein-related biological materials include at least one of the following: insect COX5B protein agonists and recombinant proteins containing the insect COX5B protein encoding gene;

[0026] Alternatively, genetic engineering techniques can be used to cultivate transgenic crops that overexpress the COX5B protein of the white-backed planthopper, thereby enhancing the crop's resistance to viral infections.

[0027] The aforementioned crops can include rice, etc.

[0028] This invention reveals that COX5B protein expression is significantly upregulated in white-backed planthoppers infected with SRBSDV, particularly in the midgut and salivary glands, key tissues for viral invasion and transmission. Furthermore, COX5B protein accumulates extensively in the intestinal tissue and exhibits significant co-localization with the SRBSDV viral protein P10. This indicates that COX5B protein actively participates in host defense mechanisms at the SRBSDV replication site, and that COX5B protein in white-backed planthoppers is upregulated and inhibits viral proliferation during SRBSDV replication.

[0029] Further research in this invention revealed that the COX5B protein is a key anti-SRBSDV factor in the white-backed planthopper. The COX5B protein inhibits the acquisition and replication of SRBSDV, thereby inhibiting the accumulation of SRBSDV and enhancing host survival. Furthermore, the COX5B protein can inhibit the spread of SRBSDV in the white-backed planthopper.

[0030] It was found that the COX5B protein can inhibit the massive replication of SRBSDV in the midgut of the white-backed planthopper, thereby reducing the viral load transferred to the salivary glands and thus blocking the spread of the virus to the plant host; the upregulation of the COX5B protein exacerbates mitochondrial damage, thereby activating autophagy and clearing the virus through selective degradation.

[0031] COX5B protein or related biomaterials can be used to prepare products for treating black-streaked dwarf virus infection and products for controlling the spread of black-streaked dwarf virus in insects, thereby improving the resistance of insects and crops to black-streaked dwarf virus. This has broad application prospects in pesticide production. Furthermore, specific antibodies against insect COX5B protein can also be used to detect black-streaked dwarf virus in insects. Attached Figure Description

[0032] Figure 1 The results are from Example 1, where A is the RT-qPCR analysis result, B is the Western blot detection result, C is the COX5B protein detection result in the midgut and salivary glands of the white-backed planthopper, D is the immunofluorescence imaging detection result, V and Viruliferous are the infection group, NV and Non-Viruliferous are the control group, Midgut is the midgut, and Salvary glands are the salivary glands.

[0033] Figure 2The results are for Example 2. In this example, A represents the RT-qPCR analysis results of the infected group, B represents the Western blot detection results of the SRBSDV viral capsid protein P10 in the infected group, C represents the survival rate of white-backed planthoppers in each group, D represents the positive rate of rice seedling virus in the dsCOX5B and dsGFP groups in the infected group, E represents the immunofluorescence imaging detection results of the midgut tissue in the dsCOX5B and dsGFP groups in the infected group, and NV represents the uninfected group.

[0034] Figure 3 AF represents the transmission electron microscopy results of Sf9 cells in each group. Among them, Mito refers to Mitochondria, AL refers to autolysosomes, AP refers to autophagosomes, Damaged mitochondrion refers to damaged mitochondria, blue arrows indicate mitochondria, and white arrows indicate autophagosome-like vesicles and lysosomes.

[0035] Figure 3 GP represents transmission electron micrographs of midgut epithelial cells from infected and uninfected white-backed planthoppers. G represents midgut cells from the uninfected group, HJ represents midgut cells from the infected group, K represents the results of immunogold labeling with ATG8 antibody, and LP represents the results of immunogold labeling with COX5B antibody. Detailed Implementation

[0036] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0037] Example 1

[0038] To investigate the effect of COX5B protein on SRBSDV replication in white-backed planthoppers, this invention infected white-backed planthoppers with SRBSDV virus (there were 10 treatment groups, each with 10 white-backed planthoppers, and uninfected white-backed planthoppers were used as the control group). The transcription and protein levels of COX5B protein in white-backed planthoppers during the infection period were periodically detected by RT-qPCR, Western blot experiment, and immunofluorescence microscopy.

[0039] The specific detection steps of the RT-qPCR method include: To quantify gene expression levels, total RNA was extracted from 10 treatment groups (10 planthoppers per group), and RT-qPCR was performed using ToloScript All-in-one RT EasyMix (TOLOBIO, 22107). Tubulin transcripts from the white-backed planthopper were used as internal controls, and the RNA was analyzed using a 2-step PCR method. -ΔΔCT The relative expression levels of genes were calculated using a method. Primers for each substance are shown in Table 1.

[0040] Table 1 Primers for each substance in the RT-qPCR method

[0041]

[0042] Western blot experiments used COX5B, SRBSDV-P10, and SRBSDV-P5-2 specific IgG as primary antibodies, and horseradish peroxidase-labeled goat anti-rabbit / mouse IgG (Sangon Biotech, D110058) as secondary antibody. The COX5B specific IgG was a rabbit-derived polyclonal antibody prepared by GenScript Biotech in Nanjing; the SRBSDV-P5-2 specific IgG was a rabbit-derived polyclonal antibody prepared by Sinocare Biotech in Beijing; and the SRBSDV-P10 specific IgG was a mouse-derived monoclonal antibody prepared by the Chinese Academy of Agricultural Sciences in Beijing.

[0043] The specific steps of immunofluorescence microscopy include:

[0044] To locate COX5B protein expression in the midgut of the white-backed planthopper, the midgut tissue was first dissected and fixed in 4% paraformaldehyde-PBS solution (Coolaber, PM5090) for 2 h, followed by permeabilization with 2% Triton X-100 (Vetec, V900502) for 0.5 h. Immunostaining was then performed using FITC-labeled COX5B-specific IgG (COX5B-FITC) and TRITC / Alexa Fluor-labeled SRBSDV-P10 and SRBSDV-P5-2-specific IgG (P10-TRITC / P5-2-647). Since both P5-2 and COX5B-specific IgG are rabbit-derived polyclonal antibodies, a stepwise labeling strategy was employed: first, incubation with P5-2-specific IgG (primary antibody), followed by binding with a fluorescent secondary antibody, then blocking non-specific sites with rabbit serum, and finally, addition of FITC-labeled COX5B-specific IgG followed by DAPI nuclear staining for fluorescence observation.

[0045] The results are as follows Figure 1 As shown.

[0046] Figure 1 The results are from Example 1, where A is the RT-qPCR analysis result, B is the Western blot detection result, C is the COX5B protein detection result in the midgut and salivary glands of the white-backed planthopper, D is the immunofluorescence imaging detection result, V and Viruliferous are the infection group, NV and Non-Viruliferous are the control group, Midgut is the midgut, and Salvary glands are the salivary glands.

[0047] Depend on Figure 1The results show that: RT-qPCR analysis revealed significant upregulation of COX5B mRNA at 2, 3, 5, 7, and 8 days post-infection; Western blot confirmed a significant increase in COX5B protein at these times; tissue-specific expression analysis showed significant upregulation of COX5B expression in key tissues for viral invasion and transmission (midgut and salivary glands); and immunofluorescence imaging further revealed a large accumulation of COX5B protein in the intestinal tissue of SRBSDV-infected white-backed planthoppers, with significant co-localization with the SRBSDV viral protein P10. These results demonstrate that SRBSDV infection can induce increased transcription and translation levels of COX5B, and that COX5B protein actively participates in host defense mechanisms and inhibits viral replication at the SRBSDV replication site.

[0048] Example 2

[0049] RNAi interference effect of dsRNA on viral infection

[0050] A T7 promoter (5'-TAATACGACTCACTATAGGG-3') was added to the 5' end of the specific sequence of the COX5B gene (i.e., 89-294 bp as shown in SEQ ID NO: 2), and T7 RiboMAX Express was used. The RNAi system (Promega, P1700) synthesized dsRNA (dsCOX5B and dsGFP were obtained, with dsGFP serving as a negative control). dsRNA (approximately 20 ng / bird) was injected into the intersegmental thorax of 2nd-3rd instar white-backed planthopper nymphs. These nymphs were then fed SRBSDV-infected rice for 2 days to obtain the virus, and then transferred to healthy rice for 5 days, designated as the infected group (6 white-backed planthoppers per group). Similarly, dsRNA (approximately 20 ng / bird) was injected into the intersegmental thorax of 2nd-3rd instar white-backed planthopper nymphs, and these nymphs were transferred to healthy rice for 7 days, designated as the uninfected group (6 white-backed planthoppers per group). RNA was then extracted from 6 white-backed planthoppers from each group (10 replicates), and gene expression was detected by RT-qPCR (tubulin was used as an internal control, and the expression level of the dsGFP group was set to 1).

[0051] For protein level detection, 30 worms were collected from each group, and the protein concentration was adjusted to 1-2 μg / μL before Western blot analysis and immunofluorescence microscopy detection. Results are as follows: Figure 2 As shown.

[0052] Figure 2The results are for Example 2. In this example, A represents the RT-qPCR analysis results of the infected group, B represents the Western blot detection results of the SRBSDV viral capsid protein P10 in the infected group, C represents the survival rate of white-backed planthoppers in each group, D represents the positive rate of rice seedling virus in the dsCOX5B and dsGFP groups in the infected group, E represents the immunofluorescence imaging detection results of the midgut tissue in the dsCOX5B and dsGFP groups in the infected group, and NV represents the uninfected group.

[0053] Depend on Figure 2 The results of AB show that knocking down COX5B expression in third-instar nymphs via RNAi significantly promotes SRBSDV replication. The RNA expression of P9-1 and P5-2 of the SRBSDV virus is increased, and the capsid protein P10 of the SRBSDV virus is significantly increased. This indicates that COX5B has an inhibitory effect on SRBSDV virus replication.

[0054] Depend on Figure 2 As can be seen from C, in the uninfected group, there was no significant difference in survival rate between the dsCOX5B group and the dsGFP group, indicating that COX5B is not essential for the basic survival of insects; however, in the infected group, the survival rate of white-backed planthoppers in the dsCOX5B group was significantly reduced, and the individual mortality rate was significantly increased; indicating that COX5B can inhibit SRBSDV replication and promote the survival of infected hosts.

[0055] Depend on Figure 2 As can be seen from D, RT-PCR detection showed that the positive rate of rice seedling virus in the dsCOX5B group (60.1%) was significantly higher than that in the dsGFP group (45.7%), indicating that silencing COX5B would improve the virus transmission efficiency of infected white-backed planthoppers.

[0056] Depend on Figure 2 E shows that in the infection group, the viral particles in the dsGFP group were limited to only some midgut epithelial cells, while in the dsCOX5B group, the virus accumulated extensively throughout the midgut.

[0057] These results confirm that the COX5B protein is a key anti-SRBSDV factor in the white-backed planthopper. The COX5B protein inhibits the acquisition and replication of SRBSDV, thereby inhibiting the accumulation of SRBSDV and enhancing host survival. Furthermore, the COX5B protein can inhibit the spread of SRBSDV in the white-backed planthopper.

[0058] Example 3

[0059] Sf9 cells were transfected with a vector to obtain three groups: Sf9 cells overexpressing COX5B protein (COX5B group), Sf9 cells overexpressing SRBSDV P5-2 protein (P5-2 group), and Sf9 cells overexpressing both COX5B and SRBSDV P5-2 protein (P5-2+COX5B group). Sf9 cells transfected with an empty vector (EV group) and untransfected Sf9 cells (WT group) served as controls. Transmission electron microscopy was performed to observe mitochondrial ultrastructure and autophagy-related features. Results are as follows: Figure 3 As shown in AF.

[0060] Figure 3 AF represents the transmission electron microscopy results of Sf9 cells in each group. Among them, Mito refers to Mitochondria, AL refers to autolysosomes, AP refers to autophagosomes, Damaged mitochondrion refers to damaged mitochondria, blue arrows indicate mitochondria, and white arrows indicate autophagosome-like vesicles and lysosomes.

[0061] Depend on Figure 3 AF showed that, compared with control cells (i.e., EV group or WT group), Sf9 cells overexpressing COX5B protein and overexpressing SRBSDV P5-2 protein exhibited changes in mitochondrial morphology and autophagy, and mitochondria were damaged, with COX5B overexpression alone causing the most severe mitochondrial damage.

[0062] Electron microscopy was performed on midgut epithelial cells of white-backed planthoppers infected with SRBSDV for 7 days and uninfected white-backed planthoppers to observe mitochondrial integrity and autophagy markers. The specific steps are as follows:

[0063] Midgut tissue from white-backed planthoppers of each group was collected, fixed, dehydrated, and embedded. Ultrathin sections were observed using a Tecnai T12 transmission electron microscope (120 kV, equipped with an Eagle CCD and CryoBox). Immunoelectron microscopy experiments used COX5B-specific IgG as the primary antibody and 15 nm gold-labeled goat anti-rabbit IgG (Sangon Biotech, D111094) as the secondary antibody. Results are as follows: Figure 3 As shown in GP.

[0064] Figure 3 GP represents transmission electron micrographs of midgut epithelial cells from infected and uninfected white-backed planthoppers. G represents midgut cells from the uninfected group, HJ represents midgut cells from the infected group, K represents the results of immunogold labeling with ATG8 antibody, and LP represents the results of immunogold labeling with COX5B antibody.

[0065] Figure 3In GP, ​​Mito refers to Mitochondria, AL refers to autolysosomes, AP refers to autophagosomes, Damaged mitochondrion refers to damaged mitochondria, Vi or Virus refers to SRBSDV virus, blue arrows refer to mitochondria, red arrows refer to virus particles, orange arrows refer to ATG8, black arrows refer to COX5B, white arrows refer to autophagosome-like vesicles and lysosomes, and the selected area is enlarged on the right.

[0066] Depend on Figure 3 G shows intact mitochondria in the midgut cells of the uninfected group of white-backed planthoppers.

[0067] Depend on Figure 3 HJ observed damaged mitochondria, lysosomes, and autophagosomes near SRBSDV virus particles in the midgut cells of the infected white-backed planthopper.

[0068] Depend on Figure 3 As can be seen from K, immunogold labeling with ATG8 antibody confirmed the existence of double-membrane autophagosomes.

[0069] Depend on Figure 3 M shows that COX5B is in a healthy mitochondrial distribution state in intestinal cells in the uninfected group.

[0070] Depend on Figure 3 As observed in L and NP, in the intestinal cells of the infected group, COX5B antibody markers were located in mitochondria and autophagosome-like vesicles containing mitochondrial remnants, and SRBSDV viral particles were adjacent to or encapsulated within damaged mitochondria.

[0071] Depend on Figure 3 It can be seen that in the uninfected group, the mitochondria of the white-backed planthopper exhibited normal ultrastructure, while the infected group showed widespread mitochondrial abnormalities, autophagic vesicles, and a large accumulation of lysosomes.

[0072] Notably, in the infected group, many lysosomes and autophagosomes contained both mitochondrial fragments and viral particles. Immunogold labeling further revealed that COX5B protein and the autophagy marker protein ATG8 were specifically enriched on damaged mitochondria encapsulated by autophagosomes. These findings suggest that upregulation of COX5B protein exacerbates mitochondrial damage, thereby activating autophagy and achieving clearance through selective viral degradation.

[0073] All quantitative data in Examples 1-3 above were analyzed by two-tailed t-test using GraphPad Prism 9.5 (GraphPad Software, USA).

[0074] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of an insect COX5B protein or related biological material in the preparation of products for preventing black-streaked dwarf virus infection and in the preparation of products for controlling the spread of black-streaked dwarf virus by insects; in, Insect COX5B protein-related biological materials include at least one of the following: insect COX5B protein agonists, expression cassettes containing insect COX5B protein encoding genes, recombinant vectors containing insect COX5B protein encoding genes, transgenic cell lines containing insect COX5B protein encoding genes, transgenic plants containing insect COX5B protein encoding genes, transgenic microorganisms containing insect COX5B protein encoding genes, and recombinant proteins containing insect COX5B protein encoding genes.

2. The application according to claim 1, characterized in that, The insect is the white-backed planthopper.

3. The application according to claim 1 or 2, characterized in that, The black-streaked dwarf virus is the Southern Rice Black-Streaked Dwarf Virus.

4. Application of a specific antibody against insect COX5B protein in the detection of black-streaked dwarf virus in insects.

5. The application according to claim 4, characterized in that, The insect is the white-backed planthopper.

6. The application according to claim 4 or 5, characterized in that, The black-streaked dwarf virus is the Southern Rice Black-Streaked Dwarf Virus.

7. A method for controlling the spread of Southern Rice Black-Streaked Dwarf Virus by the white-backed planthopper, characterized in that, By spraying or injecting COX5B protein or related biological materials into the white-backed planthopper, the antiviral ability of the planthopper can be enhanced, thus blocking the transmission chain of Southern Rice Black-Streaked Dwarf Virus. Among them, insect COX5B protein-related biological materials include at least one of the following: insect COX5B protein agonists and recombinant proteins containing the insect COX5B protein encoding gene; Alternatively, genetic engineering techniques can be used to cultivate transgenic crops that overexpress the COX5B protein of the white-backed planthopper, thereby enhancing the crop's resistance to viral infections.

Images