Function and application of virus source tyrosine phosphatase in plutella xylostella male testis development

Abstract

The invention discloses a function and an application of virus source tyrosine phosphatase in development of male testis of plutella xylostella, namely an application of a protein tyrosine phosphatase gene CvBV22-9 coded by cotesia vestalis virus in induction of apoptosis of testis cells of larvae of plutella xylostella. According to the invention, the CvBV22-9 gene is screened and identified as a main factor for inducing testis apoptosis, and the gene is continuously and highly expressed in a parasitized testis tissue and has tissue specificity. After the expression of the CvBV22-9 is knocked down, the testis apoptosis rate is obviously reduced; and about 90.80% of testis cell apoptosis can be induced by using recombinant baculovirus to over-express the gene. The low-concentration recombinant baculovirus can also destroy the testis structure and lead to decrease of the egg laying amount of mating female insects. According to the invention, a transgenic fruit fly model is successfully constructed, specific expression of CvBV22-9 in fruit fly testis is realized by using a UAS-Gal4 system, and 97.30% of testis apoptosis is induced.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for agricultural pests, and provides a multi-dimensional and systematic research system for verifying the phenomenon of castration in insect testes. Specifically, it relates to the function of the protein tyrosine phosphatase gene CvBV_22-9 encoded by the diamondback moth worm virus (CvBV) in inducing apoptosis in the testes of diamondback moth larvae and its application in pest control. Background Technology

[0002] Diamondback moth ( Plutella xylostella Diamondback moth (Prunella vulgaris) is a major lepidopteran pest affecting cruciferous vegetables worldwide. It has a high reproductive rate, a short generation cycle, and is highly susceptible to developing resistance to chemical pesticides. Currently, the control of diamondback moth still heavily relies on chemical pesticides, leading to a series of problems such as environmental pollution, pesticide residues, and pest resurgence. Therefore, developing efficient and green new control strategies has become an urgent need and a major challenge in this field.

[0003] Biological control using parasitic wasps is an important part of green pest control. After parasitizing their hosts, parasitic wasps affect multiple life processes of the diamondback moth, especially immunosuppression and growth and development. Among them, the phenomenon of "parasitic castration," that is, the strategy of parasitic wasps to destroy the reproductive organs of the host and steal its reproductive energy to benefit the development of their own offspring, is a key mechanism for successful parasitism. For example, the research results of He Fan et al. show that parasitism by the diamondback moth parasitoid wasp Cotesiave stalis (Haliday) carrying Bracovirus PDV or the semi-closed ichneumon wasp Diadegma semiclausum Hellén carrying Ichvovirus PDV has a significant inhibitory effect on the spermatogenesis and formation process of diamondback moths of different ages (He Fan, Bai Sufen, Li Xin, et al. Effects of parasitism by two parasitic wasps on the testes of diamondback moths of different ages [J]. Entomological Knowledge, 2010(3):7.). Cotesia vestalisCotesiavestalis bracvirus (CvBV) is a dominant endoparasitic enemy of the diamondback moth. During oviposition, it injects its domesticated endogenous virus into the host, CvBV. Once inside the host, CvBV integrates its genome and expresses numerous virulent genes. Among these, the protein tyrosine phosphatase (PTP) gene family is the most numerous and plays a crucial role in regulating cell signal transduction, proliferation, and apoptosis. However, the specific functions of most CvBV-PTP genes, particularly their role in regulating host reproduction, remain unknown. Previous studies have found that parasitism by *Plutella xylostella* larvae can cause testicular atrophy in its host, the diamondback moth, and CvBV is an important parasitic factor causing this phenomenon (Bai SF, Cai DZ, Li X, Chen XX. Parasitic castration of *Plutella xylostella* larvae induced by polydnaviruses and venom of *Cotesia vestalis* and *Diadegma semiclausum*. Arch Insect Biochem Physiol. 2009 Jan;70(1):30-43. doi: 10.1002 / arch.20279. PMID: 18949808), but its underlying molecular mechanism remains unclear. Furthermore, Yang Yanyan's research showed that injecting a certain concentration of CvBV prolongs the development time of diamondback moth larvae (Yang Yanyan. Effects of *Plutella xylostella* parasitism on the growth, development, and endocrine activity of diamondback moth larvae [D]. Zhejiang University, 2010.). This invention aims to identify the key regulatory genes that cause parasitic castration by parasitic wasps, reveal the molecular mechanism by which CvBV causes castration of the testes of lepidopteran hosts, and develop pest control technologies based on male sterility. Summary of the Invention

[0004] This invention clarifies, through multiple methods including testis size changes and TUNEL apoptosis staining, that parasitic wasps induce male castration in diamondback moths by triggering testis apoptosis. Furthermore, this invention screened a key virulence gene, CvBV_22-9, that induces testis apoptosis in diamondback moths. Using various techniques such as viral particle infection and RNA interference, its important regulatory role in testis castration was identified, and its function was verified to be conserved in the model insect, fruit fly.

[0005] This invention, by comparing the size of the diamondback moth's testes before and after parasitism, found that the testicular area of ​​the diamondback moth larvae was significantly reduced after parasitism. TUNEL staining revealed a large number of fragmented nucleic acids within the testicular tissue. Compared to unparasitized diamondback moths, parasitized larvae showed obvious apoptotic signals in their testes during the mid-3rd instar (3M), late 3rd instar (3L), and early 4th instar (4E) stages, and the proportion of apoptotic individuals increased with increasing instar. Therefore, parasitism can induce castration and apoptosis in the testes of diamondback moth larvae.

[0006] Furthermore, this invention screens key toxic genes that may be involved in regulating parasitic apoptosis. As the largest gene family encoded by BV, CvBV-PTP was analyzed in this study at various time points in the testis tissue after parasitism. The results showed that CvBV_22-9 (nucleotide sequence shown in SEQ ID NO. 3) was highly expressed in the testis at different post-parasitism stages.

[0007] This invention designs specific primers targeting the open reading frame sequence of the CvBV_22-9 gene and synthesizes dsCvBV_22-9 to interfere with the expression of the CvBV_22-9 gene in diamondback moths after parasitism by the diamondback moth *Papilionophora stylosa*. Results showed that the interference efficiency reached 89% 24 hours after injection of dsCvBV_22-9. Dissection of the testes to examine their size and apoptosis revealed that the testes were 17% larger in size and 38% lower in apoptosis compared to the control group, indicating that CvBV_22-9 participates in the parasitism-induced testicular apoptosis process.

[0008] Therefore, this invention provides an application of the protein tyrosine phosphatase gene CvBV_22-9 encoded by the diamondback moth worm *Pteris vittata* virus in inducing apoptosis in the testis cells of diamondback moth larvae.

[0009] Preferably, the nucleotide sequence of the CvBV_22-9 gene comprises the sequence shown in SEQ ID NO. 3 or a functional fragment thereof. The amino acid sequence encoded by the CvBV_22-9 gene is as shown in SEQ ID NO. 4 or a functional fragment thereof.

[0010] Furthermore, the application is achieved by expressing the CvBV_22-9 gene. This expression is accomplished through infection with a baculovirus expression vector.

[0011] Specifically, this invention utilizes a recombinant baculovirus infection system to express the CvBV_22-9 gene in normal diamondback moth larvae, exploring its important induction of testicular apoptosis. Recombinant baculovirus DNA is used via a baculovirus expression system. Further... sf9The recombinant plasmid was expressed in eukaryotic cells and propagated through multiple passages. After gradient centrifugation, a high concentration of recombinant baculovirus NPV-CvBV_22-9 was obtained. Third-instar diamondback moth larvae of uniform size were selected, and each was injected with 1.0 × 10⁻⁶ ppm. 5 Copy. Twenty-four hours after injection, the testes of diamondback moth larvae were dissected to examine testis size and apoptosis staining. The results showed a significant 8% reduction in testis size, and the proportion of apoptotic individuals increased from 23.53% to 90.80%, indicating that CvBV_22-9 expression can effectively induce testis cell apoptosis. Furthermore, the egg production and hatching rate of diamondback moth offspring after low-dose injection of NPV-CvBV_22-9 were examined. The results showed significant changes in testis morphology after NPV-CvBV_22-9 treatment, and a significant 33% reduction in offspring egg production.

[0012] Based on this, the present invention also provides the application of the protein tyrosine phosphatase gene CvBV_22-9 encoded by the diamondback moth worm parasitic wasp virus in the preparation of a formulation for biological control of pests, wherein the formulation achieves population control by inducing male sterility in pests.

[0013] Preferably, the pests include lepidopteran or dipteran pests.

[0014] More preferably, the lepidopteran pest is the diamondback moth, and the dipteran pest is the fruit fly, but not limited thereto.

[0015] Based on this, the present invention successfully constructed a transgenic model capable of specifically expressing and inducing apoptosis in the testes of Drosophila, elucidating the regulatory function of CvBV_22-9 on larval testicular apoptosis. Using molecular cloning technology, the target gene CvBV_22-9 was constructed into the pUASt-attb vector, and G0 generation Drosophila integrating this gene were obtained via embryo microinjection. Through a series of genetic hybridizations (using the UAS-Gal4 system to hybridize the transgenic Drosophila with a testis-specific driver strain (such as Nos-Gal4), a recombinant strain specifically expressing the CvBV_22-9 gene in the larval testes was successfully obtained. Analysis of the testes of late 3rd instar larvae of Drosophila using apoptosis staining techniques showed that in the control group of Drosophila larvae (W... 1118 In the Drosophila testis, the proportion of apoptotic signals was extremely low, only 1.92% (1 / 52). However, in the CvBV_22-9 expression group, the incidence of testicular apoptosis was as high as 97.30% (36 / 37). Expression of the CvBV_22-9 gene in the Drosophila testis strongly induces apoptosis, verifying the conservation of the CvBV_22-9 gene in insect testicular apoptosis.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Verification of parasitism-induced castration in insect testes: This invention, through morphological observation and TUNEL staining (detection of DNA breaks), reveals the dynamic process and severity of apoptosis in diamondback moth testes induced by parasitic wasps. The research conclusions are solid and reliable.

[0017] 2. Discovery and identification of a key gene CvBV_22-9 regulating insect testicular apoptosis: This invention, for the first time, screened and identified the key gene CvBV_22-9, which is continuously highly expressed in testicular tissue, from CvBV tyrosine phosphatase (CvBV-PTP) through spatiotemporal expression profiling and functional studies. It is a core functional factor for inducing testicular castration. Through two reverse genetics methods, namely recombinant baculovirus overexpression (gain-of-function experiment, proving that its expression can directly induce a high proportion of apoptosis), it was confirmed in the original host, diamondback moth, that CvBV_22-9 is a necessary and sufficient condition for inducing testicular apoptosis, providing a new approach for developing sterility technology for lepidopteran pests.

[0018] 3. A precisely controllable transgenic fruit fly model was constructed, confirming the cross-species conservation of its function: This invention successfully extended the functional study of CvBV_22-9 to the classic model organism, the fruit fly. By constructing tissue-specific transgenic lines, apoptosis was efficiently induced (97.30%) in the fruit fly testes. This not only provides a convenient and stable in vivo model for gene function research but also demonstrates the conservation of the gene's mechanism of action among different insects, greatly expanding its potential application scope.

[0019] 4. This invention provides a direct theoretical basis and core tools for developing new green pest control strategies: It not only elucidates a new mechanism for regulating the reproduction of pests, but more importantly, by constructing a plasmid carrying the CvBV_22-9 recombinant virus and constructing a transgenic model, it can be directly used to develop pest control agents that induce male sterility in pests, transgenic sterile insects, or other genetic regulation technologies, which have clear practical value and broad industrialization prospects.

[0020] In summary, existing technologies have significant gaps and bottlenecks in understanding parasitic castration mechanisms, identifying and validating the functions of key genes, and translating them into applied technologies. This invention aims to fill these technological gaps by systematically revealing the core role of the CvBV_PTP gene in inducing insect testicular apoptosis and verifying its cross-species functionality, providing a theoretical basis and genetic resources for developing novel insect sterility agents. Attached Figure Description

[0021] Figure 1Morphological observation and area statistical analysis of diamondback moth larvae testes before and after parasitism. (A) Observation of the tissue morphology of diamondback moth larvae testes before and after parasitism. NP: 4th instar unparasitized diamondback moth larvae; P: 4th instar parasitized diamondback moth larvae. (B) Analysis of testis area measurement data. 60 biological replicates were counted in the unparasitized group and 58 biological replicates were counted in the parasitized group. (C) Detection of apoptosis signals in the testes of diamondback moth larvae. NP-3M: mid-3rd instar unparasitized diamondback moth larvae; P-3M: mid-3rd instar parasitized diamondback moth larvae; NP-3L: late-3rd instar unparasitized diamondback moth larvae; P-3L: late-3rd instar parasitized diamondback moth larvae; NP-4E: early-4th instar unparasitized diamondback moth larvae; P-4E: early-4th instar parasitized diamondback moth larvae; Red: fragmented DNA; Blue: 4',6-diamidinyl-2-phenylindole. (D) Statistical analysis of apoptotic individuals. NP: non-parasitic; P: parasitic. Yellow: no apoptotic signal; red: apoptotic signal. Data were analyzed using Pearson Chi-Square (***, p < 0.001).

[0022] Figure 2 Screening and expression pattern analysis of CvBV_22-9, a key apoptosis-inducing factor. (A) Analysis of CvBV-PTP gene in the testes of post-parasitic diamondback moth larvae. (BD) Analysis of CvBV_22-9 expression in different tissues of post-parasitic diamondback moth larvae at different time points. P-3M: mid-3rd instar parasitic diamondback moth larvae; P-3L: late-3rd instar parasitic diamondback moth larvae; P-4E: early-4th instar parasitic diamondback moth larvae. HC: hemolymph; CNS: central nervous system; FB: fat body; MG: Malpighian tubules; CT: epidermis; TS: testes; SG: accessory glands; MT: Malpighian tubules. Data are based on three independent experiments and are presented as mean ± standard deviation (SD). Differences between samples were tested by one-way ANOVA, followed by Tukey's test. Different letters indicate that p-values ​​< 0.05 indicate significant differences.

[0023] Figure 3To partially restore the castration of diamondback moth larvae induced by parasitism after interfering with CvBV_22-9 expression. (A) The interference efficiency of CvBV_22-9 was detected by qPCR. Data are presented as mean ± standard deviation, and Tukey's test was used for differential analysis (***, p < 0.001). (BC) After interfering with CvBV_22-9 expression, the tissue morphology of the diamondback moth larvae testes before and after parasitism was observed, and their area was statistically analyzed. dsGFP-P: control group of diamondback moth larvae immediately parasitized after injection of GFP gene dsRNA; dsCvBV_22-9-P: treatment group of diamondback moth larvae immediately parasitized after injection of CvBV_22-9 gene dsRNA. Data are presented as mean ± standard deviation, and Tukey's test was used for differential analysis (***, p < 0.001). (D) Statistical analysis of apoptotic individuals. Yellow: no apoptotic signal; red: apoptotic signal. Data were analyzed using Pearson Chi-Square (***, p < 0.001).

[0024] Figure 4 To investigate the effect of NPV infection on castration of the testes of diamondback moth larvae expressing CvBV_22-9. (AB) After CvBV_22-9 expression, the tissue morphology of the testes of diamondback moth larvae was observed, and their area was statistically analyzed. NPV-GFP: Recombinant NPV expressing GFP was injected; NPV-CvBV_22-9: Recombinant NPV expressing CvBV_22-9 was injected. Data are presented as mean ± standard deviation, and Tukey's test was used for difference analysis (*, p < 0.05). (C) Observation of apoptotic signals in the testes of diamondback moth larvae after NPV injection. Red: Broken DNA; Blue: 4',6-diamidinyl-2-phenylindole. (D) Statistical analysis of apoptotic individuals. Yellow: No apoptotic signal; Red: Apoptotic signal. Data were analyzed using Pearson Chi-Square (***, p < 0.001). (E) Morphology of the testes of male diamondback moth adults after NPV injection. (F) Difference in oviposition rate between male diamondback moths and wild-type females after NPV injection. Data are presented as mean ± standard deviation. Tukey's test was used for difference analysis (*, p < 0.05). (G) Difference in egg hatching rate between male diamondback moths and wild-type females after NPV injection. Data are presented as mean ± standard deviation. Tukey's test was used for difference analysis (ns, no significant difference).

[0025] Figure 5Detection of apoptotic signals in the testes of Drosophila larvae after heterologous expression of CvBV_22-9. (A) Observation of apoptotic signals in the testes of Drosophila larvae after CvBV_22-9 expression. Red: fragmented DNA; Blue: 4',6-diamidinyl-2-phenylindole. (B) Statistical analysis of apoptotic individuals. Yellow: no apoptotic signal; Red: apoptotic signal present. Data were analyzed using Pearson Chi-Square (***, p < 0.001). Detailed Implementation

[0026] Example 1 Parasitism by the diamondback moth parasitoid wasp causes microscopic morphological changes in the testes of the host diamondback moth larvae, thereby inducing apoptosis.

[0027] 1.1 Rearing of Diamondback Moth and Diamondback Moth Parasitic Wasp The test plant was the Jingfeng No. 1 cabbage bred by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. Brassica oleracea L. var. capitata Cabbage is grown year-round in enclosed greenhouses, without the use of pesticides during its growing season; pest control is done manually. Leaves with 5-8 leaves that haven't yet formed a head, or the entire plant, are used to feed diamondback moths.

[0028] Diamondback moth Plutella xylostella Collected outdoors in Hangzhou (30.3009° N, 120.0870° E), the population was maintained and reared in an artificial incubator. The temperature of the artificial incubator was set at 25±1°C, the light intensity at L14:D10 (>1000 lux), and the humidity at 60-80%. Diamondback moth pupae were placed in oviposition boxes, and after emerging as adults, they were fed a 10% (w / v) sucrose solution or honey water. Adults laid eggs on special oviposition paper, and the collected eggs were then transferred to fresh cabbage leaves or plants. After hatching, the larvae fed directly on the cabbage for growth and development, eventually pupating and completing one generation cycle.

[0029] Pine moth filamentous parasitic wasp Cotesia vestalis Collected outdoors in Hangzhou (30.3009° N, 120.0870° E), the population was maintained and reared in an artificial incubator. The *Papilionophora chinensis* wasp was propagated and degenerated using colony breeding methods. 1) Select third-instar diamondback moth larvae of uniform size and place them in the parasitizing box; 2) Add fully mated female wasps (1 / 10 of the number of larvae) and parasitize for 2 hours; 3) Rearing parasitized diamondback moth larvae; 4) Collect the bee cocoons after 7-9 days and wait for them to emerge for use in experiments or the next generation.

[0030] Samples were collected using a single-parasitic method: Five female wasps, 2-3 days after emergence and fully mated, were placed in a glass finger tube, followed by one diamondback moth larva. After confirming oviposition parasitism, the diamondback moth larva was quickly removed, and the process was repeated with the next larva. The parasitized diamondback moth larvae were collected for subsequent experiments.

[0031] 1.2 Observation of testicular morphology Parasitized and unparasitized diamondback moth larvae in their early fourth instar (4E) were prepared. The testis tissue of the diamondback moth larvae was dissected in PBS buffer, and other attached tissues were removed. The collected testis tissue was photographed under a Keyence camera, and its size was measured using ImageJ software. The results showed that parasitism significantly reduced the size of the testis by 54%. Figure 1 (A and 1B).

[0032] 1.3 Testicular apoptosis staining Male diamondback moth larvae at different instars before and after parasitism were collected. Their testes were dissected in PBS, and attached fat bodies and other tissues were removed. Apoptosis signal staining was performed using the TUNEL BrightRed Apoptosis Detection Kit (Vazyme). After slide preparation, the slides were observed and photographed under a Zeiss LSM 800 laser confocal microscope. The specific methods are as follows: 1) Fixation: The dissected testes were placed in a nine-well dissecting plate and fixed with 200 μl of 4% paraformaldehyde for 30 min; 2) Washing: Aspirate the liquid, add 200 μl PBST and wash on a shaker for 5 min, repeat 3 times; 3) Permeabilization: Prepare a 20 μg / ml proteinase K solution (diluted with PBS at a ratio of 1:100), add 100 μl to the sample, and permeabilize at room temperature for 20 min; 4) Washing: Aspirate the liquid, add 200 μl PBST and wash on a shaker for 5 min, repeat 3 times; 5) Equilibration: Prepare 1 × Equilibration Buffer (diluted with 4 times the volume of ddH2O), add 100 μl to the sample, and equilibrate at room temperature for 20 min; 6) Prepare the labeling solution, as shown in Table 1: Table 1 7) Remove the equilibration solution and add 100 μl of TdT-containing labeling solution. 8) After equilibration, absorb the 1× Equilibration Buffer with absorbent paper, and add 100 μl of TdT incubation buffer to each sample. Place the samples in a humidity chamber and incubate at 37°C for 60 min; 9) Remove the labeling solution, add 200 μl PBST and wash on a shaker for 5 min, repeat 3 times; 10) Mount the slide with DAPI-containing glue, fix at room temperature for 5-10 min, then photograph using a laser confocal microscope or store at -20℃. (BrightRed observation: 594nm; DAPI observation: 460nm).

[0033] Experimental results showed that, compared with unparasitized diamondback moths, the testes of parasitized diamondback moth larvae contained a large number of fragmented nucleic acids in the mid-3rd instar (3M), late-3rd instar (3L), and early-4th instar (4E), and obvious apoptosis signals were observed. Figure 1 C), and the proportion of individuals undergoing apoptosis increases with age, with the detected proportion of apoptosis increasing from 80.56% to 95.45%. Figure 1 D). This indicates that parasitism can induce castration, including testicular apoptosis, in diamondback moth larvae, and this trend increases with age.

[0034] Example 2: CvBV_22-9 induces parasitic castration. 2.1 Screening for key genes inducing apoptosis in host testicular cells and cloning their cDNA sequences Analysis of the expression patterns of tyrosine phosphatase family genes encoded by wasp viruses in the published testis transcriptome revealed the expression of 24 CvBV-PTP genes in the testes after parasitism. Among them, CvBV_22-9 showed the highest expression level, and its expression level continued to increase at 6, 12, 24, 72, and 120 hours post-parasitism. Therefore, we hypothesize that it may play an important function in the testes. The steps for cloning the cDNA sequence of CvBV_22-9 are as follows: 1) First, a single parasitized a third-instar diamondback moth larva. Total RNA was extracted from the diamondback moth larvae at 12, 24, 48 and 96 hours after parasitism using the TRIzol method. cDNA was then synthesized using SuperScript™ III Reverse Transcriptase (Invitrogen, USA).

[0035] 2) Cloning primers (SEQ ID NO. 1 and SEQ ID NO. 2) were designed based on the CvBV_re22 sequence MZ645202.1 in the CvBV genome.

[0036] 3) Prepare the following reaction solution in a 200 μL EP tube: 10 μL 2 × KOD One TMPCR Master Mix (TOYOBO, Japan), 0.6 μL upstream primer CvBV_22-9-clone-F, 0.6 μL downstream primer CvBV_22-9-clone-R, 0.2 μL cDNA, 8.6 μL double-distilled water; gently mix the above reaction system, and then perform the following PCR reaction: 98°C pre-denaturation for 3 min, followed by 98°C denaturation for 10 s, 58°C annealing for 5 s, 68°C extension for 10 s, for 35 cycles, 72°C extension for 2 min for one cycle, and finally cooling to 4°C.

[0037] 4) After electrophoresis in a 1% agarose gel, the target nucleic acid fragment was recovered using the FastPure Gel DNA Extraction Mini Kit (Vazyme, China).

[0038] 5) The recovered fragment was ligated into the vector pCE2_TA-Blunt-Zero (Vazyme, China) using a 5-min TA / Blunt-Zero Cloning Kit (Vazyme, China), and then transformed into TG1 E. coli. The E. coli were sequenced, and the 945 bp CvBV_22-9 open reading frame sequence was verified (as shown in SEQ ID NO. 3), whose encoded amino acid sequence is shown in SEQ ID NO. 4.

[0039] 6) After the sequencing is confirmed to be correct, the plasmid is extracted using the FastPure Plasmid Mini Kit (Vazyme, China). The obtained plasmid is stored in a -20°C freezer for later use.

[0040] 2.2 Detection of spatiotemporal expression pattern of CvBV_22-9 gene Third-instar mid-stage diamondback moth larvae (12 h post-molting) were used for single-head parasitism. The parasitized larvae were placed in food and allowed to continue developing for 24 h (3M), 48 h (3L), and 72 h (4E). Afterward, their hemolymph, central nervous system, fat body, midgut, epidermis, testes, silk glands, and Malpighian tubules were dissected, with at least three biological replicates for each tissue. RNA was extracted and reverse transcribed. Using cDNA as a template, upstream and downstream primers (SEQ ID NO. 5 and SEQ ID NO. 6) were designed, and the PCR reaction was performed according to the system shown in Table 2.

[0041] Table 2 Reaction procedure: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, extension for 30 s, for a total of 40 cycles. Fluorescence intensity was recorded. (Selected) Px-β-tubulin (SEQ ID NO. 7 and SEQ ID NO. 8) and Px-β-actin (SEQ ID NO. 9 and SEQ ID NO. 10) were used as internal reference genes. Using 2 -ΔΔCt The relative differences in transcription levels among different treatments were analyzed using a one-way ANOVA method, and the differences between different samples were statistically analyzed. The results showed that... CvBV_22-9 Gene expression levels were highest in testes at all ages.

[0042] 2.3 Detection of testis size and apoptosis in diamondback moth larvae after RNA interference with CvBV_22-9 Primers for dsRNA synthesis were designed based on the CvBV_22-9 gene sequence (sequences shown in SEQ ID NO. 13-20). The template for dsRNA was synthesized by PCR following the steps in 2.1. After electrophoresis on a 1% agarose gel, the target nucleic acid fragment was recovered using the FastPure Gel DNAExtration Mini Kit (Vazyme, China). Using this recovered product as a template, dsRNA was synthesized according to the instructions of the T7RNAi Transcription Kit (Vazyme). CvBV_22-9 Perform microinjection as follows.

[0043] Microinjection: 1) Cut open the pre-prepared 1% agarose gel with a blade, place the diamondback moth in the crevices of the gel, and fix its body in place; 2) Use an Eppendorf micropipette to aspirate 10 μl of the injection solution into the capillary; 3) Insert the capillary tube into the injection connector and fix it to the microinjection robotic arm; 4) Adjust the parameters of the microinjector, set the initial injection pressure to 1200 Pa and the injection time to 0.1 s; 5) Use tweezers to break the capillary needle tip and adjust the injection pressure according to the injection volume (the needle tip should not be too large, and the injection pressure should not be lower than 500 Pa). 6) Insert the intersegmental membrane into the abdomen of the diamondback moth larva to prevent hemolymph leakage, and then proceed with normal feeding.

[0044] Tests showed that the interference efficiency could reach 89%. Figure 3 A). 3M diamondback moth larvae were selected for the above-mentioned interference experiment involving injection of double-stranded RNA followed by single-head parasitism. Twenty-four hours after CvBV_22-9 interference, the testes of the diamondback moth larvae were dissected for apoptosis staining (refer to 1.3 in Example 1). After reducing CvBV_22-9 expression, the testis area increased by 17% compared to the control group. Figure 3BC) can partially salvage this phenotype. Meanwhile, knocking down CvBV_22-9 resulted in a 38% lower rate of testicular apoptosis in post-parasitic diamondback moth larvae compared to the control group. Figure 3 (D) indicates that CvBV_22-9 is involved in the parasitic-induced testicular apoptosis process.

[0045] 2.4 Construction and synthesis of recombinant baculovirus CvBV_22-9 1) Construction of the recombinant vector: Enzyme digestion primers (SEQ ID NO. 11 and SEQ ID NO. 12) were designed based on the CvBV_22-9 ORF, and the CvBV_22-9 ORF sequence carrying the enzyme digestion primers was obtained by PCR. The ORF sequence of the target gene was ligated into the pFastBac-HTB vector using a double enzyme digestion and ligation method, and then transformed into TG1 E. coli for sequencing verification. A plasmid carrying GFP was also prepared as a negative control.

[0046] 2) After verifying the sequence is correct, extract the recombinant plasmid and transform it into DH10 Bac. TM E. coli In the transformation process, after adding antibiotic-free medium, a long incubation period (2-4 hours) with shaking at 37℃ and 180 rpm is required. The solid medium should contain 50 μg / ml Kan (K). + ), 7 μg / ml Gentamicin (G + ), 10 μg / ml Tetracycline (T + ), 100 μg / ml X-gal and 40 μg / ml IPTG.

[0047] 3) After incubation at 37℃ for 2-3 days, pick white spots for bacterial culture testing.

[0048] 4) Take 5 μl of fresh DH10 Bac TM E. coli The bacteria were inoculated into 30 ml of liquid culture medium (containing the above-mentioned concentration of K). + T + G + (For resistance), incubate overnight at 37°C with shaking at 250 rpm. Recombinant baculovirus DNA was extracted using the PureLink™ HiPure Plasmid Miniprep Kit (Thermo Scientific).

[0049] 5) Transfect the recombinant plasmid into sf9 cells according to the Cellfectin™ II Reagen reagent instructions.

[0050] 6) Resuspend the successfully transfected cells (approximately 5-7 days after transfection and exhibiting a clear transfection phenotype), centrifuge at 800 rpm for 2 min, and the supernatant will contain recombinant baculovirus particles (P1). Transfer 200 μl to a cell culture dish with 70% cell confluence for passage of the virus particles (P2). Repeat this cycle multiple times for propagation.

[0051] 7) Collect P5 generation NPV, centrifuge at 5000× g for 10 min, discard the precipitate, and transfer the supernatant to a new centrifuge tube; 8) After centrifuging at 10000× g for 60 min, centrifuge at 20000× g for 60 min, aspirate the supernatant, leaving only 20-50 μl of liquid near the bottom, mix well by pipetting, and this is the concentrated recombinant baculovirus NPV-CvBV_22-9. Recombinant baculovirus NPV-GFP serves as a negative control.

[0052] 2.5 Using baculovirus infection and staining for apoptosis in diamondback moth larvae testes The NPV titer was determined using an absolute quantitative method: Plasmids containing known concentrations and sizes of the target gene were serially diluted and used as templates for qPCR. Plasmid copy numbers were calculated and correlated with Ct values ​​to create a standard curve. Then, recombinant baculovirus NPV-CvBV_22-9 was used as a template for qPCR, and the recombinant baculovirus copy number was calculated from the Ct values ​​using the standard curve.

[0053] The collected NPV was diluted to 1.0 × 10⁻⁶. 5 Copy / μl concentration. Select 3rd instar diamondback moth larvae of uniform size and inject each with 1.0 × 10⁻⁶ copies / μl. 5 Copy NPV. Twenty-four hours after injection, the testes of diamondback moth larvae were dissected for size measurement and apoptosis staining analysis. The results showed that, compared to NPV_GFP, expression of CvBV_22-9 using NPV-CvBV_22-9 significantly reduced the size of the diamondback moth testes by 8%. Figure 4 (AB), the proportion of individuals undergoing apoptosis increased from 23.53% to 90.80% ( Figure 4 CD), indicating that the expression of CvBV_22-9 can effectively induce apoptosis in testicular cells.

[0054] 2.6 Effects of low-concentration NPV-CvBV_22-9 baculovirus infection on diamondback moth offspring Inject 1×10 into unparasitized mid-3rd instar male diamondback moth larvae 4Copyable NPV-GFP and NPV-CvBV_22-9 virus particles were used to dissect the testes of adult moths within 24 hours of emergence, and changes in the testes were photographed and recorded. Furthermore, 10 male moths injected with NPV virus particles were each mated with 10 wild-type female moths for 24 hours. All female moths were then transferred to a new plastic box containing cabbage leaf extract wrapped in Parafilm as their oviposition substrate. Diamondback moth females laid eggs on this substrate for 3 days, with the Parafilm membrane being changed every 24 hours, and the number of eggs laid and hatched was recorded. During the experiment, a 10% honey solution was provided to supplement their nutrition. The results showed that low-dose injection of NPV-CvBV_22-9 significantly altered the morphology of the diamondback moth testes. Figure 4 E). Further analysis of the egg-laying quantity and hatching rate of the diamondback moth offspring revealed that the egg-laying quantity of the offspring was significantly reduced by 33% ( Figure 4 FG).

[0055] Example 3: Functional verification of CvBV_22-9 in the model insect Drosophila melanogaster 3.1 Test Insects To verify the conservation of CvBV_22-9 function, this study used the model insect *Drosophila melanogaster*. Drosophila melanogaster Further verification was conducted. The *Drosophila melanogaster* used was... D. melanogaster The strain includes W, which is bred in our laboratory. 1118 The control strain, the Nos-Gal4 strain (FlyBase ID: FBtp0056928), and the constructed UAS-CvBV_22-9 strain were included.

[0056] 3.2 Construction of the homozygous transgenic fruit fly strain UAS-CvBV_22-9 The ORF sequence of CvBV_22-9 was ligated into the pUASt-attb vector using a double enzyme digestion ligation method (enzyme digestion primers SEQ ID NO. 21 and SEQ ID NO. 22), and the recombinant plasmid was sequenced for verification. The verified recombinant plasmid and helper plasmid were co-microinjected into white-eyed wild-type Drosophila melanogaster W. 1118 In the embryo, the gene is inserted into chromosome 3. The embryo develops into an adult, which is the G0 generation. The red-eyed fruit flies in the G0 generation are the successful UAS-CvBV_22-9 (+ / +, CvBV_22-9 / CvBV_22-9) transgenic strain (the G0 generation transgenic fruit flies were obtained by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences).

[0057] Screening for homozygous transgenic fruit fly strains: 1) Cross the UAS-CvBV_22-9 transgenic fruit fly strain with the balanced fruit fly strain (w- / w-; Sp / Cyo; TM2b / TM6b); 2) Select virgin fruit flies with curly wings and large halteres (w- / w-; + / Cyo; TM2b / CvBV-PTP24) and male fruit flies with two or more long bristles on the sides of the thorax and many hairs on the shoulders (w- / w-; Sp / +; TM6b / CvBV_22-9) for hybridization; or select virgin fruit flies with two or more long bristles on the sides of the thorax and many hairs on the shoulders (w- / w-; Sp / +; TM6b / CvBV_22-9) and male fruit flies with curly wings and large halteres (w- / w-; + / Cyo; TM2b / CvBV_22-9) for hybridization; 3) Select homozygous UAS-CvBV_22-9 transgenic fruit flies (w- / w-;Sp / Cyo; CvBV_22-9 / CvBV_22-9) with curled wings and more than two long bristles on the sides of the thorax for preservation.

[0058] 3.3 Construction of recombinant strains that specifically express CvBV_22-9 in the testes of Drosophila larvae This experiment mainly utilized the UAS-Gal4 system to achieve target gene expression in specific tissues through hybridization of two strains. Two hundred homozygous UAS-CvBV_22-9 virgin fruit flies were selected and crossed with 30 Nos-Gal4 strains (to specifically express CvBV_22-9 in the fruit fly testes) and 30 W strains, respectively. 1118 (Control) Male fruit flies were hybridized. One day later, the fruit flies were placed in fresh food every 2 hours after laying eggs and cultured at 25°C for subsequent experiments.

[0059] 3.4 Staining for apoptosis in the testes of fruit fly larvae Apoptosis staining was performed on testicular tissues of late 3rd instar Drosophila larvae under different treatments, following the specific staining procedure described in 1.3 of Example 1. The results showed that expression of the CvBV_22-9 gene in the Drosophila testes also triggered a strong apoptotic signal. Figure 5 Further statistical analysis of the number of individuals undergoing apoptosis revealed that the proportion of testes showing apoptotic signals in the control group was extremely low, at only 1.92% (1 / 52). In contrast, the incidence of testicular cell apoptosis in the CvBV_22-9 expression group was as high as 97.30%. Figure 5 In conjunction with Implementation Case 2, this invention clarifies that CvBV_22-9 can simultaneously induce testicular apoptosis in both diamondback moth (lepidopteran insect) and fruit fly (diptera insect) larvae, demonstrating the conservation of CvBV_22-9 in inducing testicular apoptosis during insect development. This makes it a potential candidate for developing pest-effect molecules based on male sterility.

Claims

1. Application of a protein tyrosine phosphatase gene CvBV_22-9 encoded by a diamondback moth worm virus in inducing apoptosis in the testis cells of diamondback moth larvae.

2. The application as described in claim 1, characterized in that, The nucleotide sequence of the CvBV_22-9 gene contains the sequence shown in SEQ ID NO. 3 or a functional fragment thereof.

3. The application as described in claim 1, characterized in that, The amino acid sequence encoded by the CvBV_22-9 gene is as shown in SEQ ID NO. 4 or a functional fragment thereof.

4. The application as described in claim 1, characterized in that, The application is achieved by expressing the CvBV_22-9 gene.

5. The application as described in claim 4, characterized in that, The expression is achieved through infection with a baculovirus expression vector.

6. The application as described in claim 5, characterized in that, The application involves synthesizing and using recombinant baculovirus NPV-CvBV_22-9 to infect diamondback moth larvae in order to induce apoptosis in male diamondback moth testis cells.

7. The application of a protein tyrosine phosphatase gene CvBV_22-9 encoded by a diamondback moth lacewing wasp virus in the preparation of formulations for biological control of pests, characterized in that, The formulation achieves population control by inducing male sterility in pests.

8. The application as described in claim 7, characterized in that, The pests include Lepidoptera or Diptera pests.

9. The application as described in claim 8, characterized in that, The lepidopteran pest is the diamondback moth, and the dipteran pest is the fruit fly.

10. A method for constructing a model for inducing apoptosis in insect testes, characterized in that, The steps include: constructing the CvBV_22-9 gene coding sequence into a vector for Drosophila transgenesis, and obtaining transgenic Drosophila through embryo microinjection; The transgenic fruit flies were hybridized with a testis-specific driver strain using the UAS-Gal4 system to obtain a fruit fly strain that specifically expresses CvBV_22-9 in the testes.

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