Use of citrus psyllid ubiquitin ligase UFD4 gene and inhibitor thereof in prevention and control of citrus huanglongbing
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- GANNAN NORMAL UNIV
- Filing Date
- 2025-12-08
- Publication Date
- 2026-06-25
Smart Images
Figure CN2025140738_25062026_PF_FP_ABST
Abstract
Description
Application of the ubiquitin ligase UFD4 gene and its inhibitor in the control of citrus Huanglongbing (HLB)
[0001] This application claims priority to Chinese Patent Application No. CN 202411875391.1, filed on December 19, 2024, entitled "Application of the UFD4 Gene of Citrus Psyllid and its Inhibitor in the Control of Citrus Huanglongbing", the entire contents of which are incorporated herein by reference. Technical Field
[0002] This application belongs to the field of genetic engineering technology, specifically relating to the application of the citrus psyllid ubiquitin ligase UFD4 gene and its inhibitor in the control of citrus Huanglongbing (HLB). Background Technology
[0003] Huanglongbing (HLB) is one of the most devastating diseases affecting the global citrus industry. Because there is currently no effective treatment for infected trees, HLB is often referred to as the "cancer" of citrus. While the HLB pathogen cannot be artificially cultured, it can grow and reproduce for extended periods within the citrus psyllid. Once infected, the psyllid can carry the pathogen for life and spread it rapidly, posing significant challenges to HLB research and control, and representing one of the biggest problems plaguing the citrus industry in recent years. Current international citrus industry control of HLB primarily relies on chemical pesticides to control the psyllid. However, the frequent use of chemical pesticides has led to numerous problems, including pesticide residues, environmental pollution, biodiversity loss, and pesticide resistance in pests. Therefore, it is essential to research new methods for rapid and long-term control of HLB. Summary of the Invention
[0004] The purpose of this application is to provide the application of the ubiquitin ligase UFD4 gene of citrus psyllid and its inhibitor in the control of citrus Huanglongbing (HLB). By regulating the ubiquitin ligase UFD4 gene, the infection of citrus psyllids can be reduced, thereby effectively controlling the infection and spread of citrus HLB.
[0005] This application provides the application of biomaterials related to the ubiquitin ligase UFD4 gene of citrus psyllid in the control of citrus Huanglongbing (HLB), wherein the amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of citrus psyllid is shown in SEQ ID NO.1.
[0006] As a preferred embodiment, the biological material comprises the citrus psyllid ubiquitin ligase UFD4 gene and / or the protein encoded by the citrus psyllid ubiquitin ligase UFD4 gene.
[0007] As a preferred embodiment, the nucleotide sequence of the citrus psyllid ubiquitin ligase UFD4 gene is shown in SEQ ID NO.2.
[0008] This application provides a primer pair for amplifying the ubiquitin ligase UFD4 gene of the citrus psyllid, the primer pair comprising: PT-F and PT-R as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; and the amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of the citrus psyllid as shown in SEQ ID NO.1.
[0009] This application provides an inhibitor of the expression of the ubiquitin ligase UFD4 gene in citrus psyllids, wherein the active ingredient of the inhibitor includes: an E3-HECT type inhibitor;
[0010] The amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of the citrus psyllid is shown in SEQ ID NO.1.
[0011] This application provides the application of the above-mentioned inhibitors in the study of ubiquitination degradation function during Huanglongbing infection.
[0012] This application provides the application of the above-mentioned inhibitors in the control of citrus Huanglongbing (HLB).
[0013] This application provides a method for controlling citrus Huanglongbing (HLB), comprising the following steps: silencing the expression of the citrus psyllid ubiquitin ligase UFD4 gene.
[0014] As a preferred embodiment, the silencing includes silencing using the aforementioned inhibitors.
[0015] As a preferred embodiment, silencing the citrus psyllid using the inhibitor includes the following steps: placing the inhibitor on a portion of the psyllid that it can be consumed by.
[0016] As a preferred embodiment, the edible parts for citrus psyllids include: branches, buds, young leaves, or sap from tender shoots.
[0017] This application provides a method for detecting the expression level of the ubiquitin ligase UFD4 gene of citrus psyllid, comprising the following steps: performing RNAi analysis using dsRNA synthesized based on the ubiquitin ligase UFD4 gene of citrus psyllid to obtain the expression level; the amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of citrus psyllid is shown in SEQ ID NO.1;
[0018] The primer pairs for dsRNA synthesis include: RNAi-F with nucleotide sequences as shown in SEQ ID NO.5 and RNAi-R with SEQ ID NO.6;
[0019] The primer pairs for the RNAi analysis include qPCR-F with nucleotide sequences as shown in SEQ ID NO.7 and qPCR-R with SEQ ID NO.8.
[0020] Beneficial Effects: This application provides the application of biomaterials related to the ubiquitin ligase UFD4 gene of the citrus psyllid in the control of citrus Huanglongbing (HLB). The amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of the citrus psyllid is shown in SEQ ID NO.1. This application is the first to identify and successfully clone the ubiquitin ligase UFD4 gene from the transcriptome of the citrus psyllid. This application found that the expression level of UFD4 was significantly upregulated after infection with CLas pathogens, thus suggesting that the ubiquitin ligase UFD4 gene of the citrus psyllid is likely involved in the response to CLas pathogen infection. The ubiquitin ligase UFD4 gene of this application contains a typical HECT domain. During ubiquitination modification, the UFD4 gene provides a recognition site for ubiquitin-binding enzyme E2 and binds to it; on the other hand, the UFD4 gene can specifically recognize target proteins, forming an E2-HECT-target protein complex, thereby promoting the transfer of ubiquitin molecules from E2 to the target protein, and thus completing the ubiquitination modification process, thereby effectively controlling citrus Huanglongbing.
[0021] This application provides an inhibitor of the ubiquitin ligase UFD4 gene in citrus psyllids, comprising an E3-HECT type inhibitor. This application is the first to utilize an E3-HECT type inhibitor to silence the ubiquitin ligase UFD4 gene and investigate its ubiquitination function during Huanglongbing (HLB) infection. The study revealed that the E3-HECT type inhibitor successfully inhibited the ubiquitin ligase UFD4 gene. In the ubiquitination degradation pathway, ubiquitin ligase E3 binds to the substrate protein in two ways. This is because E3 contains two different protein structures (HECT type and RING type). In the HECT type, the ubiquitin molecule Ub binds sequentially to E2, E3, and the substrate protein to complete degradation. In the RING type, the ubiquitin molecule does not bind to E3 but is directly transferred from E2 to bind to the substrate protein. Therefore, the E3-HECT type inhibitor can effectively silence the ubiquitin ligase UFD4 gene and possesses ubiquitination degradation function during HLB infection. Attached Figure Description
[0022] Figure 1 shows the RNAi results of the ubiquitin ligase UFD4 gene of the citrus psyllid;
[0023] Figure 2 shows the results of fungal harvesting from citrus psyllids after RNA interference.
[0024] Figure 3 shows the standard curve of Huanglongbing bacteria copy number;
[0025] Figure 4 shows the expression of the DcUFD4 gene in the citrus psyllid after treatment with the E3-HECT inhibitor.
[0026] Figure 5 shows the results of citrus psyllid acquisition after treatment with the E3-HECT inhibitor.
[0027] Figure 6 shows the expression of the DcUFD4 gene in the citrus psyllid after treatment with the E3-RING inhibitor. Detailed Implementation
[0028] This application provides the application of biological materials related to the ubiquitin ligase UFD4 gene of the citrus psyllid (Diaphorina citri) in the control of citrus Huanglongbing (HLB), wherein the amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of the citrus psyllid is shown in SEQ ID NO.1:*.
[0029] In one preferred embodiment, the biological material includes the citrus psyllid ubiquitin ligase UFD4 gene and / or the protein encoded by the citrus psyllid ubiquitin ligase UFD4 gene. For control purposes, edible parts of the citrus plant, such as branches, buds, young leaves, and sap from tender shoots, can be selected. The inhibitor is then placed on these edible parts. In a specific embodiment, citrus psyllids are fed citrus branches containing the inhibitor of the citrus psyllid ubiquitin ligase UFD4 gene and / or the protein encoded by the citrus psyllid ubiquitin ligase UFD4 gene to investigate the effect of the ubiquitin ligase UFD4 gene on Huanglongbing (CLas) infection.
[0030] In a preferred embodiment, the nucleotide sequence of the citrus psyllid ubiquitin ligase UFD4 gene is shown in SEQ ID NO.2:
[0031] Note: Horizontal lines indicate open reading frames, and bold text indicates dsRNA interference sequences. The full-length gene was not amplified in the experiment. During the amplification of gene open reading frames, Takara brand rTaq enzyme and pMD19-t vector were used, and no additional sites were required for rapid ligation.
[0032] This application provides a primer pair for amplifying the citrus psyllid ubiquitin ligase UFD4 gene. The primer pair comprises: PT-F with nucleotide sequences as shown in SEQ ID NO.3 and PT-R with SEQ ID NO.4; the amino acid sequence of the protein encoded by the citrus psyllid ubiquitin ligase UFD4 gene is shown in SEQ ID NO.1. Specifically, PT-F (SEQ ID NO.3): 5'-ACCTTGGTTTGCGGGTAT-3'; PT-R (SEQ ID NO.4): 5'-CGTCCACTGTGGGTTTTG-3'.
[0033] This application provides a method for detecting the expression level of the ubiquitin ligase UFD4 gene of citrus psyllid, comprising the following steps: performing RNAi analysis using dsRNA synthesized based on the ubiquitin ligase UFD4 gene of citrus psyllid, wherein the amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of citrus psyllid is shown in SEQ ID NO.1;
[0034] The primer pairs for dsRNA synthesis include: RNAiF with nucleotide sequences as shown in SEQ ID NO.5 and RNAi-R with SEQ ID NO.6; wherein, RNAi-F (SEQ ID NO.5): 5'-TAATACGACTCACTATAGGACCTTGGTTTGCGGGTAT-3'; RNAi-R (SEQ ID NO.6): 5'-TAATACGACTCACTATAGGCGTCCACTGTGGGTTTTG-3' (the underlined portion represents the T7 promoter);
[0035] The primer pairs for the RNAi analysis include qPCR-F and qPCR-R with nucleotide sequences as shown in SEQ ID NO.7 and SEQ ID NO.8, respectively; wherein, qPCR-F (SEQ ID NO.7): 5'-AACTGTACTTCGTTCGGTGCC-3'; qPCR-R (SEQ ID NO.8): 5'-AGCGTAAAACTCCAGGGTCG-3'.
[0036] In one specific implementation method, total RNA was extracted from adult citrus psyllids and reverse transcribed into cDNA. Using this cDNA as a template, PCR was performed with PT-F / R primers to amplify the open reading frame of the DcUFD4 gene. The amplified product was recovered using an Omega gel extraction kit. The amplified product was ligated into the pMD19-T plasmid, transformed into *E. coli* competent cells Trans5α, plated, and the next day, single colonies were picked and amplified in LB broth containing ampicillin. The bacterial culture was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Bacterial cultures with low mutation rates were selected for amplification and plasmid extraction (Sigma Rapid Small-Scale Plasmid Extraction Kit). Using the plasmid as a template, amplification was performed using primers containing the T7 promoter, and the amplified product was recovered via gel extraction. The concentration of the recovered product was measured, and dsRNA was synthesized using the PromegaT7 RiboMAX Express RNAi System with RNAi-F / R primers.
[0037] In one specific implementation, the synthesized dsRNA was diluted with enzyme-free water to a concentration of 2000 ng / μL and injected into 4th-5th instar citrus psyllid nymphs using microinjection. In one embodiment, the injection volume of dsRNA into a single psyllid nymph was 20 nL, and the injection rate was 15 nL / s. In another specific implementation, total RNA was extracted from psyllid nymphs 24 h and 48 h after injection treatment (TRizol method), reverse transcribed into cDNA, and real-time quantitative PCR was performed using qPCR-F / R primers to detect changes in the expression level of the citrus psyllid ubiquitin ligase UFD4 gene.
[0038] The expression level detection method described in this application may include real-time quantitative PCR detection. As a preferred embodiment, when using real-time quantitative PCR detection, the reaction system is as follows: ddH₂O 8 μL; SYBR 10 μL; F / R 0.5 μL; cDNA template 1 μL; total system 20 μL; the reaction program is: 95℃ pre-denaturation for 10 min; 95℃ for 10 s, 60℃ for 10 s, 72℃ for 10 s, 40 cycles. The experiment is repeated 6 times with 6 independent samples; the detection reaction is performed using a Roche LightCycler 480Ⅱ real-time quantitative PCR instrument.
[0039] This application describes a method for raising citrus psyllid nymphs treated with RNA interference on young citrus branches infected with Huanglongbing (HLB) and detecting their susceptibility. As a preferred embodiment, this application screened the citrus psyllid ubiquitin ligase UFD4 gene (DcUFD4) from a transcriptome database, designed specific primers to synthesize dsRNA, silenced the DcUFD4 gene using RNA interference, and injected it into the psyllid's abdomen via microinjection. The psyllids were then fed HLB-infected citrus branches to investigate the effect of this gene on HLB infection.
[0040] This application provides an inhibitor of ubiquitin ligase UFD4 gene expression in citrus psyllids, the inhibitor comprising an E3-HECT type inhibitor. This application describes a method for inhibiting ubiquitin ligase by injecting an E3-HECT type inhibitor, followed by quantitative real-time detection of the ubiquitin ligase UFD4 gene expression level.
[0041] This application provides a method for controlling citrus Huanglongbing (HLB), comprising the following steps: silencing the citrus psyllid ubiquitin ligase UFD4 gene. As a preferred embodiment, the silencing includes using the aforementioned inhibitor. The E3-HECT type inhibitor described in this application has ubiquitination degradation function, which can promote the degradation of ubiquitin ligase, providing a theoretical basis for the control of HLB targeting this disease.
[0042] To further illustrate this application, the following detailed description, in conjunction with embodiments, illustrates the application of the citrus psyllid ubiquitin ligase UFD4 gene and its inhibitor in the control of citrus Huanglongbing (HLB), but these descriptions should not be construed as limiting the scope of protection of this application.
[0043] Example 1: Synthesis of dsRNA and RNAi Experiment
[0044] (1) dsRNA synthesis
[0045] Total RNA was extracted from adult citrus psyllids in the laboratory and reverse transcribed into cDNA. Using the cDNA as a template, the open reading frame of the DcUFD4 gene was amplified by PCR using PT-F (SEQ ID NO. 3) and PT-R (SEQ ID NO. 4) as primers. The amplification product was recovered using an Omega gel extraction kit. The amplification product was ligated into the pMD19-t plasmid, transformed into E. coli competent cells Trans5α, plated, and the next day, single colonies were picked and amplified in LB broth containing ampicillin. The bacterial culture was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Bacterial cultures with low mutation rates were selected for amplification and plasmid extraction (Sigma Rapid Small-Scale Plasmid Extraction Kit). Using the plasmid as a template, amplification was performed using primers containing the T7 promoter, and the amplification product was recovered by gel extraction. The concentration of the recovered product was measured, and dsRNA, RNAi-F (SEQ ID NO. 5), and RNAi-R (SEQ ID NO. 4) were synthesized using the Promega T7 RiboMAX Express RNAi System (according to the manufacturer's instructions). NO.6) is used as a primer for synthesizing dsRNA.
[0046] (2) RNAi analysis
[0047] The synthesized dsRNA was diluted to a concentration of 2000 ng / μL with enzyme-free water and treated with 4th-5th instar citrus psyllid nymphs using microinjection. Specifically, the dsRNA was injected into the abdomen of the psyllid via microinjection. The injection volume of the double-stranded RNA per nymph was 20 nL, and the injection rate was 15 nL / s. Simultaneously, dsGFP treatment served as a control group, with the same amount of dsGFP injected. The green fluorescent protein gene sequence was used as a control only; the protein was not expressed, and therefore no green fluorescence was emitted. Total RNA was extracted from eight citrus psyllid nymphs after 24 h and 48 h of treatment (TRizol method), reverse transcribed into cDNA, and the expression level was detected by real-time quantitative PCR. The results are shown in Figure 1.
[0048] Real-time quantitative primers: qPCR-F (SEQ ID NO.7) and qPCR-R (SEQ ID NO.8).
[0049] Reaction system: ddH2O 8μL; SYBR 10μL; F / R 0.5μL; cDNA template 1μL; total system 20μL.
[0050] Reaction program: 95℃ pre-denaturation for 10 min; 95℃ for 10 s, 60℃ for 10 s, 72℃ for 10 s, 40 cycles. The experiment was repeated 6 times with 6 independent samples, and all reactions were performed on a Roche LightCycler 480Ⅱ real-time quantitative PCR instrument.
[0051] Figure 1 shows the relative expression of the DcUFD4 gene after RNAi, where blue represents the dsGFP treatment group and red represents the dsUFD4 treatment group. At 24h: P = 0.00446 < 0.01, indicating a highly significant difference; at 48h: P = 0.00003 < 0.01, indicating a highly significant difference. "**" indicates a highly significant difference. The results show that a dsRNA concentration of 2000 ng / μL has a good inhibitory effect, significantly inhibiting UFD4 gene expression at both 24h and 48h.
[0052] Example 2
[0053] The citrus psyllids were treated with RNAi interference by microinjecting dsRNA into their abdomens, with an equal volume of water used as a control. After 48 hours, the nymphs were collected and reared on young citrus branches infected with Huanglongbing (HLB). Every 24 hours, 15 surviving psyllids were collected to determine their CLAs pathogen copy number. The results are shown in Figure 2.
[0054] Detection method: The presence of CLAs pathogens in citrus psyllids at different time points after interference treatment was detected using laboratory-preserved double-labeled probes and detection primers (see Table 1).
[0055] Table 1 Absolute Quantitative Probes and Primers Note: FAM and TAMRA are labeled molecules.
[0056] The pMD19-t plasmid was ligated into *C. chrysogenum* CLas, following the method described in the literature Zhang JB, Zou X J, Zhang Q, et al. Quantitative ubiquitylome crosstalk with proteome analysis revealed cytoskeleton proteins influence CLas pathogen infection in Diaphorina citri[J]. International Journal of Biological Macromolecules, 2023, 232:123411. The recombinant plasmid pMD19-t-CLas was constructed (preserved in the laboratory) and used to prepare a standard curve. Serial dilutions of 10-1 were performed. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 10 -9Using diluted plasmids as templates, reaction systems were prepared, and the component ratios are shown in Table 2.
[0057] Table 2 Components of the reaction system
[0058] Absolute quantitative PCR was performed according to the reaction program. The program was: 95℃ for 30s; 95℃ for 15s, 60℃ for 60s, for a total of 40 cycles; a standard curve was constructed with lg10 (copy number) as the X-axis and Ct value as the Y-axis, as shown in Figure 3. The absolute quantitative standard system for the standard curve was established, and the standard curve was y = -1.3916x + 33.674, R0. 2 The value is 0.9929, and the horizontal axis represents the CLas pathogen copy number to base 10. The copy number of CLas pathogen in the citrus psyllid is calculated using the following formula.
[0059] Copy number = [nucleic acid concentration (ng / μL) × 10] -9 [×6.02×1023] / (number of bases×660).
[0060] Figure 2 shows the bacterial collection status of citrus psyllids after RNA interference; the ordinate represents the Clas pathogen copy number to base 10. At 24 h: P = 0.032 < 0.05, indicating a significant difference; at 48 h: p = 0.005 < 0.01, indicating a highly significant difference. "*" represents a significant difference, and "**" represents a highly significant difference. The results indicate that at both 24 h and 48 h of collection time, the experimental group showed a lower dsUFD4 pathogen copy number compared to the control group dsGFP.
[0061] Example 3
[0062] Protein structure prediction of the UFD4 gene revealed the presence of a HECT-type structure. To verify the impact of this structure on ubiquitination degradation function, a commercially available reagent, E3-HECT-type inhibitor (trade name: Heclin, purchased from Taoshu Biotechnology Co., Ltd.), was purchased to specifically inhibit the HECT-type structure.
[0063] The HECT inhibitor was diluted 10% using H2O as a solvent. 2 Microinjection was performed on 4th-5th instar citrus psyllid nymphs at a volume of 20 nL. H2O was used as a control. Total RNA (TRNA) was extracted from 8 nymphs at 24 h and 48 h after treatment (TRizol method), reverse transcribed into cDNA, and the expression level of DcUFD4 was detected by real-time quantitative PCR. The results are shown in Figure 4.
[0064] Figure 4 shows the relative expression of the DcUFD4 gene after RNAi, with blue representing the H2O treatment group and red representing the E3-HECT inhibitor treatment group. At 24h: P = 0.5023 > 0.05, no significant difference was observed; at 48h: P = 0.0032 < 0.01, a highly significant difference was observed. "ns" indicates no significant difference, and "**" indicates a highly significant difference. The results indicate that diluting the HECT inhibitor by 10... 2 After doubling, it failed to inhibit the expression of UFD4 gene after 24 hours, but it was able to significantly inhibit the expression level of UFD4 gene after 48 hours.
[0065] Fifth instar nymphs of the citrus psyllid, which were successfully inhibited by the E3-UFD4 inhibitor, were fed to infected citrus branches after 48 hours. Every 24 hours, 15 adult citrus psyllids were collected to extract total RNA (TRizol method), which was reverse transcribed into cDNA and the copy number of Clas pathogen was detected. The results are shown in Figure 5.
[0066] Figure 5 shows the bacterial collection status of citrus psyllids after treatment with the E3-HECT inhibitor; the ordinate represents the Clas pathogen copy number to base 10. At 24 h: P = 0.0071 < 0.01, indicating a highly significant difference; at 48 h: P = 0.0024 < 0.01, indicating a highly significant difference. "**" represents a highly significant difference. The results indicate that at both 24 h and 48 h after collection, the bacterial copy number in the experimental group was significantly lower than that in the control group (H2O).
[0067] Comparative Example 1
[0068] The E3-RING inhibitor (trade name: Disulfiram, purchased from: Taoshu Biotechnology Co., Ltd.) was treated in the same way as in Example 3 above, and the change in DcUFD4 expression level was detected by real-time quantitative PCR. The results are shown in Figure 6.
[0069] Figure 6 shows the expression level of DcUFD4 in the citrus psyllid after treatment with the E3-RING inhibitor; blue represents the DcUFD4 expression level in the H2O-treated group, and red represents the DcUFD4 expression level in the citrus psyllid after treatment with the E3-RING inhibitor. At 24 h: P = 0.9114 > 0.05, no significant difference was observed; at 48 h: P = 0.9892 > 0.05, no significant difference was observed, and "ns" indicates no significant difference. The results indicate that there was no significant change in the UFD4 gene expression level at both 24 h and 48 h after E3-RING inhibitor injection; therefore, the RING conformation cannot inhibit the UFD4 gene expression level.
[0070] Therefore, this application first screened the citrus psyllid ubiquitin ligase UFD4 gene (DcUFD4) from the transcriptome database, designed specific primers to synthesize dsRNA, and used RNA interference to silence the DcUFD4 gene. The gene silencing efficiency was then detected. Subsequently, the gene was placed on citrus branches infected with Huanglongbing (HLB) and fed to investigate the effect of this gene on HLB infection. Based on the above results, we conclude that the ubiquitin ligase UFD4 gene is positively correlated with HLB Clas, and that substrate proteins in its ubiquitin pathway can mediate and influence Clas's immune defense mechanisms, providing more methods and pathways for further research on the control of HLB.
[0071] Simultaneously, by injecting an E3-HECT type inhibitor to inhibit ubiquitin ligase, the inhibition efficiency was measured. Subsequently, the samples were placed on branches infected with Huanglongbing (HLB) and fed to investigate the effect of ubiquitin ligase on HLB infection. According to the experimental results, among the various ubiquitin ligase conformations, the UFD4 gene belongs to the HECT type. Inhibiting the entire HECT conformation successfully suppressed the acquisition efficiency of HLB Clas, indicating that this conformation is a key factor in the control of HLB Clas.
[0072] Although the above embodiments have provided a detailed description of this application, they are only some embodiments of this application, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of this application.
Claims
1. The use of biological material related to the citrus psylla ubiquitin ligase UFD4 gene in the prevention and control of citrus huanglongbing, characterized in that, The amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of the citrus psyllid is shown in SEQ ID NO.
1.
2. Use according to claim 1, characterized in that, The biomaterial includes the citrus psyllid ubiquitin ligase UFD4 gene and / or the protein encoded by the citrus psyllid ubiquitin ligase UFD4 gene.
3. Use according to claim 1, characterized in that, The nucleotide sequence of the citrus psyllid ubiquitin ligase UFD4 gene is shown in SEQ ID NO.
2.
4. A primer pair for amplifying the ubiquitin ligase UFD4 gene of citrus psyllid, characterized in that, The primer pair includes: PT-F and PT-R with nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; and the amino acid sequence of the protein encoded by the citrus psyllid ubiquitin ligase UFD4 gene is shown in SEQ ID NO.
1.
5. An inhibitor of expression of the Citrus Huanglongbing Ubiquitin Ligase UFD4 gene, characterized in that, The active ingredient of the inhibitor includes: an E3-HECT type inhibitor; The amino acid sequence of the protein encoded by the ubiquitin ligase UFD4 gene of the citrus psyllid is shown in SEQ ID NO.
1.
6. The application of the inhibitor according to claim 5 in the study of ubiquitination degradation function during Huanglongbing infection.
7. The application of the inhibitor according to claim 5 in the control of citrus Huanglongbing.
8. A method for preventing and controlling citrus huanglongbing, characterized by, Includes the following steps: Expression of the ubiquitin ligase UFD4 gene in citrus psyllids was silenced.
9. The method of claim 8, wherein, The silencing includes: silencing using the inhibitor of claim 5.
10. The method of claim 9, wherein, When using the inhibitor to induce silencing, the following steps are included: placing the inhibitor in a portion that is edible for the citrus psyllid.
11. The method of claim 10, wherein, The parts that can be consumed by citrus psyllids include: branches, buds, young leaves, or sap from tender shoots.
12. A method for detecting the expression level of the ubiquitin ligase UFD4 gene in citrus psyllids, characterized in that, Includes the following steps: RNAi analysis was performed using dsRNA synthesized from the citrus psyllid ubiquitin ligase UFD4 gene to obtain the expression level; the amino acid sequence of the protein encoded by the citrus psyllid ubiquitin ligase UFD4 gene is shown in SEQ ID NO.1; The primer pairs for dsRNA synthesis include: RNAi-F with nucleotide sequences as shown in SEQ ID NO.5 and RNAi-R with SEQ ID NO.6; The primer pairs for the RNAi analysis include qPCR-F with nucleotide sequences as shown in SEQ ID NO.7 and qPCR-R with SEQ ID NO.8.