Application of dcPDF gene and dsRNA in controlling citrus psylla

Abstract

The application discloses DcPDF The application discloses application of a gene and a dsRNA in preventing and treating citrus psylla DcPDF The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The application can significantly inhibit the development of the ovary of a female citrus psylla and significantly reduce the oviposition amount by feeding the citrus psylla with a dsRNA targeting a nucleotide sequence shown as SEQ ID NO. 13 to reduce the expression level of a neuropeptide pigment dispersing factor gene. The application provides a new efficient target for green prevention and treatment of citrus psylla based on RNA interference technology, and helps to realize pesticide reduction and efficiency increase, and delay development of pest resistance.

Description

Technical Field

[0001] This invention belongs to the field of green control of agricultural pests, specifically involving DcPDF Application of genes and their dsRNA in the control of citrus psyllids. Background Technology

[0002] Candidate phloem bacilli as pathogens of citrus Huanglongbing (HLB) Candidatus Liberibacter asiaticus, C Citrus psyllids (Las) cannot be cultured in vitro and lack effective control agents. Diaphorina citri Citrus psyllids are the natural vector of this disease, therefore, effective control of citrus psyllids is crucial to curbing the spread of Huanglongbing (HLB) and ensuring the healthy and sustainable development of the citrus industry. Currently, the control of citrus psyllids mainly relies on chemical pesticides, supplemented by agricultural management, physical control, and biological control measures. While chemical control is fast-acting, long-term use can lead to pesticide resistance in pests, and also causes pesticide residues and environmental pollution. Agricultural and physical control methods (such as uniform shoot growth and hanging yellow sticky traps) are often costly to manage and difficult to eradicate on a large scale. Biological control methods utilizing natural enemies or microbial pesticides are highly dependent on environmental conditions and act relatively slowly, making them difficult to control rapidly when citrus psyllid infestations occur on a large scale. Overall, existing control technologies have significant limitations in terms of environmental safety, sustainability, and control efficiency.

[0003] Studies have shown that it carries Huanglongbing bacteria. C Las significantly enhances the reproductive capacity of female citrus psyllids, increasing their oviposition by 30%-50%, creating a vicious cycle of "pathogen-driven vector proliferation," but its underlying molecular regulatory mechanism remains unclear. Pigment-dispersing factor (PDF) is an evolutionarily conserved neuropeptide that has been shown in model insects such as fruit flies to regulate reproductive development (e.g., activating vitellogenin synthesis) through signaling pathways such as cAMP / PKA. However, the function of PDF in the reproductive regulation of vector insects (especially citrus psyllids), particularly its role in their interaction with pathogens, has not been reported. In recent years, RNA interference (RNAi) technology has shown great potential in agricultural pest control due to its high target specificity and environmental friendliness. Summary of the Invention

[0004] Based on the shortcomings and deficiencies of existing technologies, this invention aims to provide... DcPDF Application of genes and their dsRNA in the control of citrus psyllids.

[0005] This invention is the first to propose the use of citrus psyllids. PDFThis gene serves as a novel target for RNAi control. It may be deeply involved in regulating key physiological processes of psyllids. This invention not only provides a target for this gene... DcPDF The highly efficient and specific dsRNA sequence of the gene and its synthesis method also provide an innovative solution for developing a green, precise, and efficient new strategy for the control of citrus psyllids, which is expected to overcome many shortcomings of existing control methods.

[0006] The first objective of this invention is to provide a method for inhibiting citrus psyllids. DcPDF The application of gene expression in the control of citrus psyllids, as described above DcPDF The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Preferably, the application is for suppressing citrus psyllids. DcPDF Application of gene expression in regulating the reproduction of female citrus psyllids.

[0008] Preferably, the application is for suppressing citrus psyllids. DcPDF Application of gene expression in inhibiting ovarian development or reducing egg production in female citrus psyllids.

[0009] Preferably, the method for inhibiting citrus psyllids DcPDF Gene expression is achieved through targeted feeding DcPDF Reduced dsRNA of genes DcPDF Genes, the targeted DcPDF The nucleotide sequence of the target region of the gene's dsRNA is shown in SEQ ID NO.13.

[0010] Preferably, the target DcPDF The concentration of the gene's dsRNA was 500 ng / µL.

[0011] The second objective of this invention is to provide a targeted DcPDF The dsRNA of the gene, and the nucleotide sequence of its target region are shown in SEQ ID NO.13.

[0012] Preferably, the method for preparing the dsRNA includes the following steps:

[0013] S1. Total RNA was extracted from citrus psyllids and reverse transcribed into cDNA;

[0014] S2. Design a specific primer pair with the T7 sequence, and perform PCR amplification using the cDNA obtained in step S1 as a template to obtain a DNA fragment containing the T7 promoter sequence; the nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID NO.10.

[0015] S3. Using the DNA fragment containing the T7 promoter sequence obtained in step S2 as a template, perform in vitro transcription using an in vitro transcription kit and purify the DNA to obtain the targeted DNA fragment. DcPDF dsRNA of genes.

[0016] A third objective of this invention is to provide a formulation for controlling citrus psyllids, containing the aforementioned targeted... DcPDF The dsRNA of the gene is the active ingredient.

[0017] A fourth object of the present invention is to provide the application of the described formulation in the control of citrus psyllids.

[0018] Preferably, the application includes the following steps: feeding citrus psyllids with the preparation.

[0019] The beneficial effects of this invention are:

[0020] The above-mentioned targets can be achieved through methods such as feeding. DcPDF The dsRNA of the gene was introduced into the citrus psyllid, effectively silencing its gene. DcPDF This invention utilizes genes to inhibit the development of ovaries in female citrus psyllids and reduce their egg production, thereby controlling citrus psyllids and reducing the occurrence of Huanglongbing (HLB). This invention provides an innovative solution for developing green, precise, and efficient new strategies for citrus psyllid control, helping to reduce pesticide use while increasing efficacy and delaying the development of pesticide resistance in pests. Attached Figure Description

[0021] Figure 1 Multiple comparisons of the amino acid sequence of neuropeptide pigment dispersion factor DcPDF for citrus psyllid with four other closely related insect species.

[0022] Figure 2 Phylogenetic analysis of the amino acid sequence of neuropeptide pigment dispersion factor DcPDF of the citrus psyllid with nine other insect species.

[0023] Figure 3 Neuropeptide pigment dispersing factor for citrus psyllids DcPDF Spatiotemporal expression profiles of genes; where A represents the expression of genes in citrus psyllids at different developmental stages. DcPDF The relative expression level of genes, B is DcPDF Relative expression levels of genes in different tissues of 8-day-old female citrus psyllids; different letters indicate significant differences ( p <0.05).

[0024] Figure 4 Female citrus psyllids treated with dsDcPDF for 1, 2, and 3 days. DcPDF Relative gene expression levels; ** and *** represent the expression levels of female citrus psyllids after treatment with dsDcPDF. p <0.01、p The level <0.001 decreased significantly. DcPDF Relative gene expression levels; Independent t tests, SPSS 20.0 software.

[0025] Figure 5 Effects of dsDcPDF treatment on ovarian development morphology in female citrus psyllids; scale bar is 200 µm.

[0026] Figure 6 The effect of dsDcPDF treatment on the oviposition rate of female citrus psyllids; *** indicates that dsDcPDF treatment significantly reduced the oviposition rate per female citrus psyllid at P < 0.001; Independent t tests, SPSS 20.0 software. Detailed Implementation

[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0028] Example 1

[0029] 1. Bioinformatics analysis of neuropeptide pigment dispersion factor DcPDF in citrus psyllids

[0030] Based on transcriptome sequencing data of citrus psyllids and comparison information from the NCBI database, citrus psyllids were obtained. DcPDF The coding sequence of the gene (SEQ ID NO.1, accession number: MG550183.1) was identified, and specific primers (SEQ ID NO.2 and SEQ ID NO.3) were designed for PCR amplification and verification. Using DNAMAN software, the verified amino acid sequence of the citrus psyllid DcPDF (SEQ ID NO.4) was compared with that of four other insect species (Ls: planthopper). Laodelphax striatellus NI: Brown planthopper Nilaparvata lugens Ld: Spotted Lanternfly Lycorma delicatula Lh: Bean Pod Mirid Bug Lygus hesperus Multiple alignments were performed on the PDF amino acid homologous sequences of the citrus psyllid DcPDF gene. The alignment results showed that the open reading frame (ORF) length of the DcPDF gene was 249 bp. Figure 1 Furthermore, using MEGA 7.0 software, the Neighbor-Joining (NJ) method was employed to study the citrus psyllid and nine other closely related insect species (including Ma: wheat aphid). Macrosiphum avenae Sg: Wheat aphid Schizaphis graminum Dm: Drosophila melanogaster Drosophila melanogaster Ag: Cotton aphidAphis gossypii Ld: Spotted Lanternfly Lycorma delicatula Lh: Bean Pod Mirid Bug Lygus hesperus NI: Brown planthopper Nilaparvata lugens Ls: Gray planthopper Laodelphax striatellus And Bt: whitefly Bemisia tabaci Cluster analysis was performed on the PDF amino acid sequences of [the data] to construct a phylogenetic tree. The constructed phylogenetic tree is shown below. Figure 2 As shown.

[0031] 2. Real-time quantitative PCR (qRT-PCR) analysis

[0032] (1) Sample preparation

[0033] Different tissues (midgut, fat body, head, bacterial-containing body, and ovary) of citrus psyllids at different developmental stages (eggs, 1st-5th instar nymphs, and adults on days 1, 5, and 10 after emergence) and 8-day-old females were collected. Females were briefly anesthetized with CO2 and then dissected in PBS buffer. The obtained tissue samples were rapidly placed in 1.5 mL RNase-free centrifuge tubes containing 30–50 μL of lysis buffer, with each sample tested in triplicate.

[0034] (2) Total RNA extraction

[0035] Using Takara's MiniBEST Universal RNA Extraction Kit (catalog number 9767), follow these steps:

[0036] 1) Place the tissue sample in a 1.5 mL RNase-free centrifuge tube, add 600 μL Buffer RL, and grind on ice for 5 min until fully lysed and no obvious precipitation.

[0037] 2) Centrifuge the lysate at 4 °C and 12,000 rpm for 5 min. Carefully transfer the supernatant to a new RNase-free centrifuge tube.

[0038] 3) Place the gDNA Eraser Spin Column onto a 2 mL collection tube, transfer the supernatant to the column, and centrifuge at 12,000 rpm for 1 min. Discard the column and retain the filtrate in the collection tube.

[0039] 4) Add an equal volume of 70% ethanol to the filtrate and mix well. Immediately transfer the mixture to an RNA Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate. Return the column to the collection tube.

[0040] 5) Add 500 μL of Buffer RWA to the column, centrifuge at 12,000 rpm for 30 s, and discard the filtrate. Then add 600 μL of Buffer RWB, centrifuge at 12,000 rpm for 30 s, and discard the filtrate.

[0041] 6) Reposition the column onto the collection tube and centrifuge at 12,000 rpm for 2 min to thoroughly dry the membrane. Finally, transfer the column to a 1.5 mL RNase-Free collection tube, add 20–30 μL of RNase-free water to the center of the membrane, incubate at room temperature for 5 min, and centrifuge at 12,000 rpm for 2 min to elute RNA.

[0042] (3) cDNA synthesis

[0043] Using Takara's PrimeScript™ RT reagent Kit with gDNA Eraser (PerfectReal Time) (catalog number RR047Q), follow these steps:

[0044] 1) Genomic DNA removal reaction: Prepare the reaction system according to Table 1 and incubate at 42℃ for 2 min. After the reaction, store the sample at -20℃.

[0045] Table 1 Genomic DNA Removal Reaction System

[0046]

[0047] 2) Reverse transcription reaction: The reaction system was prepared according to Table 2 using Takara's Prime Script RT reagent Kit (RR047A). After thorough mixing, the mixture was placed in a PCR instrument and reacted under the following conditions: 37℃ for 15 min; 85℃ for 5 s. The reaction product was stored at -20℃ for later use.

[0048] Table 2 Reverse transcription reaction system

[0049]

[0050] (4) qRT-PCR detection

[0051] 1) The reaction was performed using ChamQ SYBR qPCR Master Mix (catalog number Q331) from Nanjing Novizan Biotechnology Co., Ltd. on an ABI 7500 real-time quantitative PCR instrument. The reaction system configuration is shown in Table 3.

[0052] Table 3. Real-time PCR reaction system

[0053]

[0054] 2) Used for detection DcPDF The nucleotide sequences of the upstream and downstream primers for gene expression modulation are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. DcActin This is an internal reference gene, and the nucleotide sequences of its upstream and downstream primers for qRT-PCR are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively. Three technical replicates were set up for each sample.

[0055] 3) The PCR reaction program is as follows: pre-denaturation at 95℃ for 10 min; followed by 40 cycles of amplification (denaturation at 95℃ for 10 s, annealing / extension at 60℃ for 32 s).

[0056] qRT-PCR results showed that DcPDF The gene is expressed from the egg stage to the adult stage of the citrus psyllid, and the expression level gradually increases from the larval stage to the adult stage. Figure 3 A in the molecule is expressed in different tissues, with the highest expression level in the head, followed by a relatively high expression level in the ovary. Figure 3 (B in the middle).

[0057] 3. Gene silencing experiments

[0058] (1) Design and synthesis of dsRNA

[0059] Designing targets using the E-RNAi online platform DcPDF Primers for amplifying the target region of gene-specific dsRNA (the nucleotide sequences of the upstream and downstream primers are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively), and a targeted-enhanced green fluorescent protein were designed. EGFP Primers (SEQ ID NO. 11 and SEQ ID NO. 12) for amplifying the target region of the dsRNA of the gene (accession number: LC336961.1) were used as negative controls.

[0060] Using 2×SuperTaq PCR Star Mix (Dye) (Catalog No. A002) from Kangrun Biotechnology Co., Ltd., PCR amplification was performed using female citrus psyllid cDNA and a recombinant plasmid carrying the EGFP gene as templates, and the aforementioned designed specific targeting region amplification primers. DcPDF and EGFPThe nucleotide sequences of the target regions of the genetically engineered dsRNAs (dsDcPDF and dsEGFP) are SEQ ID NO.13 and SEQ ID NO.14, respectively. The reaction system is shown in Table 4. Reaction conditions: 98℃ for 2 min; followed by 30 cycles (98℃ for 15 s, 60℃ for 15 s, 72℃ for 30 s); and a final extension at 72℃ for 5 min. After verification by agarose gel electrophoresis, the PCR products were purified by gel extraction to obtain the gel-recovered products.

[0061] Table 4 PCR reaction system

[0062]

[0063] Table 5 In vitro transcription reaction system

[0064]

[0065] In vitro transcription was performed using the T7 High Yield Transcription Kit (catalog number: TR101-01) from Nanjing Novizan Biotechnology Co., Ltd. The reaction system is shown in Table 5. The purification steps for dsRNA are as follows:

[0066] 1) Transfer the transcription reaction solution to a 1.5 mL RNase-free centrifuge tube and add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) mixture.

[0067] 2) After mixing thoroughly, centrifuge at 12,000 rpm for 2 min at 4 °C. Transfer the supernatant to a new tube, add 2.5 times the volume of anhydrous ethanol to the supernatant, mix well, and then incubate on ice for 5 min.

[0068] 3) Centrifuge at 4℃ and 12,000 rpm for 10 min, and discard the supernatant. Briefly open the lid to air dry the precipitate.

[0069] 4) Add 20 μL of RNase-free water to dissolve the precipitate.

[0070] 5) Take 1 µL of the purified product for concentration determination and verification by agarose gel electrophoresis.

[0071] (2) dsRNA feeding treatment

[0072] RNAi experiments were conducted using a Parafilm membrane-clamped nutrient solution feeding method. dsDcPDF or dsEGFP was diluted to 500 ng / µL with a 20% sucrose solution, and an appropriate amount was added dropwise between two layers of Parafilm sealing film to create a feeding chamber. Emergent female citrus psyllids (10 individuals per group, 3 biological replicates) were introduced into the feeding device and fed continuously for 1, 2, and 3 days in an artificial climate chamber.

[0073] (3) Detection of the silence effect

[0074] Whole insect samples were collected 1, 2, and 3 days after feeding with dsRNA, and RNA was extracted for qRT-PCR to detect the silencing effect. qRT-PCR results showed that, compared with the dsEGFP control group, the silencing effect was significantly reduced in female citrus psyllids treated with dsDcPDF. DcPDF Gene expression was significantly reduced ( Figure 4 ).

[0075] (4) DcPDF Effects of gene silencing on ovarian development of citrus psyllids

[0076] Female adult worms fed with dsDcPDF for 2 days were dissected in 1×PBS buffer and fixed in 4% paraformaldehyde solution (Beyotime, catalog number: P0099) at 25℃ for 30 min. The fixed ovaries were washed twice with 1×PBS buffer (10 min each time), mounted, and observed and photographed under a super depth-of-field microscope (VHX-500). Ten ovaries were observed in each treatment group, with three replicates. The results showed that compared with the dsEGFP control group, ovarian development in the dsDcPDF treatment group was significantly delayed. Figure 5 ).

[0077] (5) DcPDF Effects of gene silencing on oviposition of citrus psyllids

[0078] After two days of dsRNA feeding, male and female adults were paired 1:1 and placed in white mesh bags (15×20 cm) along with healthy Murraya paniculata shoots. These bags were then placed in an artificial climate incubator (temperature 26±1℃, relative humidity 55±5%, photoperiod 14L:10D). The number of eggs laid by each female was recorded every other day, and new shoots were replaced until all females died. The experiment was repeated three times, with 15 pairs of psyllids observed in each replicate. The results showed that the number of eggs laid per female was significantly reduced in the dsDcPDF treatment group compared to the dsEGFP control group. Figure 6 ), to prove the target DcPDF The dsRNA of the gene can effectively inhibit the reproductive capacity of the citrus psyllid.

Claims

1. Suppressing citrus psyllids DcPDF The application of gene expression in inhibiting ovarian development or reducing oviposition in female citrus psyllids, as described above. DcPDF The nucleotide sequence of the gene is shown in SEQ ID NO.1; the gene described for inhibiting citrus psyllids DcPDF Gene expression is achieved through targeted feeding DcPDF Reduced dsRNA of genes DcPDF Gene expression, the targeted DcPDF The nucleotide sequence of the target region of the gene's dsRNA is shown in SEQ ID NO.

13.

2. The application according to claim 1, characterized in that, The target DcPDF The concentration of the gene's dsRNA was 500 ng / µL.

3. The application according to claim 1, characterized in that, The target DcPDF The method for preparing dsRNA of a gene includes the following steps: S1. Total RNA was extracted from citrus psyllids and reverse transcribed into cDNA; S2. Design specific primer pairs containing the T7 sequence, and perform PCR amplification using the cDNA obtained in step S1 as a template to obtain a DNA fragment containing the T7 promoter sequence; prepare the targeted... DcPDF The nucleotide sequence of the upstream primer of the primer pair for the dsRNA of the gene is shown in SEQ ID NO.9, for the preparation of the targeted gene. DcPDF The nucleotide sequence of the downstream primer of the primer pair for the dsRNA of the gene is shown in SEQ ID NO.10; S3. Using the DNA fragment containing the T7 promoter sequence obtained in step S2 as a template, perform in vitro transcription using an in vitro transcription kit and purify the DNA to obtain the targeted DNA fragment. DcPDF dsRNA of genes.

4. A preparation for controlling citrus psyllids, characterized in that, Contains targeted DcPDF The dsRNA of the gene is the active ingredient, as described above. DcPDF The nucleotide sequence of the gene is shown in SEQ ID NO.1; the target... DcPDF The nucleotide sequence of the target region of the gene's dsRNA is shown in SEQ ID NO. 13; preparation of the target... DcPDF The nucleotide sequence of the upstream primer of the primer pair for the dsRNA of the gene is shown in SEQ ID NO.9, for the preparation of the targeted gene. DcPDF The nucleotide sequence of the downstream primer of the primer pair for the gene's dsRNA is shown in SEQ ID NO.

10.

5. The application of the formulation according to claim 4 in inhibiting ovarian development or reducing the egg production of female citrus psyllids.

6. The application according to claim 5, characterized in that, Includes the following steps: The aforementioned preparation was used to feed citrus psyllids.

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