Dcfaxo gene and its application in preventing and treating citrus psylla

Abstract

The application discloses DcFoxO A gene and application thereof in preventing and treating Toxoptera citricola. DcFoxO The nucleotide sequence of the gene is shown as SEQ ID NO. 1. The application discloses a gene of Toxoptera citricola DcFoxO The gene is highly expressed in the midgut of Toxoptera citricola, and the gene is targeted by feeding dsRNA to Toxoptera citricola DcFoxO It is found that the oviposition period of a female Toxoptera citricola is shortened, the oviposition amount is reduced, the ovary development is abnormal, and the titre of Candidatus Liberibacter asiaticum is reduced, the prevention and treatment effect on Toxoptera citricola is remarkable, and the gene has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pest control technology, specifically involving DcFoxO Genes and their application in the control of citrus psyllids. Background Technology

[0002] Citrus Huanglongbing (HLB) is a devastating disease of the citrus industry, mainly caused by Asian species of bacteria belonging to the genus *Phyllostachys*. Candidatus Liberibacter asiaticus, C This is caused by Las, and transmitted through its vector insect, the Asian citrus psyllid (Las). Diaphorina citri The Asian citrus psyllid (Kuwayama) spreads rapidly in the field. Domestically, it is usually referred to as the citrus psyllid. The citrus psyllid primarily acquires and spreads Huanglongbing (HLB) pathogen by sucking sap from the phloem of citrus trees. Reports indicate that infected citrus psyllids exhibit significantly increased reproductive capacity and impaired physiological activity, but the underlying mechanisms are currently unclear. Furthermore, since HLB pathogens cannot be artificially cultured, it is difficult to propose effective strategies for controlling HLB based solely on the pathogen itself.

[0003] Therefore, the main direction at present is to explore and apply the reproductive-related genes of citrus psyllids and develop targeted environmentally friendly pesticides to achieve the goal of controlling citrus psyllids, which is of great significance for the prevention and control of Huanglongbing (HLB). Summary of the Invention

[0004] Based on the shortcomings and defects of existing technologies, this invention aims to provide... DcFoxO Genes and their application in the control of citrus psyllids.

[0005] Forkhead box transcription factor FoxO ( Forkhead Box O ) are a class of conserved transcription factors that regulate gene expression by binding to the promoters of target genes. They can interact with JH (juvenile hormone) and 20E (ecdysone). The interaction of hydroxyecdysone, vitellogenin (Vg), and other proteins jointly regulates the growth and development of insects, utilizing the citrus psyllid. FoxO ( DcFoxO The control of citrus psyllids has good development prospects.

[0006] The first objective of this invention is to provide citrus psyllids. DcFoxO The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0007] A second objective of this invention is to provide a way to suppress the aforementioned DcFoxOApplication of gene expression in suppressing the reproductive development of citrus psyllids.

[0008] Preferably, the inhibition of the reproductive development of citrus psyllids involves prolonging the pre-oviposition period of female citrus psyllids, shortening the oviposition period of female citrus psyllids, reducing the number of eggs laid by female citrus psyllids, inhibiting the development of female ovaries of female citrus psyllids, or reducing the titer of Huanglongbing bacteria in the ovaries of female citrus psyllids.

[0009] Preferably, the inhibition DcFoxO Gene expression was achieved by feeding citrus psyllids targeted gene expression. DcFoxO The target is achieved through the dsRNA of the gene. DcFoxO The nucleotide sequence of the target region of the gene's dsRNA is shown in SEQ ID NO.8.

[0010] Preferably, the target DcFoxO The concentration of the gene's dsRNA was 100 ng / µL.

[0011] The third objective of this invention is to provide a targeted DcFoxO The dsRNA of the gene, and the nucleotide sequence of its target region, as shown in SEQ ID NO.8.

[0012] The fourth objective of this invention is to provide a method for preparing targeted [treatments]. DcFoxO The primer pair for the dsRNA of the gene, wherein the nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO.6, and the nucleotide sequence of the downstream primer of the primer pair is shown in SEQ ID NO.7.

[0013] The fifth objective of this invention is to provide a formulation for controlling citrus psyllids, wherein the formulation contains the dsRNA as an active ingredient.

[0014] A sixth object of the present invention is to provide the application of the described formulation in the control of citrus psyllids.

[0015] Preferably, the application includes the following steps: feeding citrus psyllids with the preparation. The beneficial effects of the present invention are:

[0016] This invention discovered the citrus psyllid DcFoxO The gene is highly expressed in the midgut of the citrus psyllid. Targeted gene expression was detected through feeding. DcFoxO The dsRNA analysis revealed that the oviposition capacity of the citrus psyllid decreased, ovarian development was abnormal, and the titer of Huanglongbing fungus in citrus was reduced. The results showed that the dsRNA had a significant effect on the control of the citrus psyllid, providing a new approach for the development of new pesticides for the control of the citrus psyllid and showing good application prospects. Attached Figure Description

[0017] Figure 1 It is a female citrus psyllid. DcFoxOSpatiotemporal expression profiles of genes; where A is... DcFoxO Gene expression levels in different tissues of female citrus psyllids, B represents... DcFoxO The expression levels of the gene in the ovaries of infected and uninfected psyllids at different developmental stages are plotted, with different letters indicating significant differences (p<0.05).

[0018] Figure 2 It is a feeding target DcFoxO The graph shows the changes in the reproductive capacity of female citrus psyllids after dsRNA administration; where A represents the changes in female citrus psyllids before oviposition, B represents the changes during the oviposition period, and C represents the changes in the number of eggs laid. C Las-: Uninfected citrus psyllid population C Las+: A population of citrus psyllids carrying pathogens; ds GFP For feeding ds GFP As a comparison, ds FoxO Targeted feeding DcFoxO dsRNA; * and *** indicate significant differences at p<0.05 and p<0.001 levels, respectively.

[0019] Figure 3 It is a feeding target DcFoxO A diagram showing the changes in ovarian development in female citrus psyllids after dsRNA administration.

[0020] Figure 4 It is a feeding target DcFoxO Graph showing the change in titer of citrus Huanglongbing bacteria in the ovaries of female citrus psyllids after dsRNA administration. Detailed Implementation

[0021] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0022] Example 1

[0023] 1. Rearing of the test insects

[0024] Citrus psyllid populations not carrying Huanglongbing fungus (uninfected psyllids, C Las-): Laboratory-bred on lemons that do not carry Huanglongbing (HLB).

[0025] Citrus psyllid populations carrying Huanglongbing fungus (fungi-carrying psyllids) C Las+: Laboratory-bred on lemons carrying Huanglongbing (HLB).

[0026] Both populations of citrus psyllids and lemons were placed in an indoor artificial climate chamber (RXA-436) with the following parameters: temperature 26 ± 1℃, relative humidity 60 ± 5%, and photoperiod L:D = 14:10h.

[0027] 2.DcFoxO spatiotemporal expression pattern analysis

[0028] Different tissues (head, midgut, fat body, ovary) from 9-day-old female citrus psyllids and ovaries from females at different ages (5, 9, and 13 days post-emergence) were collected by dissection. Thirty worms were dissected in each group, with three replicates. RNA was extracted using Trizol lysis buffer (Takara, catalog number 9109); first-strand cDNA was synthesized using the PrimeScript™ RT reagent Kit (Takara, catalog number RR047A); Actin As an internal reference gene, it is used for detection. DcFoxO (The nucleotide sequence is shown in SEQ ID NO. 1) expression levels in different tissues and developmental stages of female citrus psyllids. Specifically, the qRT-PCR reaction system was prepared using ChamQSYBR qPCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd., catalog number Q311) and qRT-PCR primers, and the expression levels were measured using a real-time quantitative PCR instrument. DcFoxO The level of expression.

[0029] Actin The qRT-PCR primer sequences are: q Actin -F (SEQ ID NO.2): 5'-TGTTCCAACCTTCCTTCCTG-3', q Actin -R (SEQ ID NO.3): 5'-GTGTTGGCGTACAGGTCCTT-3'; DcFoxO The qRT-PCR primer sequences are: q DcFoxO -F (SEQ ID NO.4): 5'-AGGGCTTCGATTGTAATGTT-3', q DcFoxO -R (SEQ ID NO. 5): 5'-GCTGAGATGTAGACGATGAC-3'.

[0030] The results showed that DcFoxO High expression in the midgut of female citrus psyllids ( Figure 1 A in DcFoxO The expression levels of the gene in both infected and uninfected psyllid ovaries increased with development, and the expression level in infected psyllid ovaries was consistently higher than that in the uninfected group. Figure 1 (B in the middle).

[0031] 3. Changes in the reproductive capacity of citrus psyllids after feeding with dsRNA

[0032] Take samples with bacteria ( C Las+) and not carrying bacteria ( CSeven-day-old female citrus psyllids from two populations (Las-) were fed with ds at a concentration of 100 ng / µL. DcFoxO (targeted) DcFoxO The dsRNA, whose nucleotide sequence is shown in SEQ ID NO.8, has the following synthesis primers: ds DcFoxO -F: 5'-gatcactaatacgactcactatagggGATATCGCAAGCCATCCAGT-3', SEQ ID NO.6; ds DcFoxO -R: 5'-gatcactaatacgactcactatagggGTCCGGACTGAGCTGAAAAT-3', SEQ ID NO.7) lowered DcFoxO The expression level, in order to feed ds GFP (Its nucleotide sequence is shown in SEQ ID NO.11; its synthesis primer is: ds GFP -F: 5'-gatcactaatacgactcactatagggACTCCAGCAGGACCATGTGATC-3', SEQ ID NO.9; ds GFP -R: 5'-gatcactaatacgactcactatagggACCTGAAGTTCATCTGCACCAC-3' (SEQ ID NO.10) served as a control. Each population and each treatment was repeated three times, with 30 insects collected from each treatment. After feeding for 48 h, each female insect was paired with a male insect at a 1:1 ratio and placed on the tender shoots of healthy lemon trees. Each shoot containing insects was covered with a mesh bag. After completion, the lemon trees were placed in an artificial climate incubator with consistent conditions. Oviposition was observed every 24 h, and each pair of psyllids was transferred to a new shoot to continue oviposition. The pre-oviposition period, oviposition period, and number of eggs laid were recorded until all female insects died. The results showed that knocking down... DcFoxO The expression of this substance can inhibit the reproductive capacity of the citrus psyllid. Figure 2 Specifically, this manifests as a prolonged pre-spawning period ( Figure 2 A) Shorten the spawning period ( Figure 2 (B) Reduce egg production ( Figure 2 (C in the middle).

[0033] 4. Changes in ovarian development in citrus psyllids after feeding with dsRNA

[0034] Nine-day-old female citrus psyllids were collected and fed with ds at a concentration of 100 ng / µL. DcFoxO In one study, ovaries were dissected and mounted on slides 48 h after feeding with the same concentration of dsGFP as a control. Each treatment was repeated three times, with 10 ovaries collected each time. The samples were then observed and photographed using a super-depth-of-field 3D microscope (VHX-500). The results showed that knockdown...DcFoxO The expression of this substance can inhibit the development of ovaries in citrus psyllids. Figure 3 ).

[0035] 5. Changes in Huanglongbing bacteria titer after feeding dsRNA

[0036] Using fluorescence in situ hybridization (FISH), fluorescently labeled nucleic acid probes are mixed with... C The Las gene specifically binds, and fluorescence signals are detected under a confocal microscope to determine the feeding method for ds. DcFoxO The ovaries of female citrus psyllids C The location and quantity of Las, etc., are used to feed ds GFP The ovaries of female citrus psyllids were used as a control. C The GenBank accession number for the Las gene is L22532.1, and the nucleotide sequence of the nucleic acid probe is shown in SEQ ID NO. 12. The results indicate that knockdown... DcFoxO The expression of this substance can reduce the titer of Huanglongbing fungus in the ovaries of female citrus psyllids. Figure 4 ).

[0037] The specific steps of fluorescence in situ hybridization technology are as follows:

[0038] a. Dissection yielded samples of dsRNA (ds DcFoxO Ovaries of female psyllids (or dsGFP) were collected after 48 h (10 complete ovaries were obtained from each, repeated 3 times). They were placed in 1.5 mL enzyme-free centrifuge tubes containing Carnoy's fixative (according to the volume ratio of glacial acetic acid: anhydrous ethanol: chloroform = 1:3:6) and fixed at 4°C in the dark for 48 h.

[0039] b. Use a pipette to aspirate as much Carnos fixative as possible, add 1 mL of 50% ethanol solution to rinse the ovary for 5 min, repeat 3 times to ensure that the Carnos fixative is completely removed;

[0040] c. After removing 50% ethanol solution from the tube, add 6% hydrogen peroxide ethanol decolorizing solution, let stand at room temperature in the dark until completely decolorized, and then remove the decolorizing solution.

[0041] d. Add 1 mL of PBST and rinse for 10 min, repeat 3 times.

[0042] e. Aspirate PBST, add 500 µL of hybridization buffer without fluorescent probe (5M NaCl, 10% SDS, 30% Formanide, the remainder is sterile water), let stand for 5 min, repeat 3 times, remove the hybridization buffer without fluorescent probe, add hybridization buffer containing 10 pmol / ml fluorescent probe, and let stand at room temperature in the dark for 12 h.

[0043] f. Remove the hybridization buffer containing 10 pmol / ml fluorescent probe, then rinse with PBST for 10 min, repeat 4 times, add hybridization buffer containing 0.1 mg / mL DAPI to the removed liquid, and let stand at room temperature in the dark for 30 min.

[0044] g. Aspirate the hybridization buffer containing 0.1 mg / mL DAPI, rinse 3 times with PBST for 10 min each time, aspirate the ovary and place it on a glass slide, adjust the position of the ovary, mount the slide, and detect the fluorescence signal under a confocal microscope.

Claims

1. Inhibition DcFoxO The application of gene expression in suppressing the reproductive development of citrus psyllids is characterized by, The aforementioned DcFoxO The nucleotide sequence of the gene is shown in SEQ ID NO.1; the described inhibition DcFoxO Gene expression was achieved by feeding citrus psyllids targeted gene expression. DcFoxO The target is achieved through the dsRNA of the gene. DcFoxO The nucleotide sequence of the target region of the gene's dsRNA is shown in SEQ ID NO.8; the inhibition of the reproductive development of the citrus psyllid is to prolong the pre-oviposition period of female citrus psyllids, shorten the oviposition period of female citrus psyllids, reduce the number of eggs laid by female citrus psyllids, inhibit the ovarian development of female citrus psyllids, or reduce the titer of Huanglongbing bacteria in the ovaries of female citrus psyllids.

2. The application according to claim 1, characterized in that, The target DcFoxO The concentration of the gene's dsRNA was 100 ng / µL.

3. The application according to claim 1, characterized in that, Preparation of the target DcFoxO The nucleotide sequence of the upstream primer for the dsRNA of the gene is shown in SEQ ID NO.6, and the targeted gene is prepared accordingly. DcFoxO The nucleotide sequence of the downstream primer of the gene's dsRNA is shown in SEQ ID NO.

7.

4. A preparation for controlling citrus psyllids, characterized in that, The formulation contains targeted DcFoxO The dsRNA of the gene is the active ingredient, as described above. DcFoxO The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the target... DcFoxO The nucleotide sequence of the target region of the gene's dsRNA is as shown in SEQ ID NO. 8; preparation of the target... DcFoxO The nucleotide sequence of the upstream primer for the dsRNA of the gene is shown in SEQ ID NO.6, and the targeted gene is prepared accordingly. DcFoxO The nucleotide sequence of the downstream primer of the gene's dsRNA is shown in SEQ ID NO.

7.

5. The application of the formulation according to claim 4 in the control of 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.

Images