Dialeurodes citri effect protein DcPDI1 and application thereof

Abstract

The application discloses a diaphorina citri effector protein DcPDI1 and application thereof. The amino acid sequence of the diaphorina citri effector protein DcPDI1 is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2. After the diaphorina citri DcPDI1 is knocked out, the oviposition and the development duration of the diaphorina citri are significantly reduced, which indicates that the DcPDI1 plays an important role in the growth and development of the diaphorina citri. The diaphorina citri effector protein DcPDI1 of the application can be a new molecular target, plays a key role in activating the defense response of a host plant and helping the diaphorina citri to feed, and provides new insights into the molecular mechanism of the interaction between the diaphorina citri and the plant.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bio-agriculture, and particularly relates to a citrus psyllid effector protein DcPDI1 and application thereof. BACKGROUND

[0002] The citrus psyllid (Diaphorina citri Kuwayama) belongs to the family of Psyllidae in the order of Hemiptera. Diaphorina citri Currently, the citrus psyllid is mainly controlled by using chemical pesticides. Long-term use of chemical pesticides causes the citrus psyllid to develop drug resistance. Therefore, it is crucial to find a new strategy for preventing and controlling the citrus psyllid.

[0003] Phytophagous insects secrete saliva into host plants during feeding. The components in the saliva play a crucial role in the feeding of the insects on the plants, and not only pre-digest and interpret the nutrients of the plants, but also may contain components, i.e. effector proteins, involved in the early defense response of the host plants. The citrus psyllid is a piercing-sucking insect that feeds on the phloem sap of plants, and injects saliva carrying the Huanglongbing pathogen into the plants during feeding. Identifying potential effector protein genes in the citrus psyllid helps to understand the mechanism of the citrus psyllid in breaking through the defense of the host plants, and helps to develop a new type of plant immune regulator for preventing and controlling the citrus psyllid. SUMMARY

[0004] In order to screen potential effector proteins in the genome of the citrus psyllid, we analyzed the LC-MS / MS data of the sweet orange leaves after the citrus psyllid fed on the sweet orange, and found a gene encoding an effector protein, named DcPDI1. In order to study the function of DcPDI1, we introduced DcPDI1 into E. coli by constructing a vector, and transformed Agrobacterium. We found that the protein can cause programmed cell death (PCD) in the leaves of N. benthamiana grown for 4 weeks. The expression of key genes related to the defense pathway in the tobacco overexpressing DcPDI1 changed significantly, indicating that the protein plays a key role in regulating the defense response of the plants.

[0005] The first object of the application is to provide a citrus psyllid effector protein DcPDI1, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0006] The second object of the application is to provide a gene encoding the citrus psyllid effector protein DcPDI1, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0007] The third object of the application is to provide the application of the citrus psyllid effector protein DcPDI1 or the gene in preventing and controlling the citrus psyllid.

[0008] Preferably, the application of the preparation of silencing the gene of DcPDI1 of Diaphorina citri in preventing Diaphorina citri.

[0009] Preferably, the preparation is dsRNA of the gene encoding DcPDI1 of Diaphorina citri.

[0010] Preferably, the prevention of Diaphorina citri is to reduce the oviposition and developmental duration of Diaphorina citri.

[0011] The fourth object of the present application is to provide the application of the preparation of inhibiting DcPDI1 of Diaphorina citri or the above-mentioned gene in preparing the product for preventing Diaphorina citri.

[0012] The fifth object of the present application is to provide the application of DcPDI1 of Diaphorina citri in regulating the defense response of plants.

[0013] Preferably, the application is to hinder the promotion of DcPDI1 of Diaphorina citri to ROS, the inhibition of DcPDI1 of Diaphorina citri to the related gene of salicylic acid pathway and / or the promotion of DcPDI1 of Diaphorina citri to the related gene of jasmonic acid pathway.

[0014] Preferably, the related gene of salicylic acid pathway is NPR1, PAL and PR1a, the related gene of jasmonic acid pathway is PR3, PR4, LOX2, MYC2 and JIP21, and the plant is tobacco.

[0015] Advantages of the present application:

[0016] The present application studies the function of DcPDI1 in Diaphorina citri, and shows that when DcPDI1 is knocked down, the oviposition and developmental duration of Diaphorina citri are significantly reduced, indicating that DcPDI1 plays an important role in the growth and development of Diaphorina citri. DcPDI1 of Diaphorina citri may be a new molecular target, which plays a key role in activating the defense response of host plants and helping Diaphorina citri to feed, and provides new insights into the molecular mechanism of the interaction between Diaphorina citri and plants. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 DcPDI1 of Diaphorina citri induces necrosis in tobacco leaves.

[0018] Figure 2 H2O2 level changes in tobacco leaves after injection of DcPDI1.

[0019] Figure 3 DcPDI1 of Diaphorina citri sequence information.

[0020] Figure 4 Expression of defense-related genes in tobacco after injection of DcPDI.

[0021] Figure 5 is the citrus psylla DcPDI1 expression of the gene in different age stages.

[0022] Figure 6 is the citrus psylla DcPDI1 expression of the gene in different tissues.

[0023] Figure 7 is the citrus psylla DcPDI1 expression of the gene before and after feeding.

[0024] Figure 8 is the function of DcPDI1 of the citrus psylla DETAILED DESCRIPTION

[0025] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0026] Example:

[0027] 1. LC-MS / MS analysis of citrus psylla saliva

[0028] The citrus psylla and the plant sweet orange were raised in the greenhouse of the Fruit Tree Research Institute of Guangdong Academy of Agricultural Sciences.

[0029] The sweet orange seedlings grown for 2 months were placed in a 15 cm*15 cm*15 cm nylon mesh cage. 50 citrus psylla of different age stages were starved for 2 h, and then placed in the cage to feed on sweet orange leaves for 48 h. Then all the citrus psylla were removed, cleaned, and the fed sweet orange leaves were collected, wrapped in tin foil paper, and placed in liquid nitrogen. The samples were then sent to Shanghai ZK New Life Biotechnology Co., Ltd. for LC-MS / MS analysis.

[0030] 2. Screening of citrus psylla candidate effector proteins

[0031] After LC-MS / MS sequencing of sweet orange leaves, the reference genomes of sweet orange and citrus psylla were downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / datasets / genome / ) through comparison, the sweet orange gene data in the original sequencing data was filtered out, and the remaining data was analyzed, and finally the candidate saliva proteins of citrus psylla were obtained. All candidate proteins were predicted for signal peptide and transmembrane domain, and proteins with signal peptide and without transmembrane domain were left as candidate effector proteins.

[0032] Through LC-MS / MS analysis, 32 potential saliva effector proteins of citrus psylla were predicted.

[0033] 3. Total RNA was extracted from citrus psyllids and reverse transcribed into cDNA. The experimental procedures were performed according to the kit instructions. The RNA extraction kit (DP451) and reverse transcription kit (KR126-02) were both purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0034] 4. Cloning potential effector proteins

[0035] Based on the obtained candidate effector protein gene sequences, specific primers were designed using the NCBI database Primer-BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ), and the primers were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. (Table 1). PCR was performed using citrus psyllid cDNA as a template. High-fidelity enzymes were purchased from Nanjing Novizan Biotechnology Co., Ltd. The PCR amplification system was as follows: 2 × Phanta Max Master Mix 25 μL, upstream primer 2 μL, downstream primer 2 μL, cDNA 2 μL, ddH2O 19 μL. The PCR amplification program was as follows: pre-denaturation 95℃ for 3 min, denaturation 95℃ for 15 sec, annealing 55℃ for 15 sec, extension 72℃ for 15 sec, 34 cycles, and a final extension 72℃ for 7 min. After PCR, the amplification products were electrophoresed on a 1% agarose gel, the gel strips were cut off, and the gel was recovered using a gel extraction kit. The gel extraction kit (DP204) was purchased from Tiangen Biotech (Beijing) Co., Ltd. The extracted gel products were sent to Beijing Tianyi Huiyuan Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the predicted sequences to obtain the full-length cDNA of the effector protein genes.

[0036] By designing specific primers, 27 complete open read frames (ORFs) were cloned using citrus psyllid cDNA as a template.

[0037] Table 1. PCR primer sequences for 27 candidate citrus psyllid effector protein genes.

[0038] ;

[0039] .

[0040] Lowercase letter bases represent homologous arm sequences.

[0041] 5. Construction of candidate effector protein expression vectors

[0042] The homologous recombination reagent (C115-01) was purchased from Nanjing Novizan Biotechnology Co., Ltd. The reaction system was as follows (operated on ice): 5 μL of 2 × ClonExpress Mix, 1 μL of linearized pCAMBIA1300-EGFP vector, 1 μL of insert fragment, and 3 μL of ddH2O. The centrifuge tubes containing the reaction solution were incubated at 50°C for 15 min. The pCAMBIA1300-EGFP vector (V013557) was purchased from Shanghai Newp Biotechnology Co., Ltd. The restriction enzymes were purchased from NEB, with restriction sites of BamHI (R0136V) and XbaI (R0145V). The reaction solution from the previous step was added to 100 μL of DH5α competent cells, incubated on ice for 30 min, then incubated at 42°C for 45 sec, and then incubated on ice for 3 min. 700 μL of antibiotic-free LB liquid was added, and the mixture was incubated at 37°C for 1 h using a shaker at 200 rpm.

[0043] Centrifuge the reaction solution from the previous step at 5000 rpm for 3 min, discard 700 μL of supernatant, resuspend the remaining liquid, and gently spread it onto LB agar plates containing cannabinoids (concentration of 50 μg / mL) using a sterile spreader. Incubate the plates upside down in a 37°C incubator for 15 h.

[0044] Positive clones were selected and sequenced. The sequencing results were compared with the predicted sequences to obtain the recombinant expression plasmids for the effector proteins. Plasmid extraction was performed according to the kit instructions. The plasmid extraction kit (P1001-02) was purchased from Shanghai Maigen Biotechnology Co., Ltd.

[0045] Expression vectors for 27 candidate effector protein genes were successfully constructed.

[0046] The recombinant expression plasmid was transformed into Agrobacterium GV3101. The main steps are as follows: 0.5 μg of the recombinant plasmid was added to GV3101 competent cells, and the cells were incubated on ice for 5 min, in liquid nitrogen for 5 min, at 37℃ for 5 min, and on ice for 5 min. 700 μL of antibiotic-free LB broth was added, and the cells were incubated in a shaker at 28℃ for 2 h. GV3101 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0047] Centrifuge the reaction solution from the previous step at 5000 rpm for 3 min, discard 700 μL of supernatant, resuspend the remaining liquid, and spread it onto LB agar plates containing Cannabidiol (concentration 50 μg / mL). Incubate the plates upside down in a 28℃ incubator for 24 h.

[0048] Select positive clone strains for culture PCR (using a single clone strain as a template, the PCR system, procedure and primers are as in step 4) to obtain Agrobacterium containing recombinant plasmids of effector proteins.

[0049] 6. Transient expression of candidate effector protein genes in Nicotiana benthamiana

[0050] Agrobacterium containing the recombinant plasmid of the effector protein was cultured in 50 mL of LB liquid medium containing Kanamycin (50 μg / mL) at 28°C for approximately 24 h. The cells were collected by centrifugation at 5000 rpm for 10 min, and resuspended in a pre-prepared infection solution (10 mM MES, 10 mM MgCl2). Then, 100 μM acetylsyringone (AS) was added. 600 =0.5-1.0, let stand at room temperature for 2 h. Inject the bacterial solution into the leaves of tobacco plants that have grown for 4 weeks using a 1 mL disposable syringe, injecting 5 sites on each leaf, repeating the injection for 3-5 tobacco plants, injecting 4 leaves per plant. Use Phytophthora infestans ( Phytophthora infestans elicitin (INF1, GenBank: AAV92919.1) and apoptosis regulator (BCL2 associated X, BaX, GenBank: JAAKGM020000011.1) were used as positive controls, and green fluorescent protein (GFP) was used as a negative control.

[0051] Twenty-seven candidate effector proteins were injected into Nicotiana benthamiana cultured for four weeks, with five injection sites per leaf. The results showed that 36 hours after injection, one candidate effector protein (named DcPDI1) induced a necrotic phenotype in the tobacco leaves. Necrotic spots were observed after staining with 3,3'-diaminobenzidine (DAB) and destaining with anhydrous ethanol, indicating that DcPDI1 induced necrosis in tobacco leaves accompanied by the production of reactive oxygen species (ROS). Even after removing the signal peptide sequence of DcPDI1 (MRRLSLLFLLISPLVVFA), it still induced necrosis in tobacco leaves. Figure 1 By detecting the changes in H2O2 content in tobacco leaves after DcPDI1 injection, it was found that the H2O2 level in leaves injected with DcPDI1 was significantly higher than that in leaves injected without DcPDI1, and the H2O2 level reached its peak on days 4-5. Figure 2 The results indicate that DcPDI1 promotes the accumulation of ROS in tobacco.

[0052] Using specific primers (121346 in Table 1), the complete open reading frame (ORF) of DcPDI1 was amplified, with a length of 1494 bp. The sequence is as follows: Figure 3As shown, the nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1.

[0053] The physicochemical properties of the DcPDI1 sequence were analyzed using Expasy software (https: / / www.expasy.org / ) (Table 2). The molecular weight of this gene protein is 55.8 kDa, the isoelectric point is 4.89, the instability coefficient is 37.88, the aliphatic amino acid index is 88.87, and the total hydrophilicity is -0.299, indicating that it is a hydrophilic protein.

[0054] Table 2 Physicochemical properties of the DcPDI1 sequence

[0055] .

[0056] 7. Expression of key genes in defense-related pathways in tobacco after DcPDI1 injection

[0057] To verify whether DcPDI1 induces a defense response in plants, the expression of key genes in defense-related pathways in tobacco was examined after DcPDI1 injection. These included genes related to the salicylic acid (SA) pathway (NPR1, PAL, PR1a) and genes related to the aasmonic acid (JA) pathway (PR3, PR4, LOX2, MYC2, JIP21).

[0058] Two days after injection of empty vector (EV) and Agrobacterium DcPDI1, tobacco leaves were harvested, and RNA was extracted and reverse transcribed. RNA extraction and reverse transcription were performed as in step 3. The qPCR system was as follows: 10 μL of 2 × SupRealQ Ultra HunterSYBR qPCR Master Mix (U+), 0.4 μL each of forward and reverse primers, 1 μL of cDNA template, and 8.2 μL of ddH2O. The reaction system was as follows: 95℃ pre-denaturation for 30 sec; 95℃ denaturation for 10 sec, extension for 60 sec, and 10 sec, for 40 cycles; finally, the instrument's default melting curve was added. The detection primer sequences were DcPDI1-qF: CTGAATTGGCCAAGACTTTC and DcPDI1-qR: CGGACCTTCGTAGTTGACGC. Internal reference primer sequences: NbRPL23-qF: AAGGATGCCGTGAAGAAGATGT, NbRPL23-qR: GCATCGTAGTCAGGAGTCAACC.

[0059] The expression of key genes in defense-related pathways in tobacco was detected 24 h after DcPDI1 injection. The results showed that the expression of genes related to the salicylic acid pathway was significantly inhibited. Figure 4 (a) of the study, while the expression of genes related to the jasmonic acid pathway was significantly increased. Figure 4 (b) in the middle.

[0060] 8. Expression of effector protein DcPDI1 in citrus psyllids

[0061] Citrus psyllids of different instars (eggs, nymphs, and adults) were collected, and different tissues (head, thorax, abdomen, midgut, and antennae) were dissected for RNA extraction, reverse transcription, and qPCR. RNA extraction and reverse transcription were performed as in step 3. The qPCR procedure was the same as in step 7, with the detection primer sequences being DcPDI1-qF: CTGAATTGGCCAAGACTTTC and DcPDI1-qR: CGGACCTTCGTAGTTGACGC. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, with the primer sequences being DcGAPDH-qF: CATGGCAAGTTCAACGGTGA and DcGAPDH-qR: CGATGCCTTCTCAATGGTGG.

[0062] The expression of DcPDI1 in different instars (eggs, nymphs, and adults) and different tissues (head, thorax, abdomen, midgut, antennae, and legs) of the citrus psyllid was detected by qPCR. Figure 5 and Figure 6 The results showed that the expression level of DcPDI1 varied across different larval stages and tissues. The expression level was significantly higher in the egg stage than in the nymph and adult stages; it was also relatively high in the midgut and abdomen, and significantly higher than in other tissues.

[0063] Adult citrus psyllids were collected before feeding (0 h) and at 12 h, 24 h, 36 h, and 48 h after feeding. RNA was extracted, reverse transcribed, and qPCR was performed. The RNA extraction and reverse transcription steps were the same as in step 3. The qPCR steps were the same as in step 7, and the primer sequences were the same as above (DcPDI1-qF, DcPDI1-qR, DcGAPDH-qF, DcGAPDH-qR).

[0064] The expression of DcPDI1 after consuming sweet oranges was examined. It was found that the expression level of DcPDI1 increased significantly with increasing consuming time, reaching a peak at 36 h. Figure 7 ).

[0065] 9. RNAi of DcPDI1

[0066] Following the method of Yu et al. (Yu X, Gowda S, Killiny N, 2017. Double-stranded RNA delivery through soaking mediates silencing of the muscle protein 20 and increases mortality to the Asian citrus psyllid, Diaphorina citri. Pestmanagement science, 73: 1846-1853), RNAi experiments on citrus psyllids were conducted using a feeding method. The main steps are as follows: First, dsRNA was synthesized using the T7 RiboMAX™ Express RNAi System kit (Promega, catalog number P1700) according to the manufacturer's instructions. Then, the dsRNA was diluted to 60 ng / μL. After starving adult citrus psyllids for 4 h, they were anesthetized with CO2 and 0.2 μL of the dsRNA solution was dropped onto the thorax and ventral side of the citrus psyllid between its three pairs of legs using a microsyringe. 60 ng / μL of dsRNA-GFP was used as a control. After 60 seconds of incubation for complete absorption, the samples were transferred to clean disposable petri dishes and then placed in an incubator at 26 ± 2 ℃, 60 ± 5% humidity, and a photoperiod of L:D = 16:8. Surviving adult citrus psyllids were collected at 12 h, 24 h, 36 h, 48 h, and 72 h to detect DcPDI1 expression, with five replicates. The oviposition rate of surviving female citrus psyllids after 72 h of dsRNA treatment was recorded within 24 h, with ten replicates. The developmental time of citrus psyllids after RNAi was analyzed, with a total of 30 individuals counted.

[0067] The primers for dsRNA are as follows:

[0068] dsRNA-DcPDI1-F: TAATACGACTCACTATAGGG CTGACAGCAAGGTTGTTGTTG;

[0069] dsRNA-DcPDI1-R: TAATACGACTCACTATAGGG GAGAAGAACACCAACAGATG;

[0070] dsRNA-GFP-F: TAATACGACTCACTATAGGG GTCCTCGATGTTGTGGCGGA;

[0071] dsRNA-GFP-R: TAATACGACTCACTATAGGG ACCACATGAAGCAGCACGAC;

[0072] The underlined sequence is the T7 promoter sequence. RNAi studies investigated the function of DcPDI1 in the citrus psyllid, and the results showed that knockdown of DcPDI1 significantly reduced oviposition and developmental duration in the citrus psyllid. Figure 8 This indicates that DcPDI1 plays an important role in the growth and development of citrus psyllids.

[0073] The above findings suggest that DcPDI1 may be a novel molecular target that plays a key role in activating the host plant's defense response and assisting the citrus psyllid in feeding, providing new insights into the molecular mechanisms of the interaction between the citrus psyllid and the plant.

[0074] SEQ ID NO.1 (Amino acid sequence of DcPDI1)

[0075] MRRLSLLFLLISPLVVFA DDVTEEDGVLVLTQDNFQSSIEKHDHILVEFYAPWCGHCKQLVPEYSKAALQLATDGHDIKLAKVDATQHTALAEQYGVRGYPTLKFFKKRSIIEYGGGRTAEDIVNWLLKKTGPPAKE FTSVDEIKAFIADSKVVVAGLFKDASSELAKTFNEIASKVDDLVFVTSTNADILAEYSVDDDTVAIFKKFDEGRVNYEGPASDEAALRKFLSTQSLPLVVEFNHETAQKIFGGEIKSHLL VFFSKAAGHYESHFEPVQTVAKDFREKVLFVTINTDEEDHQKILEFFGMSKDEVPSLLRLIRLEEDMAKYKPATSEISVDTVRSFVTEFLAGNLKQHLLSQPLPEDWDKNAVKVLVASNFD EIAFDKSKHVLVEFYAPWCGHCKQLAPIYDKLGEKFADRDDITIAKIDATVNELEHTKITSFPTLKLYAKDDNRVIDYNGERVLEALSNFVESGGKEGGLPSGAQEEQDEDDDQPKRDEL

[0076] The underlined part represents the signal peptide sequence.

[0077] SEQ ID NO.2 ( DcPDI1 (nucleotide sequence)

[0078] ATGCGCCGGTTATCCTTGTTCAAAGAGAGATGAGTTGTAA 。

Claims

1. A citrus psyllid effector protein, DcPDI1, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the citrus psyllid effector protein DcPDI1 as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

3. The application of the citrus psyllid effector protein DcPDI1 as described in claim 1 or the gene as described in claim 2 in the control of citrus psyllids, characterized in that, This refers to the application of a preparation that silences the gene of the citrus psyllid effector protein DcPDI1 in the control of citrus psyllids.

4. The application according to claim 3, characterized in that, The formulation is the dsRNA of the gene encoding the citrus psyllid effector protein DcPDI1.

5. The application according to claim 3, characterized in that, The control of citrus psyllids aims to reduce their egg production and development period.

6. The use of an agent that inhibits the citrus psyllid effector protein DcPDI1 as described in claim 1 or the gene as described in claim 2 in the preparation of a product for the control of citrus psyllids.

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